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https://openalex.org/W2079042468
https://escholarship.org/content/qt5wf9d6p4/qt5wf9d6p4.pdf?t=q0wsf4
English
null
Economic Liberalization and the Computer Industry: Comparing Outcomes in Brazil and Mexico
World development
2,001
cc-by
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UC Irvine UC Irvine Previously Published Works Title Economic Liberalization and the Computer Industry: Comparing Outcomes in Brazil and Mexico Permalink https://escholarship.org/uc/item/5wf9d6p4 Journal World Development, 29(7) ISSN 0305-750X Authors Dedrick, Jason Kraemer, Kenneth L Palacios, Juan J et al. Publication Date 2001-07-01 DOI 10.1016/s0305-750x(01)00038-9 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/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Economic Liberalization and the Computer Industry: Comparing Outcomes in Brazil and Mexico Permalink https://escholarship.org/uc/item/5wf9d6p4 Journal World Development, 29(7) ISSN 0305-750X Authors Dedrick, Jason Kraemer, Kenneth L Palacios, Juan J et al. Publication Date 2001-07-01 DOI 10.1016/s0305-750x(01)00038-9 Copyright Information This work is made available under the terms of a Creative Commo Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed , %             /      F G        )      $         9: !.      )   /      .    9: !.$ (    F G  (. ;  /  /   /     (    $    H      % ;  /   9(  ; %:   . *  )  ' F .    A F    7331'   %   ( +(" %& ,                !              -                 .         /     $ .        / '          /      0     $           /                1223' %            4       5 /      $        0  4 4     /   '      4       $   6   4     /     (    $ 4   4    /     - ' % 5    4      4     .         .   /            /   '  7331   .   '    . ' " #  $     $   $  $  $ /     $   $   /    Powered by the California Digital Library University of California eScholarship.org %  & '( = ' 72$ ' >$ ' 1122?171@$ 7331  7331   .   '    .  )    *     3A3B;>B3CD31DE ;  /        444'  . 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Susceptibility mapping and estimation of rainfall threshold using space based input for assessment of landslide hazard in Guwahati city in North East India
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ABSTRACT: Slopes are the most common landforms in North Eastern Region (NER) of India and because of its relatively immature topography, active tectonics, and intense rainfall activities; the region is susceptible to landslide incidences. The scenario is further aggravated due to unscientific human activities leading to destabilization of slopes. Guwahati, the capital city of Assam also experiences similar hazardous situation especially during monsoon season thus demanding a systematic study towards landslide risk reduction. A systematic assessment of landslide hazard requires understanding of two components, „where‟ and „when‟ that landslides may occur. Presently no such system exists for Guwahati city due to lack of landslide inventory data, high resolution thematic maps, DEM, sparse rain gauge network, etc. The present study elucidates the potential of space-based inputs in addressing the problem in absence of field-based observing networks. First, Landslide susceptibility map in 1:10,000 scale was derived by integrating geospatial datasets interpreted from high resolution satellite data. Secondly, the rainfall threshold for dynamic triggering of landslide was estimated using rainfall estimates from Tropical Rainfall Measuring Mission Multi-satellite Precipitation Analysis. The 3B41RT data for 1 hourly rainfall estimates were used to make Intensity-Duration plot. Critical rainfall was estimated for every incidence by analysing cumulative rainfall leading to a landslide for total of 19 incidences and an empirical rainfall intensity-duration threshold for triggering shallow debris slides was developed (Intensity = 5.9 Duration-0.479). 1. INTRODUCTION Nabagraha-Sunsali hill series situated in extreme north eastern corner (2) Japorigog hill situated in the extreme east, (3) Sonaighuli-Jutikuchi situated in the south, (4) Narakashur hill with Birubari-Dispur-Kacharibasti in the north-eastern side and Jatiya -Kahilipara- Odalbakra in the south and south-western side (5) Nilachal/Kamakhya hill situated in west central, (6) Fatasilhill situated in south-west with Santipur-Gotanagar- Teteliya in the northwestern side, Fatasil Ambari-Ganeshpara- Katabari in the eastern side andGarchuk-Teteliya at its southern side, (7) Jalukbari hill in the extreme south-western corner of the city and (8) Khanapara hill situated in the extreme south- eastern corner of the city. Nabagraha-Sunsali hill series situated in extreme north eastern corner (2) Japorigog hill situated in the extreme east, (3) Sonaighuli-Jutikuchi situated in the south, (4) Narakashur hill with Birubari-Dispur-Kacharibasti in the north-eastern side and Jatiya -Kahilipara- Odalbakra in the south and south-western side (5) Nilachal/Kamakhya hill situated in west central, (6) Fatasilhill situated in south-west with Santipur-Gotanagar- Teteliya in the northwestern side, Fatasil Ambari-Ganeshpara- Katabari in the eastern side andGarchuk-Teteliya at its southern side, (7) Jalukbari hill in the extreme south-western corner of the city and (8) Khanapara hill situated in the extreme south- eastern corner of the city. Landslides, as one of the major natural hazards, account every year for enormous damage to life and property. The down-slope movement of masses of rocks, debris or earth under the influence of gravity is termed as a landslide (Cruden and Varnes, 1996). They are natural events which may occur due to or without human intervention, invariably leading to an increase in shear stress or decrease in shear strength of the slope-forming materials. They are triggered by a variety of external stimuli, such as rainfall, earthquakes, water level change, volcanic activity, forest fires, storm waves or rapid stream erosion etc. The scenario gets further aggravated due to unscientific human activities leading to destabilization of slopes and their eventual failure. North East India falls in the medium to high and high category of the Global Landslide Susceptibility Map and is one of the major natural hazards in the region (Hong and Adler, 2008). Figure 1. Study area In Guwahati city landslide was first reported in the year 1972, Susceptibility mapping and estimation of rainfall threshold using space based input for assessment of landslide hazard in Guwahati city in North East India. Kuntala Bhusan*, Shyam Sundar Kundu$, Kasturi Goswami, S Sudhakar+ North Eastern Space Applications Centre (NESAC), Department of Space, Govt of India, Umiam, Shillong, Meghalaya, India *kuntala.bhusan@nesac.gov.in , $ss.kundu@nesac.gov.in, + s.sudhakar@nesac.gov.in KEY WORDS: Landslide , Guwahati ,Satellite image, Susceptibility map, Intensity-Duration plot The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 2014 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 20 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-8-15-2014 2. STUDY AREA AND SCOPE However, in true sense landslide hazard zonation maps are susceptibility map in absence of temporal component. from Nabagraha hill, subsequently sharp increase in frequency of landslide occurrences are observed. During 80‟s and as well as in 90‟s landslide incidences were reported eight times in each decade. However, during 2000 to 2011 landslide incidences were reported for about twenty three times. It needs to be mentioned here that systematic records of landslide incidences with precise location and time of occurrence is available with District Administration only from 2007.Hence for this study inventory and resulting damages are summarized from various sources, like news paper publications, personal communications other than the official records of District Administration (Figure 2). from Nabagraha hill, subsequently sharp increase in frequency of landslide occurrences are observed. During 80‟s and as well as in 90‟s landslide incidences were reported eight times in each decade. However, during 2000 to 2011 landslide incidences were reported for about twenty three times. It needs to be mentioned here that systematic records of landslide incidences with precise location and time of occurrence is available with District Administration only from 2007.Hence for this study inventory and resulting damages are summarized from various sources, like news paper publications, personal communications other than the official records of District Administration (Figure 2). To fulfil the research objective, preparation of Landslide Susceptibility (LS) map in 1:10,000 scale following satellite data are used, As the cost of landslide damage keeps on increasing dramatically with intensified pace of urbanization on the geologically sensitive slopes, so, a systematic study starting with large scale susceptibility mapping along with assessing the role of rainfall as a triggering factor is felt to be essential to understand the overall scenario. Aim of this study is to understand the city-specific circumstances where intensity of rainfall varies within few square kilometres (sq km). (i) IRS P5, Cartosat I stereo data of 09 January 2011 (2.5m spatial resolution). (ii) World view II multispectral imagery of 25 November 2010 (4band, 0.5m spatial resolution). 0.5m spatial resolution) Apart from this, collateral data (both spatial and non spatial) are collected from both primary and secondary sources. 0.5m spatial resolution) Apart from this, collateral data (both spatial and non spatial) are collected from both primary and secondary sources. Figure 2. 2. STUDY AREA AND SCOPE Graphical representation of landslide inventory data The LS map of Guwahati city is generated by integrating various thematic parameters (Table3) using Analytical Table 3. Details of the thematic criteria considered for LS mapping Thematic criteria Factors Remarks 1 Lithology Considered as Causative Factors Detailed mapping is done from image interpretation followed by their ground verification 2 Fault/Lineament and density 3 Drainage and density 4 Geomorphology 5 Rock weathering 6 Soil depth Soil texture 8 Slope parameter Derived from Cartosat 1 stereo data 9 Slope discontinuity relationship Detailed field mapping of joint pattern is done followed by their stereographic projection 10 Land Use/ land cover Special emphasis was given on anthropogenic factors 11 Rainfall Considered as Triggering Factors Information collected from legacy data 12 Earthquake incidences 13 Landslide Used for evaluating past incidences Contains both archive and geomorphological inventory details used mainly for validation of the result Table 3. Details of the thematic criteria considered for LS mapping Figure 2. Graphical representation of landslide inventory data A systematic assessment of landslide hazard requires understanding of two components, „location‟ and „time‟ of probable incidence under certain geo-environmental setup. In absence of high resolution susceptibility map, systematic archival of landslide inventory data, desired network of rain gauge stations etc, present study elucidates the potential of space-based inputs in addressing the problem. The whole work was divided into two components, landslide susceptibility mapping and estimation of rainfall threshold mainly for shallow landslides that involve poorly consolidated soils or colluviums, weathered materials on altered steep hill slopes. 2. STUDY AREA AND SCOPE Guwahati, the capital city of Assam and the hub for various activities in the entire North Eastern Region (NER) of India, is one of the fast developing city with a population of about 1.6 million. The Guwahati city is a crescent shaped basin, surrounded by lofty hills which are outliers of Meghalaya Plateau. The city is bounded by 26.08º to 26.17 º N latitude and 91.50º to 91.83º E longitudes. Guwahati Municipal Corporation (GMC) covers an area of 216.7sq km. (Figure 1) while the Guwahati master plan area indicates an area of 261.77 sq.km The population density is of the order of 3750 persons/sq km and out of 90 GMC wards almost 34 are completely or partially affected by landslide and related phenomena. For easy reference (in Figure 1), various hills of Guwahati city are numbered as (1) Figure 1. Study area Figure 1. Study area In Guwahati city landslide was first reported in the year 1972, This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-8-15-2014 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-8-15-2014 15 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 2014 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 2014 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 20 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India Landslide Hazard Zonation (LHZ) refers to the division of the land surface into homogenous areas or domains and their ranking according to different degrees of hazard due to mass movement (Varnes 1984). According to this definition, hazard map should both display the location of actual and potential slope-failures and provide information on the time or probability of their return period. However, in true sense landslide hazard zonation maps are susceptibility map in absence of temporal component. Landslide Hazard Zonation (LHZ) refers to the division of the land surface into homogenous areas or domains and their ranking according to different degrees of hazard due to mass movement (Varnes 1984). According to this definition, hazard map should both display the location of actual and potential slope-failures and provide information on the time or probability of their return period. 3. LANDSLIDE SUSCEPTIBILITY MAPPING Since early 70‟s the landslide event has been studied considering various aspects by the scientist‟s world over. Guzzetti et al (2012) have explained elaborately the new space based tools available to address the old problem of landslide mapping. Landslide hazard and risk zoning and mapping for urban and rural areas is widely performed around the world (Carrara et al., 1991, and 1992). In India, use of satellite imageries in landslide studies was evaluated by many experts like Lakhera (1982), Champati ray (2004); Sarkar et al., (2004) and many more. This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-8-15-2014 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-8-15-2014 16 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 2014 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 2014 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 2014 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 20 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India Hierarchal Process (AHP) depending on their role in causing slope instability. The susceptibility map is generated on the assumption that the factors which caused slope-failure in a region are the same as those which will generate landslides in the future (Carrara et al., 1995).Thematic maps representing various geo-environmental parameters of the study area are generated from visual interpretation of satellite images in consultation with collateral data followed by their ground verification during field surveys. Because of mapping scale, local variations in land use pattern, slope parameter derived from digital elevation model (DEM), variations in geological structural relationships etc could not be depicted in the map and hence in many instances these are generalized. more than 60° to almost vertical. These unscientific anthropogenic activities are one of the direct mechanical causes of landslide in the city. Exceptions are there where rock falls were reported, for example, in Shantipur, Kalapahar Pahartali area etc. Figure 5.View of extensive hill cutting and alteration of natural slope as seen in Fatasil hill near Durgasarobar area. 3. LANDSLIDE SUSCEPTIBILITY MAPPING (Data: World view II image) Interpretation and mapping of various thematic data and field survey indicate that the most dominant rock units of the study area are Quartzo Feldspathic Gneiss (QFG) followed by Grey Porphyritic Granite which occurs as an intrusive to the former. Structurally the area is complex, regional metamorphism and various phases of deformations have lead to alteration of the original rock fabric leading to the development of structural features such as folds, faults, joints, lineaments, shear zones etc. Geomorphologically the entire area is divisible into two broad units, namely denudational and depositional areas. All the hills are of denudational origin. Every where some visible sign of rock weathering is seen and heavy rainfall during monsoon season is the main source of weathering. In general, weathering of porphyritic granite is very low compared to QFG.QFG is highly weathered and in many places already converted to soil There is sufficient soil cover on the hill slopes. However, soil cover on the hill tops/ slopes has variable thickness. In general on granitic terrain soil thickness is less compared to gneissic terrain (Figure 4). Also it has been noticed that, on a hill slope, Figure 5.View of extensive hill cutting and alteration of natural slope as seen in Fatasil hill near Durgasarobar area. (Data: World view II image) The lineament map derived from image interpretation depicts that hills of the Guwahati city have moderate to dense set of randomly oriented lineaments. From this, it is implied that infiltration along these lineaments happens to be the most prominent inducing factor as it increases groundwater level following heavy rain. Another major inherent cause of the landslides is the rock type and joint orientation with respect to slope direction. It is observed that mostly slope failure is observed where slope is perpendicular to major discontinuity, which facilitates water percolation and differential weathering causing wedge failure. Most of the landslides in Guwahati city are of complex type while some are of debris slide type, a few are rock fall and rock slide. Rock fall and rock slide are common in the hills where porphyritic granite are exposed. Earth slide, debris slide are common in the hills where deeply weathered gneissic rocks are exposed. In Guwahati, rock falls were reported from Dispur Kacharibasti, Kahilipara Colony Bazar, Odalbakra, Shantipur and from Pubsarania. Debris slides were reported from Noonmati, Nabagraha, Kharguli, Narikalbari, Rupnagar, Birubari,Jutikuchi etc. This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-8-15-2014 3. LANDSLIDE SUSCEPTIBILITY MAPPING doi:10.5194/isprsarchives-XL-8-15-2014 17 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 2014 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 2014 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India namely earthquake and rainfall records. namely earthquake and rainfall records. System (AMSR-E) on Aqua, and the Advanced Microwave Sounding Unit B (AMSU-B) on the National Oceanic and Atmospheric Administration (NOAA) satellite series. The second major data source for the TMPA is the window-channel (–10.7 micron) infrared (IR) data that are being collected by the international constellation of geosynchronous-Earth-orbit satellites, which provide excellent time-space coverage (half- hourly 4x4-km equivalent lat./long. grids) after merged by the Climate Prediction Center of the National Weather Service/NOAA (Janowiak et al., 2001). The IR brightness temperatures are corrected for zenith-angle viewing effects and inter-satellite calibration differences. The TMPA is a TRMM standard product at fine time and space scales and covers the latitude band 50°N - 50°S for the period 1998 to the delayed present. A real-time version of the TMPA merged product was introduced in February 2002 and is available on the NASA TRMM web site (http://trmm.gsfc.nasa.gov). namely earthquake and rainfall records. Figure 6. Landslide Susceptibility Map of the study area There has been several studies on to develop empirical rainfall intensity-duration thresholds at regional scales (Larsen and Simon, 1993; Godt, 2004). For the current study, critical rainfall was estimated for every incidence by analysing cumulative rainfall leading to a landslide altogether for 19 landslide events other than rock fall and rock slide that occurred within Guwahati city during 2000 to 2012 was considered to develop the empirical relation. The lower bound of rainfall intensity- duration threshold was approximately identified as Intensity = 5.9 Duration-0.479 (Figure 7).When coupled with real-time rainfall data, such rainfall intensity-duration thresholds might prove useful for development of early warning systems for shallow landslides (Liritano et al., 1998). The TRMM rainfall estimates were compared with weather station measured rainfall data for one station. A near systematic underestimation was observed in the TRMM estimates particularly for high rainfall events. Figure 6. Landslide Susceptibility Map of the study area 4. ESTIMATION OF RAINFALL THRESHOLD Empirical rainfall intensity-duration threshold triggering landslides (Intensity = 5.9 Duration-0.479) for landslides (dots) that occurred in the Guwahati city during 2000-2012 period). 4. ESTIMATION OF RAINFALL THRESHOLD Early warning of landslides could be one of the most effective ways to minimize risk from landslides particularly for region like NER of India and particularly Guwahati city. Over this region, most of the landslides are rainfall triggered. Rainfall triggered landslides may be predicted by modelling the relationship between rainfall intensity-duration and landslide occurrence exclusively for a particular region (Keefer and Wilson, 1987). Currently no system exists to identify rainfall conditions and amount that may trigger landslides mainly because of the lack of in-situ rainfall measurements over this region. The situation does not seem to improve significantly in near future. Therefore remote sensing based information on rainfall data could be used effectively to develop such empirical relations for different regions (Hong and Adler, 2008). Recent advances and improvements in satellite-based precipitation observation technology and increasing availability of high- resolution geospatial products are providing an unprecedented opportunity to develop a real-time prediction system for rainfall-triggered landslides. In the case of landslides and rainfall, the minimum intensity or duration of rainfall necessary to cause a landslide of shallow soil slips, debris flows, debris slides or slumps (Varnes, 1978) is known as the rainfall threshold for landsliding. Figure 7. Empirical rainfall intensity-duration threshold triggering landslides (Intensity = 5.9 Duration-0.479) for landslides (dots) that occurred in the Guwahati city during 2000-2012 period). The duration and intensity of rainfall play the most important role in triggering landslides. The Tropical Rainfall Measuring Mission (TRMM) Multi-satellite Precipitation Analysis (TMPA) (Huffman et al., 2006) data set has been used for the proposed landslide monitoring system in this study. The near real time hourly rainfall estimates TRMM_3B41RT.007 (collection 7 at 0.25° X 0.25° resolution) have been used so that the developed empirical relation can be used for early warning of landslide using the same product. The TMPA global rainfall map is produced by using TRMM to calibrate, or adjust, the estimates from other satellite sensors, and then combining all the estimates into the TMPA final analysis (Hong and Adler, 2008). The coverage of the TMPA depends on input from different sets of sensors (First, precipitation-related passive microwave data are collected by a variety of low-Earth-orbit satellites, including the TRMM Microwave Imager (TMI) on TRMM, Special Sensor Microwave/Imager on Defense Meteorological Satellite Program (DMSP) satellites, Advanced Microwave Scanning Radiometer for the Earth Observing Figure 7. 3. LANDSLIDE SUSCEPTIBILITY MAPPING Landslide sites scattered over Guwahati city were affected by landslides of varying degrees leading to property and life loss. Old slide zones are mostly stable as these are reshaped by subsequent erosional processes and/or are modified by constructional activities or covered by dense vegetation. Figure 4. Terrain condition; Location- Garchuk Granitic terrain QFG terrain The susceptibility map generated by integrating various thematic criteria is divided into five classes, namely, Very Low Hazard Zone (VLHZ), Low Hazard Zone (LHZ), Moderate Hazard Zone (MHZ), High Hazard Zone (HHZ) and Very High Hazard Zone (VHHZ) (Figure 6) on the basis of natural breaks. Further, for the same study area, Rapid Visual Screening (RVS) for potential landslide areas was carried out by Civil Engineering Department, Assam Engineering College mainly for settlement areas. The results of RVS survey when overlaid on the LS map provides a comprehensive understanding of the result obtained from space based inputs and field based (point) information. About 85% of the locations identified by field survey which requires immediate attention to reduce landslide risk fall in the HHZ and VHHZ classes of the susceptibility map derived from space based inputs. Rest 25% fall in MHZ class, while, majority of the locations which do not require immediate attention also fall in the MHZ class of the susceptibility map. Landslide hazard assessment was carried out from historic record of both landslide occurrences as well as role of triggering factors, Figure 4. Terrain condition; Location- Garchuk type and thickness of the soil determines the type and dimension of landslide also. Hills of Guwahati city do not have steep natural slopes as majority of them fall in between 15° to 25° slope. It is observed that the frequencies of landslides, especially debris and earth slides are also more within this range as anthropogenic activities are more intense on such moderate slope conditions. The land use/ land cover pattern of Guwahati city is in general controlled by natural physical features. It is seen that because of the uneven relief, varied lithological composition of the hills, population growth etc, settlement and land use pattern is quite complex and houses are coming up even on the hill tops and slopes (Figure 5). Especially in QFG terrain, where soil thickness is more because of unscientific construction activities and slope cutting, natural slopes are locally altered to This contribution has been peer-reviewed. The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 2014 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India Ph.D dissertation, University of Colorado, Boulder, CO. Guzzetti, F., Mondini, A .C., Cardinali, M.,Fiorucci, F., Santangelo, M.,Chang K.T., 2012. Landslide inventory maps: New tools for an old problem. Earth Science Reviews, 112, 42- 66. Hong, Y. and Robert, F. Adler., 2008. Predicting global landslide spatiotemporal distribution: Integrating landslide susceptibility zoning techniques and real-time satellite rainfall estimates. Int. J. of Sediment Research, Vol. 23, pp 249-257. Guzzetti, F., Mondini, A .C., Cardinali, M.,Fiorucci, F., Santangelo, M.,Chang K.T., 2012. Landslide inventory maps: New tools for an old problem. Earth Science Reviews, 112, 42- 66. Hong, Y. and Robert, F. Adler., 2008. Predicting global landslide spatiotemporal distribution: Integrating landslide susceptibility zoning techniques and real-time satellite rainfall estimates. Int. J. of Sediment Research, Vol. 23, pp 249-257. During the course of the study, it is realised that systematic records of landslide incidences with precise location and time of incidence is lacking. Forecasting of landslide as well as precise and quantitative assessment of landslide hazard cannot be address in absence of this information. Hence, concerned organisation need to maintain these data for future scientific study and generation of site specific model required for effective management of landslide in Guwahati city. Installation of rain gauge/ AWS network within 10-15km aerial distance from each hill may also be considered to calculate rainfall threshold for landslide in Guwahati city. Huffman G. J., Adler R. F., Bolvin D. T., Gu G., Nelkin E. J., Bowman K. P., Hong Y., Stocker E. F., and Wolff D. B. 2007. The TRMM Multi-satellite Precipitation Analysis: Quasi- Global, Multi-Year, Combined-Sensor Precipitation Estimates at Fine Scale. Journal Hydrometeor, Vol. 8, No. 1, pp. 38–55. Janowiak, J. E., Joyce, R. J., and Yarosh, Y., 2001. A Real- Time Global Half-Hourly Pixel-Resolution IR Dataset and Its Applications. Bull. Amer. Meteor. Soc., 82, pp. 205217. It must be mentioned here that the space based approach followed during the present work is first of its kind to assess landslide hazard (shallow landslides) followed by assessment of rainfall triggering threshold. This type of shallow landslides also has the potential to kill people and destroy properties. At present no system exists at either local or at regional scale to identify rainfall conditions that may trigger landslides. However, a more extensive dataset providing detailed inventory would further increase the accuracy of this kind of exercise. Keefer, D. K. and Wilson, R. C., 1987. Real-time landslide warning during heavy rainfall. Science, Vol. The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 2014 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 2014 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India Particular to landslide hazard in Guwahati city, alarming unscientific anthropogenic activities along with the quantity, duration and kind of precipitation/rainfall have been considered as direct mechanical cause in triggering landslides especially debris and earth slides. For example, debris slide of Ganesh Nagar, Sunsali hills area of 2007, Narakasur hills area od 2012 and similar incidences. However, in few instances, the slope failure problem in the Guwahati city has resulted due to interactions of different geo-environmental causative factors, especially rock slide of Kalapahar, Colony Bazar Hill Side Area (Pahartali) of 1999, Shantipur-Durga Sarobar area of 2004 etc. However, no-correlation with seismic activities is observed for reported landslide incidences. Although most of the slides are shallow landslides of smaller dimension these are disastrous and leading to causalities as they occur mainly in settlement areas. Hence, LS map of Guwahati city is prepared giving main emphasis on land use land cover changes. Carrara A.,Cardinali M., Guzzetti F.,1992. Uncertainty in assessing landslide hazard and risk. ITC Jour., v. 1992:2 172- 183. Carrara, A., Gardinali, M., Guzzetti, F., and Reichenbach, P., 1995. GIS technology in mapping landslide hazard. Carrara A and Guzzetti F (Eds), Geographical Information Systems in Assessing Natural Hazards. Kluwer Pub, Dordrecht, The Netherlands, pp 135-175 and Guzzetti F (Eds), Geographical Information Systems in Assessing Natural Hazards. Kluwer Pub, Dordrecht, The Netherlands, pp 135-175 Champati ray, P.K., 2004. GIS based Landslide Modelling. In Nagarajan R (ed) Landslide disaster: Assessment and monitoring, Anmol Publications Pvt. Ltd, New Delhi. Pp 81-96 Cruden, D.M., and Varnes, D.J., 1996. Landslide Types and Processes. In: A.K. Turner and R.L. Schuster (Eds.) Landslide: Investigation and Mitigation, Transportation Research Board Special Report 247, National Academy Press, WA, pp.36-75 The precipitation intensity that forms the key shallow landslide triggering factor, can be effectively monitored using TRMM TMPA products. The availability of hourly and near real time estimates makes it possible to monitor landslide probability in near real time. The triggering threshold may be used to develop an effective landslide early warning system. However, as many intense rainfall incidents over Guwahati city are aurography induced and are localized, a satellite based rainfall estimate often are not able to recognize them. Godt, J. 2004. Observed and Modelled conditions for shallow landslide in the Seattle, Washington area. The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-8, 2014 ISPRS Technical Commission VIII Symposium, 09 – 12 December 2014, Hyderabad, India 238, No. 13, pp. 921–925. Lakhera , R.C., 1982. Geotechnical studies of Bowala- Nandprayag Hydel-Scheme Area, Chamoli District, U.P., Unpublished report of IIRS, Dehradun. Larsen, M. C., and Simon, A., 1993. A rainfall intensity- duration threshold for landslides in a humid-tropical environment. Puerto Rico: Geografiska Annaler, Vol. 75A, pp. 13–23. 6. ACKNOWLEDGEMENT Authors are thankful to Assam State Disaster Management Authority (ASDMA), Govt of Assam; as part of this research findings are from the project titled “Remote Sensing and GIS Based Input for Hazard, Risk, Vulnerability Assessment of Guwahati city, Silchar, Dibrugarh towns and Dhemaji district, Assam.” sponsored by ASDMA. Sarkar, S., Kanungo, D.P., and Chauhan, P.K.S., 2004. An integrated approach for landslide susceptibility mapping using remote sensing and GIS, Photogrammetry Engineering and Remote Sensing 70(5): 617-625 Varnes, D. J., 1978. Slope movement types and processes. In: Schuster R. L. & Krizek R. J. Ed., Landslides, analysis and control. Transportation Research Board Sp. Rep. No. 176, Nat. Acad. oi Sciences, pp. 11–33. 5. CONCLUSION This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-8-15-2014 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-8-15-2014 18 REFERENCES Carrara, A.,Cardinali, M.,Detti R., Guzzetti, F., Pasqui, V., and Reichenbach, P., 1991. GIS techniques and statistical models in evaluating landslide hazard. Earth Surface Process and Landforms, v. 16, 427-445. Varnes, D.J., 1984. Landslide Hazard Zonation: A Review of Principles and Practice. Paris: United Nations International. This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-8-15-2014 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-8-15-2014 19
https://openalex.org/W4200200165
https://link.springer.com/content/pdf/10.1007/s00432-021-03880-4.pdf
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Solitary colorectal liver metastasis: overview of treatment strategies and role of prognostic factors
Journal of cancer research and clinical oncology
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Abstract The following is an overview of the treatment strategies and the prognostic factors to consider in the therapeutic choice of patients characterized by solitary colorectal liver metastasis. Liver resection is the only potential curative option; neverthe- less, only 25% of the patients are considered to be eligible for surgery. To expand the potentially resectable pool of patients, surgeons developed multidisciplinary techniques like portal vein embolization, two-stage hepatectomy or associating liver partition and portal vein ligation for staged hepatectomy. Moreover, mini-invasive surgery is gaining support, since it offers lower post-operative complication rates and shorter hospital stay with no differences in long-term outcomes. In case of unre- sectable disease, various techniques of local ablation have been developed. Radiofrequency ablation is the most commonly used form of thermal ablation: it is widely used for unresectable patients and is trying to find its role in patients with small resectable metastasis. The identification of prognostic factors is crucial in the choice of the treatment strategy. Previous works that focused on patients with solitary colorectal liver metastasis obtained trustable negative predictive factors such as pres- ence of lymph-node metastasis in the primary tumour, synchronous metastasis, R status, right-sided primary colon tumor, and additional presence of extrahepatic tumour lesion. Even the time factor could turn into a predictor of tumour biology as well as further clinical course, and could be helpful to discern patients with worse prognosis. Keywords  Solitary colorectal liver metastasis · Liver surgery · Local ablation * A. Altendorf‑Hofmann annelore.altendorf-hofmann@gmx.de Solitary colorectal liver metastasis: overview of treatment strategies and role of prognostic factors S. Acciuffi1   · F. Meyer1 · A. Bauschke2 · R. Croner1 · U. Settmacher2 · A. Altendorf‑Hofmann2 Received: 22 August 2021 / Accepted: 7 December 2021 / Published online: 16 December 2021 © The Author(s) 2021 1 Department of General, Abdominal and Vascular Surgery, University Hospital, Leipziger Str. 44, 39120 Magdeburg, Germany 2 Department of General, Abdominal and Vascular Surgery, University Hospital, Am Klinikum 1, 07747 Jena, Germany Journal of Cancer Research and Clinical Oncology (2022) 148:657–665 https://doi.org/10.1007/s00432-021-03880-4 Journal of Cancer Research and Clinical Oncology (2022) 148:657–665 https://doi.org/10.1007/s00432-021-03880-4 REVIEW – CLINICAL ONCOLOGY Surgical treatment In the late 1990s, surgery was offered only to a high selected group of patients with liver-limited disease, confined to only one lobe. These patients should have no more than three metastasis and the larger one should not be bigger than 5 cm. Moreover, the resection had to be technically feasible with at least 1 cm tumour-free margins (Kanas et al. 2012). Following these criteria for about 90% of the patients, the disease was considered unresectable and a 5 year survival of merely 3.3% and 6.1% for syn- chronous and metachronous metastasis, respectively, was reported (Manfredi et al. 2006). Later on, it became clear that the first option to improve overall survival (OS) was to expand surgery indications. It has been evaluated if the use of neoadjuvant chemo- therapy before liver surgery could improve the oncological outcome. Chemotherapy addresses micrometastases and cir- culating tumor cells to reduce recurrent metastatic disease in resectable CLM, and downsizes liver lesions with the aim of achieving resectability in initially unresectable CLM. On the other hand, neoadjuvant treatment strategies with cytotoxic doublets and targeted agents cause time-dependent liver damage and steatohepatitis resulting in increased liver fail- ure after surgery (Aigner et al. 2017). The only controlled, phase-III trial that compared perioperative therapy with FOLFOX (before and after surgery) with surgery alone for patients with liver-only metastases showed that perioperative chemotherapy increases progression-free survival, but there was no statistically significant survival difference between the two groups (Nordlinger et al. 2008, 2013). To date, every patient with a disease control is a candi- date for surgery. Independently of tumor burden, the most important factor that limits the surgeon is the future liver remnant that should be at least 30% or a remnant liver-to- body weight ratio > 0.5 (E Van Cutsem et al. 2016a, b). y g ( ) The presence of limited extrahepatic disease is no longer considered an absolute contraindication. The con- cept of oligometastases has gained importance in colorec- tal cancer, since it has been demonstrated that patients who underwent resection of hepatic and extrahepatic (lung or peritoneum or lymph node) metastasis had survival data exceeding the best outcomes in patients receiving systemic chemotherapy alone (Hadden et al. 2016). Introduction and surgical advances made, only about 25% of patients affected are suitable for resection (Engstrand et al. 2018). The development of surgical techniques, the increasing effi- cacy of modern chemotherapy with or without biological agents, and the emergence of multidisciplinary approaches have allowed patients with conventionally unresectable CLM to undergo surgery (Imai et al. 2019). Colorectal cancer is the third most common tumor in men and the second in women, with an estimate of 1.8 million new cases and approximately 881,000 deaths in 2018 (Bray et al. 2018). Hepatic metastases develop in approximately 50% of colorectal cancer cases (Kanas et al. 2012). The liver, in addition to being the most common site of metastases, is also the first and only area of spread in 30–40% of patients (Hadden et al. 2016). The decision of the treatment strategy should be based on patients’ features and on trustable as well as relevant predic- tive factors to obtain the best oncologic result with as few complications as possible. The following is an overview of the treatment strategies and the prognostic factors to con- sider in the therapeutic choice of patients characterized by, in particular, solitary liver metastasis (SLM), a homogene- ous group of patients, with—therefore—reliable statements on factors and parameters relevant for early post-operative and long-term oncosurgical outcome. Surgery is considered the gold standard treatment and the only potentially curative option for colorectal liver metastases (CLM). Unfortunately, despite the oncological :(0123 1 23456789) 3 Journal of Cancer Research and Clinical Oncology (2022) 148:657–665 658 may be given to colorectal-first or liver-first strategies depending on possible complications related to the pri- mary tumour or on the progression of CLM during the treatment of the primary tumour (Abdalla et al. 2013). A recent study proposed a tumor burden-driven strategy: the liver-first approach has a clear survival advantage in patients with multiple bilobar metastases. For patients with solitary liver metastases or multiple unilobal metas- tasis, the staged procedures (liver first vs primary first) showed to be equivalent. Simultaneous resections should be cautiously considered because of increased mortality risk whenever a major hepatectomy is needed (Giuliante et al. 2021). Surgical treatment A recent retrospective study demonstrated that the presence of metastases in the future liver remnant before PVE had no impact on progression-free or OS, which supports aggressive treatment strategies with clearing of the future liver remnant by percutaneous ablation or atypical resection for patients with bilobar colorectal liver metastasis (Hitpass et al. 2020). The two-stage hepatectomy is indicated for bilateral, multinodular disease that is not amenable to complete removal by single hepatectomy. In the first stage, the less-invaded liver lobe is cleaned of its metastases in combination with contralateral PVE to induce hypertrophy of the future liver remnant. In the second stage, the tumor‐ bearing liver lobe (deportalized liver lobe) is anatomically removed (Imai et al. 2021). (Fretland et al. 2018). A recent review of Kabir et al. showed the results of nine retrospective studies: they agreed that lap- aroscopic liver resection is associated with lower blood loss and blood transfusion rates in addition to shorter length of stay and reduced post-operative complications, with similar mortality rates to open surgery (Kabir et al. 2020). Robot- based liver surgery is considered to be a further development of laparoscopic liver surgery due to its three-dimensional display, the high range of motion of the instruments, the reduction in physiological tremors, and the option of micro- surgical anastomoses. This technique allows a well exposi- tion of the liver, so that tumour location is no longer a limit for mini-invasive surgery. Moreover, robotic surgery proved to be at least comparable with laparoscopic surgery in term of short- and long-term outcomes (Rahimli et al. 2020). Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) was introduced in 2007 in Regensburg (Germany) as a procedure to induce a more rapid hypertrophy of the functional liver remnant. Briefly, ALPPS involves portal vein ligation of the to-be resected segments and in-situ split of the liver during the first stage. The rapid hypertrophic response allows removal of the deportalized liver in the second stage when volume and/ or function of the future remnant liver is sufficient, usually after 1–2 weeks (Schlitt et al. 2017). The high morbidity and mortality rate (respectively, 68% and 12%) that was first described decreased to 27% and 9%, with increasing of the surgical experience (Schadde et al. 2014). Surgical treatment Moreover, the introduction of the “partial ALPPS” that consist in pre- serving the middle hepatic vein and the transection of only 50–80% of liver parenchyma by the first operation lead to a significant reduction in perioperative morbidity (Linecker et al. 2017). The oncological effect of ALPPS for CLM was first described in a cohort study of 510 patients from 22 international centers. The 3 and 5 year cancer-specific survival after ALPPS were 59%, and 33%. The response to neoadjuvant chemotherapy was the strongest independent predictor of short- and long-term oncological outcome. T4 stage, right-sided localization of the primary colon tumor, and K/N-RAS mutation were negative predictor factors. Sur- prisingly, size of liver metastases, bilobar disease, or even concomitant lung metastases had no significant impact on oncological outcome after ALPPS (Petrowsky et al. 2020). Surgical treatment Oligometasta- ses are characterized by a few lesions (generally up to 5) localized to a few sites (up to 2 or occasionally 3), and are associated with the possibility of achieving complete ablation of all tumour masses, using surgical R0 resection and/or local ablation strategies, either initially or possibly after induction treatment with systemic therapy (Eric Van Cutsem et al. 2016a, b). For these patients, a potentially curative approach exists. The decision of which therapy strategy should be pre- ferred must be discussed and made within a multidiscipli- nary board. Furthermore, the potential resectability of the metastasis should always undergo the evaluation of an expert liver surgeon to not miss the opportunity of the resection in cases of big or central located metastasis. It is conventionally accepted that resection of up to 75% of the total liver volume or six liver segments with an adequate inflow and outflow can be safely performed in patients with normal liver paren- chyma. In the setting of steatosis, steatohepatitis, or cirrho- sis, the volume of liver that can safely be resected may have to be dramatically reduced to avoid the risk of post-operative hepatic failure. A work of Truant et al. demonstrated that a remnant liver volume-to-body weight ratio ≤ 0.5% is a trust- able tool to predict post-operative hepatic liver failure and post-operative mortality (Truant et al. 2015). In an attempt to improve the functional liver remnant, various techniques have been employed including portal vein embolization (PVE), two-stage hepatectomy, or portal vein ligation. In the portal vein embolization, after selective catheteriza- tion, one of two portal branches and its ramifications are occluded through the diffusion of embolizing agent. Two-to- four weeks after PVE, patients received a contrast-enhanced liver CT or liver MRI to assess hypertrophy of the future -In patients with synchronous resectable metastases, there are three operative options including,i -Staged resection with colon first strategy,i i -Staged resection with liver-first strategy, or i -Simultaneous resection of both primary tumour and metastases. In case of risk of primary tumour complications such as bleeding, obstruction, or perforation, the colorectal-first approach should be favored. The liver-first sequence is most suited to rectal cancers, so that the liver metasta- ses are not left untreated during the radiation portion of treatment to the rectum. The priority in staged resections 3 3 659 Journal of Cancer Research and Clinical Oncology (2022) 148:657–665 liver remnant. Local ablation strategy For patients with unresectable disease or significant comor- bidities precluding resection, there are several alternative therapies able to spare liver parenchyma. The thermal abla- tion of tumours utilizes image guidance to deliver extreme temperatures to a tumour and its surrounding tissue. The advantages include its adaptability to minimally invasive approaches, the ability to spare liver parenchyma, and a low morbidity rate. Thermal ablation can be performed percu- taneously, laparoscopically, or at laparotomy (Abdalla et al. 2013). Radiofrequency ablation (RFA) is the most com- monly used form of thermal ablation in the treatment of liver tumours. In RFA, needles placed in and around the tumours deliver alternating electrical current in the radiofrequency range that generates heat. The limitation is that it is gener- ally ineffective in tumours bigger than 3 cm, the rate of local recurrence is high, and there is a lack of pathologic evidence that the target lesion was completely ablated with sufficient tumor-negative margins. In addition, the heat generated by RFA can injure adjacent structures (Abdalla et al. 2013). A further technique is the cryoablation that involves liquid nitrogen or argon gas being delivered into the liver tumour, guided by ultrasound. Ice crystal formation during rapid freezing causes destruction of cellular structure and kills the tumour cells. Cryoablation has fallen out of favor because of a higher complication rate and recurrence rate in comparison with RFA (Clark and Smith 2014). Minimally invasive surgery Moreover, 5 year survival was significantly better in the conversion group (Ammori et al. 2013). -Ill located metastasis that did not allow a resection, or -Patient decision. -Ill located metastasis that did not allow a resection, or -Patient decision. RFA patients group received the ablation percutaneously or intraoperatively. The results about survival rates were not homogeneous: only two of six works showed a significant better OS in the surgical group and only the study of Aloia et al. demonstrated a significant better disease-free survival (DFS) for the surgical group (Aloia et al. 2006; Hur et al. 2009). The most studies agree that patient treated with RFA had significant higher local recurrence than surgical-treated patients (Aloia et al. 2006; Lee et al. 2008; Hur et al. 2009; Aliyev et al. 2013). Berber et al. analyzed the factors that influence the recurrence after RFA in a group of 1.032 tumours: tumour size, ablation margin, and blood vessel proximity demonstrate to be independent predictors for local recurrence. Due to blood vessel proximity, it is well known that hepatic blood flow leads to incomplete ablation with its cooling properties and is commonly termed ‘‘heat sink effect’’ (Pillai et al. 2015). Concerning the ablation mar- gin, a margin of 10 mm all around the target tumour is the ideal result of any ablation. However, there is a discrepancy between the success of the ablation based on imaging and real microscopic status at the ablation margin (Petre and Sofocleous 2017). A study of Sotirchos et al. performed a biopsy of the ablation zone immediately after RFA and dem- onstrated that in 24% of the cases, viable tumour cells may be present within the ablation zone, even when postproce- dural imaging displays sufficient ablation margins (Sotirchos et al. 2016). TACE is the administration of embolic particles mixed with chemotherapeutic drugs. It produces a shutdown of blood flow and the simultaneous release of high doses of the drug. The most used chemotherapy is irinotecan (DEBIRI) (Clark and Smith 2014). A comparison of DEBIRI ver- sus systemic therapy showed that DEBIRI prolonged the median survival and was associated with a greater tumour response in the liver. The regional approach was not inferior to FOLFIRI in preventing extrahepatic metastatic progres- sion (Fiorentini et al. 2012). Minimally invasive surgery Percutaneous ethanol injection (PEI) is a local treatment based on the chemical properties of alcohol. Intratumoural injection of highly concentrated ethanol induces protein denaturation, microvascular thrombosis, cellular dehydra- tion, and coagulative necrosis of the tumor. Comparatively to physical ablation techniques, PEI shows several limita- tions. First, the requirement to repeat multiple PEI proce- dures may not be tolerated as well. Second, intra-tumoural ethanol diffusion is less predictable than thermal ablation. Minimally invasive surgery is increasingly being performed on the liver. The OSLO-COMET study was the first rand- omized controlled trial that compared laparoscopic vs open liver resection for CLM. It demonstrated that laparoscopic liver resection was associated with lower post-operative complication rates and shorter post-operative hospital stay as compared to open liver resection, with no differences in blood loss, operation time, or 90 day mortality rates 1 3 660 Journal of Cancer Research and Clinical Oncology (2022) 148:657–665 Moreover, this method has high tumoural recurrence rates and worse disease-free and overall survival rates than RFA (Revel-Mouroz et al. 2017). as surgery in the treatment of small SLM. In the past years, multiple studies compared surgery to RFA for the treatment of SLM (Oshowo et al. 2003; Aloia et al. 2006; Lee et al. 2008; Hur et al. 2009; Kim et al. 2011; Aliyev et al. 2013) (Table 1). They all are retrospective works that compared liver resection versus local ablation: the last one was applied in case of CLM have been shown to depend heavily on the hepatic artery for most of their blood supply, whereas the normal liver parenchyma relies mainly on portal blood flow. Based on this concept, several techniques have been developed such as hepatic arterial infusion chemotherapy (HAI), tran- sarterial chemoembolization (TACE), and selective internal radiation therapy (SIRT). -Patient comorbidities that did not allow major surgery,fi -Patient comorbidities that did not allow major surgery,fi -Not sufficient functional liver remnant after surgery (cir- rhosis or steatohepatitis), -Not sufficient functional liver remnant after surgery (cir- rhosis or steatohepatitis), HAI is a technique that introduce chemotherapy directly through a catheter placed in the hepatic artery. A high con- centration of antimetabolite substance reaches the tumour permitting a lower systemic toxicity. Floxuridine is the most commonly used chemotherapy (Abdalla et al. 2013). A study of Ammori et al. reached a 25% of conversion to complete resection with a combination of HAI and systemic chemo- therapy. Minimally invasive surgery In the SIRT procedure, a single dose of 2.0–3.0 Gbq of ­yttrium90 microspheres is delivered into the hepatic artery that results in selective tumour uptake and radiation (Khatri et al. 2007). The SIRFLOX study is the largest phase-III ran- domized controlled trial to assess the efficacy and safety of chemotherapy plus/minus SIRT in patients with metastatic colorectal cancer. The results failed to show an improvement in median progression-free survival and OS with the addi- tion of SIRT. On the other hand, overall response rate in the liver was improved by the addition of SIRT (van Hazel et al. 2016; Wasan et al. 2017). Resection vs. RFA Regarding the tumour size, two further studies empha- sized the efficacy of RFA for metastasis smaller than 3 cm: in this subgroup of patients, the difference in OS, DFS, and local recurrence-free survival flattened out if compared with surgery (Kim et al. 2011; Hur et al. 2009). In a study of Aliyev et al. 44 laparoscopic RFA were performed and com- pared to 60 liver resections: the OS and DFS did not differ in the two groups; the local recurrence rate after RFA was higher than surgery even if it was one of the lowest reported (18%). Moreover, they reached a local tumour control rate of 82% for metastases smaller than 3 cm. Such results are not RFA is predominantly used for patients with surgery- prohibitive comorbidities or with poor functional hepatic reserve after resection. Ruers et al. demonstrated that abla- tion gives an overall- and progression-free survival benefit in addition to chemotherapy if compared to chemotherapy alone (Ruers et al. 2012, 2015). Since ablation strategy has low complications rate, is less invasive, can be repeated to treat progression or new metastases, and does not require prolonged interruption of chemotherapy, there is a growing interest whether the RFA could reach same oncologic results 1 3 Journal of Cancer Research and Clinical Oncology (2022) 148:657–665 661 Table 1   Resection versus RFA: OS, DFS, and local recurrence rate after treatment of SLM References Patients no 3 year OS (%) p 5 year OS (%) p 3 year DFS (%) p 5 year DFS (%) p Local recur- rence (%) p Oshowo et al. (2003) RFA 25 52.6 n.s – – – – – – – – Resection 20 55.4 – – – – Aloia et al. (2006) RFA 30 57  < 0.001 27  < 0.001 0 0.001 37  < 0.001 Resection 150 79 71 50 5 Lee et al. (2008) RFA 37 – – 48.5 n.s 32.2 n.s 25.7 n.s 29.7  < 0.001 Resection 116 – 65.7 34.5 30.1 6.9 Hur et al. (2009) RFA 25 60 0.026 25.5 0.026 – – – – 28 n.s Resection 42 70 50.1 – – 9.5 Kim et al. (2011) RFA 177 – – 51.1 n.s – – 33.6 n.s – – Resection 278 – 51.2 – 31.6 – Aliyev et al. Resection vs. RFA (2013) RFA 44 81 n.s 57 n.s 32 n.s – – 18 0.012 Resection 60 81 47 36 – 4 reproducible with percutaneous RFA, since the laparoscopic technique was guided by a laparoscopic ultrasonography device that allow to examine in detail the ablation margin and is able to detect additional liver tumour lesions not seen in the preoperative imaging (20% of all cases) (Aliyev et al. 2013). Since the results are very heterogeneous, two recent works performed a meta-analysis to confront local ablative techniques and surgery. Both study recognized a superior- ity in surgical resection in OS and DFS with reduced local recurrence, even in case of small (< 3 cm) resectable SLM (Di Martino et al. 2020; Gavriilidis et al. 2021). Summing up, to date, there are no study that proved the superiority of RFA over surgery and liver resection remains the best oncological treatment for solitary liver metastasis. There is only an ongoing phase-III randomized trial compar- ing thermal ablation to liver resection for patients with small (< 3 cm) CLM (COLLISION trial) (Puijk et al. 2018). The results have not been yet published. Moreover, the LAVA trial was a multicenter randomized controlled trial that wanted to compare thermal ablation versus liver resection surgery in high surgical risk patients. It was stopped after 1 year of pilot study, because only 9 patients were rand- omized (Davidson et al. 2020). Prognostic factors A meta-analysis of 2880 patients with synchronous metasta- ses proved that simultaneous resection was as efficient as a delayed procedure for the long-term oncological outcomes, since there were no significant differences of both OS and DFS, confirming that the delayed procedure is safe and fea- sible (Yin et al. 2013). An important concern is the risk of “missed opportunities” through loss of resectability because For solitary liver metastasis, the size of the liver resec- tion does not influence the post-operative outcome in case an R0 status has been reached (Acciuffi et al. 2018). The importance of the microscopic status of the resection margin is largely described and is widely accepted that a micro- scopically positive (R1) margin is strongly correlated with worse OS (Altendorf-Hofmann and Scheele 2003; Pawlik et al. 2005; Poultsides et al. 2010; Tranchart et al. 2013). Since the 1980s, there has been a general consensus that the optimal surgical margin during resection of CLM should measure ≥ 1 cm. In fact, some authors even suggested that inability to accommodate a 1 cm margin should perhaps pre- clude a patient from being considered for hepatic resection (Cady and Stone 1991; Ekberg et al. 1986). More recently, surgeons have progressively moved from the “1 cm” rule to the “1 mm” rule, and multiple reports have demonstrated that margin width does not affect the outcome as long as a negative margin is achieved (Altendorf-Hofmann and Scheele 2003; Poultsides et al. 2010; Viganò et al. 2018). This aspect increases the responsibility of the surgeon who should strive assiduously to achieve complete macro- and microscopic resection of CLM to help ensure the best out- comes for the patient. Actually, there is an additional distinc- tion that should be done between standard R1 parenchymal resection (tumour exposure along the parenchymal transec- tion line) and R1 vascular resection (detachment of CLM from major intrahepatic vessels). Although both are types of R1 resection, a recent study demonstrated that R1 vascular resection guaranteed exactly the same local control as R0 resection, confirming the hypothesis that vessels in contact with CLM limit tumor spread (Viganò et al. 2016). The role of adjuvant chemotherapy after resection of CLM has not been yet defined. Several trials that try to find an answer failed to reach trustable results due to poor recruitment, early termination, and usage of non-modern chemotherapy by today’s standards (G. Chan and Chee 2020). Prognostic factors The most recent trial JCOG0603 compared 6 months of post-operative mFOLFOX6 with observation alone. The chemotherapy arm had a significant better 3 year DFS. However, the 5 year OS was 83.0% in the control arm compared to 69.5% for patients receiving adjuvant chemotherapy (Kanemitsu et al. 2021). Prognostic factors There is a general consensus that patients with SLM have a significant better prognosis than patients with multiple metastases (K.-M. Chan et al. 2014; Brouquet et al. 2011; Abdalla et al. 2004). A meta-analysis summarized infor- mation of survival after liver resection for CLM of papers published between 1999 and 2010. For SLM, a 5 year OS of 47.4% was reported, not so far from the result of multiple liver metastases (40.3%) (Kanas et al. 2012). To improve the reliability and applicability of the results, two recent works, one of which was developed from our study group, focused on a homogeneous group of patients characterized by SLM treated with curative intent surgery. Both studies recog- nized the presence of lymph-node metastasis as a strong negative prognostic factor, with worse OS and DFS such as higher rate of tumour recurrences. The size of metastasis, the T-category, and the site of the primary tumour (colon versus rectum) did not affect the survival rates (Shin et al. 2019; Acciuffi et al. 2018). There is a strong evidence that synchronous metastasis and presence of extrahepatic tumour are factors associated with worse prognosis (Lee et al. 2008; Acciuffi et al. 2018). Bold figures show statistically significant diffenences p< 0.05 fi Moreover, right-sided primary colon tumor have worse survival rates if related to left-sided primary colon tumour (Benedix et al. 2010). Right-sided colon tumour shows more often activating mutations of RAS, BRAF, and PIK3CA genes. In contrast, left-sided colon tumour is characterized by a more frequent occurrence of chromosomal instability 1 3 3 Journal of Cancer Research and Clinical Oncology (2022) 148:657–665 662 surgery (Viganò et al. 2013). In our previous work, about half of the recurrences appeared in the first 12 months after resection (Acciuffi et al. 2018). Multiple works showed that early recurrence is highly associated with worse disease-spe- cific survival (Tan et al. 2013; Viganò et al. 2021). A recent work of Fromer et al. showed that in a group of 259 major liver resections for CLM, 12.6% had a recurrence event within 6 months after operation and experienced a futile liver resection with significantly lower OS (Fromer et al. 2021). We can suppose that patients with early recurrence probably had subclinical undetected liver lesions at the time of surgery and thus small that they could not be identified even with the intraoperative ultrasound (occult metastases). Prognostic factors In these cases, the surgeon did not resect the “only one” metastasis but rather the first detectable lesion of multiple metastases. A previous study of Yokoyama et al. demon- strated that immunohistochemically detected hepatic micro- metastases were found in about a half of patients treated with curative liver resection. The presence of micrometastases was a predictive factor for increased risk of intrahepatic recurrence and was a poor prognostic indicator of survival (Yokoyama et al. 2002). Since the modern diagnostic tools are not able to identify the micrometastases yet, only the time factor permits to evaluate the aggressiveness of the tumour and discern patients with fast tumour progression. To recognize among the SLM the one with worse biology, a “test-of-time” strategy for those patients with poor prognos- tic indicator could be taken into consideration. If after a few months, a re-stage of the patients reveals new metastases, it can be assumed that they were present as subclinical unde- tected micrometastases at the time of diagnosis. After the reevaluation, the strategy of treatment has to be discussed again to reach a complete surgical resection of the tumour or, in case of unresectable disease, another treatment strategy should be adopted such as liver-parenchyma-sparing surgical technique, local ablation, or systemic therapy or a combina- tion of the previous. Lambert et al. delayed resection for 3–6 months in 42 of 73 total patient with liver metastases, to allow occult disease to become clinically detectable and spare the morbidity and mortality of major surgery in those patients that become unresectable during the interval to reevaluation. There was no significant difference in survival between those patients who underwent immediate hepatic resection and those who underwent interval reevaluation. In the reevaluation group, 28% of the patient were spared the morbidity of noncurative surgery (Lambert et al. 2000). A meta-analysis of 2880 patients with synchronous metasta- ses proved that simultaneous resection was as efficient as a and a gene expression profile corresponding to an activation of the epithelial growth factor receptor (EGFR) pathway. Patients with RAS wild-type left-sided colon tumour had a markedly greater benefit from anti-EGFR-based therapy (Holch et al. 2017). surgery (Viganò et al. 2013). In our previous work, about half of the recurrences appeared in the first 12 months after resection (Acciuffi et al. 2018). Multiple works showed that early recurrence is highly associated with worse disease-spe- cific survival (Tan et al. 2013; Viganò et al. Prognostic factors 2021). A recent work of Fromer et al. showed that in a group of 259 major liver resections for CLM, 12.6% had a recurrence event within 6 months after operation and experienced a futile liver resection with significantly lower OS (Fromer et al. 2021). We can suppose that patients with early recurrence probably had subclinical undetected liver lesions at the time of surgery and thus small that they could not be identified even with the intraoperative ultrasound (occult metastases). In these cases, the surgeon did not resect the “only one” metastasis but rather the first detectable lesion of multiple metastases. A previous study of Yokoyama et al. demon- strated that immunohistochemically detected hepatic micro- metastases were found in about a half of patients treated with curative liver resection. The presence of micrometastases was a predictive factor for increased risk of intrahepatic recurrence and was a poor prognostic indicator of survival (Yokoyama et al. 2002). Since the modern diagnostic tools are not able to identify the micrometastases yet, only the time factor permits to evaluate the aggressiveness of the tumour and discern patients with fast tumour progression. To recognize among the SLM the one with worse biology, a “test-of-time” strategy for those patients with poor prognos- tic indicator could be taken into consideration. If after a few months, a re-stage of the patients reveals new metastases, it can be assumed that they were present as subclinical unde- tected micrometastases at the time of diagnosis. After the reevaluation, the strategy of treatment has to be discussed again to reach a complete surgical resection of the tumour or, in case of unresectable disease, another treatment strategy should be adopted such as liver-parenchyma-sparing surgical technique, local ablation, or systemic therapy or a combina- tion of the previous. Lambert et al. delayed resection for 3–6 months in 42 of 73 total patient with liver metastases, to allow occult disease to become clinically detectable and spare the morbidity and mortality of major surgery in those patients that become unresectable during the interval to reevaluation. There was no significant difference in survival between those patients who underwent immediate hepatic resection and those who underwent interval reevaluation. In the reevaluation group, 28% of the patient were spared the morbidity of noncurative surgery (Lambert et al. 2000). Conclusion Surgery remains the first choice of treatment for solitary colorectal liver metastases. Nowadays, the surgical skills and the modern multidisciplinary hypertrophy strategy lead an increased number of patients to be eligible for surgery. In case of unresectable disease, multiple local ablation treat- ments should be considered in combination to chemother- apy. Trustable negative predictive factors that can help in the interdisciplinary treatment decision are N-category of the primary tumor, synchronous metastasis, extrahepatic tumour location, right-sided primary colon tumor, and R1 status. A test-of-time is a possibility for high selected patients to reveal subclinical metastases permitting to treat all the tumour manifestations or to offer an alternative strategy of therapy. https://​doi.​org/​10.​1007/​s00432-​018-​2583-y Aigner F, Pratschke J, Schmelzle M (2017) Oligometastatic disease in colorectal cancer-how to proceed? Visc Med 33:23–28. https://​ doi.​org/​10.​1159/​00045​4688 Aliyev S, Agcaoglu O, Aksoy E, Taskin E, Vogt D, Fung J, Siperstein A, Berber E (2013) Efficacy of laparoscopic radiofrequency ablation for the treatment of patients with small solitary colo- rectal liver metastasis. 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Lancet Oncol 18(9):1159–1171. References https://​doi.​org/​10.​ 1097/​01.​sla.​00002​17629.​94941.​cf Hadden WJ, de Reuver PR, Brown K, Mittal A, Samra JS, Hugh TJ (2016) Resection of colorectal liver metastases and extra-hepatic disease: a systematic review and proportional meta-analysis of survival outcomes. HPB (oxford) 18(3):209–220. https://​doi.​org/​ 10.​1016/j.​hpb.​2015.​12.​004 Nordlinger B, Sorbye H, Glimelius B, Poston GJ, Schlag PM, Rou- gier P, Bechstein WO et al (2008) Perioperative chemotherapy with FOLFOX4 and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC Intergroup Trial 40983): a randomised controlled trial. Lancet 371(9617):1007– 1016. https://​doi.​org/​10.​1016/​S0140-​6736(08)​60455-9 Hitpass L, Heise D, Schulze-Hagen M, Pedersoli F, Ulmer F, Amygda- los I, Isfort P et al (2020) Primary tumor location is a prognostic factor for intrahepatic progression-free survival in patients with colorectal liver metastases undergoing portal vein embolization as preparation for major hepatic surgery. Cancers 12:1638. https://​ doi.​org/​10.​3390/​cance​rs120​61638 Nordlinger B, Sorbye H, Glimelius B, Poston GJ, Schlag PM, Rougier P, Bechstein WO et al (2013) Perioperative FOLFOX4 chemother- apy and surgery versus surgery alone for resectable liver metasta- ses from colorectal cancer (EORTC 40983): long-term results of a randomised, controlled, phase 3 trial. Lancet Oncol 14(12):1208– 1215. https://​doi.​org/​10.​1016/​S1470-​2045(13)​70447-9 Holch JW, Ricard I, Stintzing S, Modest DP, Heinemann V (2017) The relevance of primary tumour location in patients with metastatic colorectal cancer: a meta-analysis of first-line clinical trials. Eur J Cancer 70(January):87–98. https://​doi.​org/​10.​1016/J.​EJCA.​2016.​ 10.​007 Oshowo A, Gillams A, Harrison E, Lees WR, Taylor I (2003) Com- parison of resection and radiofrequency ablation for treatment of solitary colorectal liver metastases. Br J Surg 90(10):1240–1243. https://​doi.​org/​10.​1002/​bjs.​4264 Hur H, Ko YT, Min BS, Kim KS, Choi JS, Sohn SK, Cho CH, Ko HK, Lee JT, Kim NK (2009) Clinical surgery-international comparative study of resection and radiofrequency ablation in the treatment of solitary colorectal liver metastases. Am J Surg 197(6):728–736. https://​doi.​org/​10.​1016/j.​amjsu​rg.​2008.​04.​013 Pawlik TM, Scoggins CR, Zorzi D, Abdalla EK, Andres A, Eng C, Curley SA et al (2005) Effect of surgical margin status on survival and site of recurrence after hepatic resection for colorectal metas- tases. Ann Surg 241(5):715–722. https://​doi.​org/​10.​1097/​01.​sla.​ 00001​60703.​75808.​7d Imai K, Adam R, Baba H (2019) How to increase the resectability of initially unresectable colorectal liver metastases: a surgical 1 3 665 Journal of Cancer Research and Clinical Oncology (2022) 148:657–665 Petre EN, Sofocleous C (2017) Thermal ablation in the management of colorectal cancer patients with oligometastatic liver disease. Visc Med 33:62–68. References https://​doi.​org/​10.​1016/​S1470-​2045(17)​30457-6 Schlitt HJ, Hackl C, Lang SA (2017) ‘In-situ split’’ liver resection/ alpps—historical development and current practice’. Visceral Med 33(6):408–412. https://​doi.​org/​10.​1159/​00047​9850 Yin Zi, Liu C, Yajin Chen Yu, Bai CS, Yin R, Yin D, Wang J (2013) Timing of hepatectomy in resectable synchronous colorectal liver metastases (SCRLM): simultaneous or delayed? Hepatology 57(6):2346–2357. https://​doi.​org/​10.​1002/​hep.​26283 Shin H, Kim CW, Lee JL, Yoon YS, Park IJ, Lim SB, Chang Sik Yu, Kim JC (2019) Solitary colorectal liver metastasis after curative intent surgery: prognostic factors affecting outcomes and survival. ANZ J Surg 89(1–2):61–67. https://​doi.​org/​10.​1111/​ans.​14933 Yokoyama N, Shirai Y, Ajioka Y, Nagakura S, Suda T, Hatakeyama K (2002) Immunohistochemically detected hepatic micrometastases predict a high risk of intrahepatic recurrence after resection of colorectal carcinoma liver metastases. Cancer 94(6):1642–1647. https://​doi.​org/​10.​1002/​cncr.​10422 Sotirchos VS, Petrovic LM, Gönen M, Klimstra DS, Do RKG, Petre EN, Garcia AR et al (2016) Colorectal cancer liver metastases: biopsy of the ablation zone and margins can be used to predict oncologic outcome. Radiology 280(3):949–959. https://​doi.​org/​ 10.​1148/​radiol.​20161​51005 Publisher's Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Tan MCB, Butte JM, Gonen M, Kemeny N, Fong Y, Allen PJ, Peter Kingham T, Dematteo RP, Jarnagin WR, D’Angelica MI (2013) Prognostic significance of early recurrence: a conditional survival analysis in patients with resected colorectal liver metastasis. HPB (oxford) 15(10):803–813. https://​doi.​org/​10.​1111/​hpb.​12136 1 3
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Effects of Silicon Amendment on Soilborne and Fruit Diseases of Avocado
Plants
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Received: 1 September 2017; Accepted: 16 October 2017; Published: 20 October 2017 Received: 1 September 2017; Accepted: 16 October 2017; Published: 20 October 2017 Abstract: The effects of silicon (Si) amendment have been studied in several plant/pathogen interactions; however, studies in horticultural tree crops are limited. Effects of amendment with soluble potassium silicate (AgSil®32, approximately 30% available Si), or milled cement building board by-products (Mineral Mulch (MM) or Mineral Dust (MD), containing 5% available Si) were investigated in field and greenhouse trials with avocado. Orchard soil drench applications with potassium silicate improved yield and quality of fruit, but visual health of trees declining from Phytophthora root rot (PRR) was not affected. Orchard spray or trunk injection applications with potassium silicate were ineffective. Amendment of potting mix with MM and MD reduced root necrosis of avocado seedlings after inoculation with Calonectria ilicicola, an aggressive soilborne pathogen causing black root rot. Application of MM to mature orchard trees declining with PRR had a beneficial effect on visual tree health, and Si accumulation in leaves and fruit peel, after only 10 months. Products that deliver available Si consistently for uptake are likely to be most successful in perennial tree crops. Keywords: Phytophthora; potassium silicate; available silicon; anthracnose; stem end rot Plants 2017, 6, 51; doi:10.3390/plants6040051 plants plants www.mdpi.com/journal/plants plants plants Effects of Silicon Amendment on Soilborne and Fruit Diseases of Avocado Elizabeth K. Dann * and Duy P. Le † Elizabeth K. Dann * and Duy P. Le † Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Brisbane, QLD 4072, Australia * Correspondence: e.dann@uq.edu.au; Tel.: +61-7-3443-2455 † Current address: New South Wales Department of Primary Industries, Narrabri, NSW 2390, Australia; lephuduy08@gmail.com Elizabeth K. Dann * and Duy P. Le † Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Brisbane, QLD 4072, Australia * Correspondence: e.dann@uq.edu.au; Tel.: +61-7-3443-2455 † Current address: New South Wales Department of Primary Industries, Narrabri, NSW 2390, Australia; lephuduy08@gmail.com Queensland Alliance for Agriculture and Food Innovation, University of Queensland, * Correspondence: e.dann@uq.edu.au; Tel.: +61-7-3443-2455 † Current address: New South Wales Department of Primary Industries, Narrabri, NSW 2390, Australia; lephuduy08@gmail.com 1. Introduction Silicon (Si) has been shown to reduce disease and increase plant growth in a number of agricultural crops, predominantly cereal grains and dicotyledonous annual crops such as cucumber and tomato, and the topic has been recently reviewed [1,2]. Likely mechanisms include formation of physical barriers to pathogen ingress, activation of biochemical plant defenses, and influence on absorption of essential nutrients. The benefits of Si amendment for ameliorating toxicities and abiotic stresses is also well established [3], which may also influence plant health and disease suppression, although those interactions have not been thoroughly investigated. The reports concerning effects of amendment with soluble Si to horticultural tree crops is still scarce. Field or soil applications of various formulations and concentrations of Si to mango, citrus or apple reduce diseases caused by bacterial [4,5] or fungal pathogens [6–9]; enhance resistance to insect pests [10] and physiological disorders [11]; and increase yield [7], fruit size [12], and biomass [13]. Reports of Si amendment in avocado primarily concern effects on fruit quality including postharvest physiology and anthracnose disease, caused by Colletotrichum spp. and health of trees affected by Phytophthora root rot (PRR), caused by Phytophthora cinnamomi (Pc). Many of these reports have not been published in the mainstream literature, thus are reviewed in detail below. Studies undertaken in 2004 provided the first indication that soluble Si, applied as trunk injections (common in avocado for the delivery of phosphonate to protect roots from PRR), could reduce anthracnose development as fruit ripen after harvest. “Hass” avocado trees carrying crops of 2 of 15 Plants 2017, 6, 51 developing fruit were injected with approximately 60 mL of 1000 ppm potassium silicate, and fruit harvested for quality assessments 2, 8 and 12 weeks later. There was no significant difference in anthracnose disease when harvested two weeks after injection treatment, however, anthracnose was significantly reduced by Si injection compared with controls when fruit were harvested and ripened at the later harvests, with 80% reduction in severity, and increased shelf life, recorded in fruit harvested two months after Si injection treatment [14]. A subsequent study confirmed that injecting with 1000 or 2000 ppm potassium silicate during early fruit development (28 weeks prior to harvest), or 12 weeks prior to harvest, reduced anthracnose development compared with untreated controls by at least 40%. 1. Introduction Treatment with 1000 ppm during fruit development and 2000 ppm 12 weeks prior to harvest had greater than 60% reduction in fruit anthracnose which was significantly less than that recorded in fruit from untreated trees. Interestingly, 1000 ppm injected at each of the treatment times did not significantly reduce anthracnose, and there was no effect on stem end rot disease in fruit [15]. Five orchard sprays at intervals of approximately six weeks throughout fruit development with 1000 ppm potassium silicate were not effective at reducing postharvest disease or improving marketability of “Hass” avocado fruit, compared with industry standard fungicide applications [16]. The effects of Si soil drench applications in orchards, or as post-harvest applications, on avocado fruit physiology and accumulation of Si have been assessed. Soil drench applications (concentrations not reported) in-field during fruit development had no effect on accumulation of Si in the exocarp and mesocarp of fruit, or on CO2 production in ripe fruit [17]. However, dipping harvested fruit into high concentrations (<2500 ppm) of potassium silicate suppressed respiration (reduced CO2) and ethylene production. Energy dispersive X-ray analyses demonstrated that Si accumulated in the exocarp of such fruit approximately 14% wt, with <1% in the mesocarp. Although no data were presented, the authors stated that none of the Si treatments affected anthracnose disease in fruit, and thus concluded that, despite the suppression of ripening parameters, an enhanced shelf life was not achieved because of the severity of postharvest disease [17]. Similarly, there was no significant difference in anthracnose upon ripening when harvested fruit were dipped in 1000 ppm potassium silicate, compared with untreated control fruit, although there was an indication of enhanced defense enzyme activity [18]. Tesfay et al. [19] reported the potential of potassium silicate postharvest treatment to improve avocado fruit quality via maintenance of fruit moisture, increased catalase enzyme activity, and increased accumulation of phenolic compounds. In preliminary greenhouse tests with one-month-old avocado seedlings, several Si products, including potassium silicate), calcium silicate (wollastonite) and magnesium silicate were not as effective at reducing root necrosis caused by Pc as phosphonate, a standard treatment for PRR [20]. There were some indications of root health improvement following some treatments, but effects were inconsistent. 1. Introduction Conversely, greenhouse tests undertaken in South Africa with advanced nursery stock (12–18 months old) showed that one soil drench application of potassium silicate before and four applications after inoculation with Pc resulted in significantly healthier root systems than inoculated control plants, and were comparable to those treated with phosphonate, in two of four experiments [21]. Root mass and new root growth was increased by Si, suggesting that Si may stimulate root growth and fortification under high Pc disease pressure. A single Si soil drench application a day prior to inoculation was not effective at reducing root necrosis, and the authors suggested that for effective disease suppression, plant-available Si should be applied continuously, due to the rapid polymerization of soluble Si. A day was most likely not sufficient time for Si to be translocated and deposited and for defenses to pathogen infection to be activated. Effects of soil drench applications on canopy health and root densities were assessed in an orchard trial with mature trees. Twenty liters per tree of potassium silicate, 20 mL/L (approximately 4000 ppm), was applied once, twice or three times at four monthly intervals, in the first year, and repeated in Year 2. Two or three applications resulted in significantly healthier canopies, compared with untreated trees, which declined in health over time [22,23]. The trees receiving two or three Si treatments per year had the highest root densities (determined by image analyses of 0.5 m2 soil surface area of each tree), 3 of 15 Plants 2017, 6, 51 compared with controls or those receiving industry standard injections with phosphonate, suggesting that Si may aid in the regeneration of roots in PRR-compromised trees. One or two soil drench applications with Si reduced tree yields, however, three applications increased yields. The authors suggested that the optimal timing of applications was during flowering/fruit set, before fruit drop, and prior to the summer root flush, however the timing of applications should be determined by soil structure as Si leaches easily, i.e., sandy soil will require more regular applications. There were inconsistent effects against postharvest diseases (anthracnose and stem end rot) and physiological disorders of fruit. Stem injections of Si did not improve tree health or increase root growth [23]. There was an indication of increased leaf boron uptake in Si treated trees, but no consistent effect on leaf or root Si content [23]. Dixon et al. 1. Introduction [24] applied sodium metasilicate as a soil drench (delivering 100 or 200 ppm a.i. soluble Si), to avocado trees in severe decline due to PRR, and assessed tree health 6 and 13 months later. The single application did not significantly improve tree health at the final assessment; however, there was a slight improvement in trees treated with the higher rate. Root health was improved at six months compared with untreated controls, and the authors concluded that multiple applications may have been more effective. p pp y Phytophthora root rot remains the key challenge to orchard productivity for avocado growers in Australia, particularly in the eastern tropical and sub-tropical regions which have encountered high rainfall in recent years, favoring this root disease. PRR has significantly impacted productivity at orchard and whole-industry level due to tree decline and death, an abundance of small fruit which can be difficult to market, and poorer quality fruit from PRR affected trees [25]. The nectriaceous soilborne fungi Calonectria ilicicola and Dactylonectria spp. are common nursery pathogens infecting roots and may cause tree deaths with 12 months of planting [26,27]. The most problematic fruit diseases are anthracnose, caused by Colletotrichum spp. and stem end rots, caused by Colletotrichum spp., and/or fungi in the family Botryosphaeriaceae [25]. Currently, orchard management includes a range of cultural and chemical practices, such as optimized drainage, irrigation and fertilizer regimes, mulching and targeted sprays with fungicides for fruit disease and injections with phosphonate for PRR. It is wise for the industry to remain open-minded about alternative or complementary management options. The successful management of pests and diseases will rely on an integrated approach, which utilizes as many cultural, chemical and agronomic options as possible. The aim of the current research was to investigate effects of two products with high available Si contents, for their effects on avocado canopy health, fruit quality and accumulation of Si. Greenhouse trials were undertaken over 3–4 months where treated seedlings were inoculated with root pathogens Calonectria ilicicola or Phytophthora cinnamomi. Trials at several sites were conducted over 2–3 years in established orchards where trees were declining due to PRR. Potassium silicate (SiO2/K2O), for example AgSil®32 (PQ Australia P/L), has been available for many years, and has been used in many of the studies described above. Mineral Mulch (MM) and Mineral Dust (MD) are cement building board by-products available commercially in Australia from Shiftwaste P/L since 2014. 1. Introduction Research with MM to date has concentrated on sugarcane, a Si accumulator, where yield increases of up to 16% above untreated controls have been recorded after its application [28]. Anecdotal data also showed positive response of capsicum and macadamia to MM; however, neither MM nor MD have been evaluated in avocado. Trials in commercial orchards over multiple years are essential, where assessments of yield, tree health, fruit quality and packout data are critical for determining the role of such amendments in improving orchard productivity. 2.1. Field Trials with AgSil 32 (2014–2016) Trials were conducted on-farm at Childers, Goodwood, Beechmont (QLD) and Comboyne (NSW). At Site 1, Childers, there were no significant effects on tree health of Agsil drench applications (5000 ppm solution of Si delivered in 20 L water per tree on three occasions annually) eight months 4 of 15 Plants 2017, 6, 51 to two years after the initiation of the trial (Table 1). In April 2015, one year after commencement of the trial, images of root growth within the “windows” placed under each tree were captured, and analyzed with WinRhizo software. There were no significant differences in root lengths, area or diameter between the trees treated with Agsil drench vs. non-drenched trees (data not shown). Yields were recorded mid-way through the trial in July 2015 when yields from Agsil drenched trees were not significantly different compared with non-drenched trees (Table 1). Single bins of fruit from each treatment were run through a commercial packingshed (hence no statistical analysis was possible). There were small increases in the percentage of fruit in premium grade and total percent packouts from the Agsil drench treated trees, however, there was a 40% increase in 2nd grade fruit (and decrease in 3rd grade fruit) from Agsil-drenched trees compared with non-drenched trees (Table 2). Table 1. Tree health and yield data for 2014–2016 from Agsil amendment trial—Site 1, Childers. Table 1. Tree health and yield data for 2014–2016 from Agsil amendment trial—Site 1, Childers. Tree Health a Yield (kg/tree) April 2014 December 2014 March 2015 July 2016 May 2015 No Agsil Drench 5.36 5.81 6.18 4.55 79.9 Agsil Drench 5.39 5.34 5.52 4.74 68.1 p NS NS NS NS NS a Tree health is rated on a scale where 0 = healthy and 10 = dead [29]; n = 11 trees for No Agsil Drench, n = 23 trees for Agsil Drench. Table 2. Packout data collected May 2015 from Agsil amendment trial—Site 1, Childers. % Packout by Fruit Grade Premium 2nd 3rd Total No Agsil Drench a 50.3 21.1 26.2 97.6 Agsil Drench b 51.3 36.5 10.8 98.6 a No Agsil drench—downgrades due to limb rub (70%), sunburn (20%), insect chew (5%) and pepper spot (5%); b Agsil drench—downgrades due to limb rub (45%), hail damage (35%), sunburn (20%) and insect chew (5%). Table 2. Packout data collected May 2015 from Agsil amendment trial—Site 1, Childers. 2.1. Field Trials with AgSil 32 (2014–2016) Treatment Tree Health a May 2014 December 2014 May 2015 July 2015 December 2015 May 2016 August 2016 Untreated 5.36 5.55 5.09 5.18 4.91 4.82 5.09 Agsil drench 5.36 5.45 5.00 5.55 4.73 4.91 4.64 Agsil spray 5.42 5.92 5.17 4.83 4.17 4.08 4.67 Agsil inject 5.45 5.45 5.27 5.27 5.40 5.09 5.00 p NS NS NS NS NS NS NS a Tree health is rated on a scale where 0 = healthy and 10 = dead [29]; n = 11 for Untreated, Agsil drench and Agsil inject, n = 12 for Agsil spray. a Tree health is rated on a scale where 0 = healthy and 10 = dead [29]; n = 11 for Untreated, Agsil drench and Agsil inject, n = 12 for Agsil spray. Table 4. Postharvest quality data collected from fruit harvested in August 2015 from Agsil amendment trial—Site 3, Beechmont. Table 4. Postharvest quality data collected from fruit harvested in August 2015 from Agsil amendment trial—Site 3, Beechmont. Treatment Anthracnose Severity (%) Stem End Rot Severity (%) Anthracnose Incidence (%) Stem End Rot Incidence (%) Marketability a (%) Untreated 9.60 6.97 abc 55.9 19.1 51.4 Agsil drench 9.04 3.65 c 45.5 11.4 58.6 Agsil spray 9.03 5.09 bc 41.7 16.3 58.8 Agsil inject 9.79 9.16 a 48.6 20.9 50.5 p NS <0.05 NS NS NS Means followed by the same letter are not significantly (p < 0.05) different; n = 11 for Untreated, Agsil drench and Agsil inject, n = 12 for Agsil spray. a Fruit marketability = less than 5% severity of anthracnose and no stem end rot. Means followed by the same letter are not significantly (p < 0.05) different; n = 11 for Untreated, Agsil drench and Agsil inject, n = 12 for Agsil spray. a Fruit marketability = less than 5% severity of anthracnose and no stem end rot. Table 5. Postharvest quality data collected from fruit harvested in August 2016 from Agsil amendment trial—Site 3, Beechmont. Treatment Anthracnose Severity (%) Stem End Rot Severity (%) Anthracnose Incidence (%) Stem End Rot Incidence (%) Marketability a (%) Untreated 4.77 12.8 25.4 33.0 56.5 Agsil drench 5.34 9.48 27.8 26.8 61.7 Agsil spray 5.72 9.61 27.6 26.3 64.0 Agsil inject 3.79 8.61 20.6 26.3 63.2 p NS NS NS NS NS n = 11 for Untreated, Agsil drench and Agsil inject, n = 12 for Agsil spray. 2.1. Field Trials with AgSil 32 (2014–2016) a No Agsil drench—downgrades due to limb rub (70%), sunburn (20%), insect chew (5%) and pepper spot (5%); b Agsil drench—downgrades due to limb rub (45%), hail damage (35%), sunburn (20%) and insect chew (5%). No Agsil drench downgrades due to limb rub (70%), sunburn (20%), insect chew (5%) and pepper spot (5%); b Agsil drench—downgrades due to limb rub (45%), hail damage (35%), sunburn (20%) and insect chew (5%). The trial at Site 2, Goodwood, (QLD), consisted of two rows of trees treated with Agsil drench (5000 ppm Si per tree delivered in 20 L water) in two separate blocks, with untreated rows either side assessed as the untreated controls. Agsil did not consistently improve tree health compared with untreated trees at any time point, however tree yields from Agsil drenched trees were approximately 13% and 39% higher than trees in the untreated rows for the two blocks (data not presented). Treatments to trees at Site 3, Beechmont, (QLD), were undertaken by project staff, (not the orchard operator), and included Agsil soil drenches (5000 ppm solution of Si delivered in 20 L water per tree), foliar spray (3300 ppm solution of Si delivered in 5 L water per tree) and trunk injection (3000 ppm solution of Si delivered in 5 × 20 mL injection sites per tree). Tree health was not significantly affected by Agsil treatment at any of the assessment dates between May 2014 and August 2016 (Table 3). A tray of fruit from each trial tree was harvested at commercial maturity in 2015 and 2016 and assessed for postharvest disease. There were no significant differences in severity or incidence of anthracnose among treatments in either 2015 or 2016, however fruit from trees sprayed or drenched with Agsil had significantly less severe stem end rot than those that were injected in 2015 (Table 4). There were no significant effects of Agsil on stem end rot in 2016 (Table 5). 5 of 15 Plants 2017, 6, 51 Table 3. Tree health data for 2014–2016 from Agsil amendment trial—Site 3, Beechmont. Table 3. Tree health data for 2014–2016 from Agsil amendment trial—Site 3, Beechmont. 2.1. Field Trials with AgSil 32 (2014–2016) a Fruit marketability = less than 5% severity of anthracnose and no stem end rot. Table 5. Postharvest quality data collected from fruit harvested in August 2016 from Agsil amendment trial—Site 3, Beechmont. Agsil was applied six times to selected trees in the trial at Site 4, Comboyne, (NSW), between August 2014 and June 2016. Health of Agsil drenched trees had improved by the final assessment compared with untreated controls, but the effect was not statistically significant (Table 6). However, estimated yield was 14% higher for the Agsil drenched trees (Table 6). One bin of fruit from each treatment was transported to a commercial packingshed to obtain packout data. While the percentage of fruit in the Premium grade was similar for fruit from the two treatments, there were more 2nd grade from Agsil treated trees than from untreated controls (Table 7). 6 of 15 Plants 2017, 6, 51 Table 6. Tree health data for 2014 and 2016 and estimated yield 2016 from Agsil amendment trial—Site 4, Comboyne. Treatment Tree Health a Est. Yield kg/tree June 2014 August 2016 August 2016 Untreated 5.3 5.6 47.8 Agsil drench 5.2 4.3 55.0 p NS NS ND a Tree health is rated on a scale where 0 = healthy and 10 = dead [29]; n = 9 untreated, n = 10 Agsil drench. ND = not determined. Fruit from trees in each treatment was picked into separate bins, and individual yields per tree were not recorded. Treatment Tree Health a Est. Yield kg/tree June 2014 August 2016 August 2016 Untreated 5.3 5.6 47.8 Agsil drench 5.2 4.3 55.0 p NS NS ND a Tree health is rated on a scale where 0 = healthy and 10 = dead [29]; n = 9 untreated, n = 10 Agsil drench. ND = not determined. Fruit from trees in each treatment was picked into separate bins, and individual yields per tree were not recorded. a Tree health is rated on a scale where 0 = healthy and 10 = dead [29]; n = 9 untreated, n = 10 Agsil drench. ND = not determined. Fruit from trees in each treatment was picked into separate bins, and individual yields per tree were not recorded. Table 7. Packout data collected August 2016 from Agsil amendment trial—Site 4, Comboyne. Table 7. Packout data collected August 2016 from Agsil amendment trial—Site 4, Comboyne. 2.2.1. Experiment 1 Twenty-eight days after inoculation with C. ilicicola (Ci), seedlings were significantly shorter and had reduced leaf/stem and root biomass, and a greater proportion of necrotic roots, compared with those which were uninoculated (Table 8). There was no significant effect of MD treatment on plant height, compared with plants which did not receive MD amendment. There were indications of a MD effect amongst the uninoculated treatments, where the healthiest roots (least necrosis) were from seedlings receiving 2 g MD, and were significantly healthier than those receiving 6 g MD (Table 8). Si concentration in leaves and roots of seedlings treated with 2 g and 6 g MD were not significantly different from those receiving no MD (Figure 1). Table 8. Seedling growth, fresh and dry weight of avocado leaves and stems and roots and percent of necrotic roots after amendment of potting mix with Mineral Dust (MD) and inoculation with Calonectria ilicicola (Ci), Experiment 1. Table 8. Seedling growth, fresh and dry weight of avocado leaves and stems and roots and percent of necrotic roots after amendment of potting mix with Mineral Dust (MD) and inoculation with Calonectria ilicicola (Ci), Experiment 1. Amendment to Potting Media a Seedling Growth (cm) Leaves and Stem Biomass b (g) Root Biomass (g) % Necrotic Roots FW DW FW DW MD (2 g) + Ci 1.37 b 14.9 b 5.92 b 7.25 b 0.91 b 88.2 a MD (2 g) 10.70 a 28.5 a 8.75 a 17.0 a 2.00 a 12.5 c MD (6 g) + Ci 0.98 b 13.7 b 4.99 b 4.21 b 0.52 b 94.4 a MD (6 g) 11.00 a 29.3 a 8.98 a 17.5 a 2.11 a 24.6 b Control + Ci 1.15 b 16.0 b 5.82 b 6.52 b 0.78 b 87.7 a Control 10.50 a 26.3 a 8.20 a 15.2 a 1.72 a 17.5 bc p <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Mean values followed by the same latter are not significantly different at the p value indicated (n = 12). a Two separate doses of MD were applied. First application was 30 days before Ci inoculation and second application three days after inoculation. Control received no MD. b FW, Fresh weight; DW, Dry weight. Mean values followed by the same latter are not significantly different at the p value indicated (n = 12). a Two separate doses of MD were applied. 2.1. Field Trials with AgSil 32 (2014–2016) Treatment % Packout by Fruit Grade Premium 2nd 3rd Processing a Untreated b 56.5 27.8 13.2 2.5 Agsil Drench c 55.6 32.6 9.4 2.3 a Processing = defect; b Untreated—downgrades due to caterpillar damage (16%), hail (13%), wind rub (19%), sunburn (22%), pepper spot (8%) and anthracnose (3%); c Agsil drench—downgrades due to caterpillar damage (18%), hail (21%), wind rub (22%), sunburn (24%), and pepper spot (4%). No anthracnose damage. a Processing = defect; b Untreated—downgrades due to caterpillar damage (16%), hail (13%), wind rub (19%), sunburn (22%), pepper spot (8%) and anthracnose (3%); c Agsil drench—downgrades due to caterpillar damage (18%), hail (21%), wind rub (22%), sunburn (24%), and pepper spot (4%). No anthracnose damage. 2.2. Greenhouse Trials with Mineral Mulch and Mineral Dust Mean values followed by the same latter are not significantly different at the p value indicated (n = 12). a Two separate doses of MD were applied. First application was 30 days before Ci inoculation and second application three days after inoculation. Control received no MD. b FW, Fresh weight; DW, Dry weight. 2.2.2. Experiment 2 2.2.2. Experiment 2 Seedling growth, fresh and dry weight of avocado leaves and stems and roots and percent of necrotic roots after amendment of potting mix with Mineral Dust (MD) or Mineral Mulch (MM) and inoculation with Calonectria ilicicola (Ci), Experiment 2. Table 9. Seedling growth, fresh and dry weight of avocado leaves and stems and roots and percent of necrotic roots after amendment of potting mix with Mineral Dust (MD) or Mineral Mulch (MM) and inoculation with Calonectria ilicicola (Ci), Experiment 2. and inoculation with Calonectria ilicicola (Ci), Experiment 2. Amendment to Potting Media a Seedling Growth (cm) Leaves and Stem Biomass b (g) Root Biomass (g) % Necrotic Roots FW DW FW DW MD (2 g) + Ci 5.67 cd 32.6 bcd 10.8 16.6 b 2.01 abc 41.7 b MD (2 g) 10.50 a 41.2 a 13.5 27.4 a 2.63 a 0.64 c MM (2 g) + Ci 3.79 d 28.9 cd 10.0 15.4 b 1.82 bc 47.1 b MM (2 g) 9.33 ab 38.0 ab 11.7 27.2 a 2.57 ab 1.08 c Control + Ci 3.13 d 25.8 d 8.78 12.9 b 1.54 c 68.3 a Control 7.25 bc 35.9 abc 11.3 26.1 a 2.55 ab 1.10 c p <0.001 <0.01 NS <0.001 <0.05 <0.001 Mean values followed by the same letter are not significantly different at the p values indicated (n = 12). a Two separate doses of MD/MM were applied. First application was 30 days before Ci i l ti d d li ti th d ft i l ti C t l i d ith MD inoculation with Calonectria ilicicola (Ci), Experiment 2. 2.2.1. Experiment 1 First application was 30 days before Ci inoculation and second application three days after inoculation. Control received no MD. b FW, Fresh weight; DW, Dry weight. 7 of 15 7 of 15 Plants 2017, 6, 51 Plants 2017, 6, 51 0 20 40 60 80 100 ROOT LEAF Silicon content (mg/kg) Control MD (2g) MD (6g) Figure 1. Silicon content (mg/kg) in dried root and leaf tissue four weeks after treatment with Mineral dust (MD), Experiment 1. Only plants from uninoculated treatments were analyzed. Control plants received no MD; MD (2 g), plants received 2 g MD in total; MD (6 g), plants received 6 g MD in total. Root and leaf, No significant differences among treatments (p > 0.05, n = 6). 0 20 40 60 80 100 ROOT LEAF Silicon content (mg/kg) Control MD (2g) MD (6g) Figure 1. Silicon content (mg/kg) in dried root and leaf tissue four weeks after treatment with Mineral dust (MD), Experiment 1. Only plants from uninoculated treatments were analyzed. Control plants received no MD; MD (2 g), plants received 2 g MD in total; MD (6 g), plants received 6 g MD in total. Root and leaf, No significant differences among treatments (p > 0.05, n = 6). Figure 1. Silicon content (mg/kg) in dried root and leaf tissue four weeks after treatment with Mineral dust (MD), Experiment 1. Only plants from uninoculated treatments were analyzed. Control plants received no MD; MD (2 g), plants received 2 g MD in total; MD (6 g), plants received 6 g MD in total. Root and leaf, No significant differences among treatments (p > 0.05, n = 6). Figure 1. Silicon content (mg/kg) in dried root and leaf tissue four weeks after treatment with Mineral dust (MD), Experiment 1. Only plants from uninoculated treatments were analyzed. Control plants received no MD; MD (2 g), plants received 2 g MD in total; MD (6 g), plants received 6 g MD in total. Root and leaf, No significant differences among treatments (p > 0.05, n = 6). 2.2.2. Experiment 2 2.2.2. Experiment 2 The treatments for the second experiment were modified to replace 6 g MD treatment with 2 g MM. There was a significant effect of Ci reducing plant height over the four-week duration of the experiment, although not as severely as in Experiment 1. There were no significant differences in seedling growth among treatments when comparing respective inoculated and uninoculated seedlings (Table 9). Leaf and stem and root biomass (FW and DW) were not significantly reduced by fungal inoculation within treatment comparisons (Table 9). Both MM and MD significantly reduced the percentage of necrotic roots compared with non-amended seedlings when assessed four weeks after inoculation (Table 9). Inoculation with Ci reduced Si accumulation in roots compared with respective non-inoculated plants, however, there were no significant differences in Si accumulation in leaves or roots of MM or MD treated seedlings compared with respective Ci- inoculated or uninoculated controls (Figure 2). The treatments for the second experiment were modified to replace 6 g MD treatment with 2 g MM. There was a significant effect of Ci reducing plant height over the four-week duration of the experiment, although not as severely as in Experiment 1. There were no significant differences in seedling growth among treatments when comparing respective inoculated and uninoculated seedlings (Table 9). Leaf and stem and root biomass (FW and DW) were not significantly reduced by fungal inoculation within treatment comparisons (Table 9). Both MM and MD significantly reduced the percentage of necrotic roots compared with non-amended seedlings when assessed four weeks after inoculation (Table 9). Inoculation with Ci reduced Si accumulation in roots compared with respective non-inoculated plants, however, there were no significant differences in Si accumulation in leaves or roots of MM or MD treated seedlings compared with respective Ci-inoculated or uninoculated controls (Figure 2). Table 9. Seedling growth, fresh and dry weight of avocado leaves and stems and roots and percent of necrotic roots after amendment of potting mix with Mineral Dust (MD) or Mineral Mulch (MM) and inoculation with Calonectria ilicicola (Ci), Experiment 2. Table 9. Seedling growth, fresh and dry weight of avocado leaves and stems and roots and percent of necrotic roots after amendment of potting mix with Mineral Dust (MD) or Mineral Mulch (MM) and inoculation with Calonectria ilicicola (Ci), Experiment 2. Table 9. p <0.001 <0.01 NS <0.001 <0.05 <0.001 Mean values followed by the same letter are not significantly different at the p values indicated (n = 12). a Two separate doses of MD/MM were applied. First application was 30 days before Ci Mean values followed by the same letter are not significantly different at the p values indicated (n = 12). a Two separate doses of MD/MM were applied. First application was 30 days before Ci inoculation and second application three days after inoculation. Control received neither MD nor MM. b FW, Fresh weight; DW, Dry weight. 2 2 3 Experiment 3 2.2.3. Experiment 3 p Phytophthora cinnamomi, the oomycete pathogen causing PRR, was included as a second inoculation treatment in Experiment 3. There were significant differences amongst treatments for all measured parameters. Inoculation with either Ci or Pc significantly reduced seedling growth, root biomass and increased severity of root rot (Table 10), with effects of Pc inoculation generally (but not always statistically) worse than Ci. Amendment with either MD or MM did not affect plant growth (height), biomass or root necrosis compared with respective uninoculated or inoculated controls. Root necrosis was approximately 16% or 24% less severe in plants treated with MD or MM, respectively, before inoculation with Ci compared with untreated controls, however the difference was not statistically significant (Table 10). There were no leaf and root nutrient analyses obtained for this experiment Phytophthora cinnamomi, the oomycete pathogen causing PRR, was included as a second inoculation treatment in Experiment 3. There were significant differences amongst treatments for all measured parameters. Inoculation with either Ci or Pc significantly reduced seedling growth, root biomass and increased severity of root rot (Table 10), with effects of Pc inoculation generally (but not always statistically) worse than Ci. Amendment with either MD or MM did not affect plant growth (height), biomass or root necrosis compared with respective uninoculated or inoculated controls. Root necrosis was approximately 16% or 24% less severe in plants treated with MD or MM, respectively, before inoculation with Ci compared with untreated controls, however the difference was not statistically significant (Table 10). There were no leaf and root nutrient analyses obtained for this experiment. p Table 10. Plant growth and severity of root rot after amendment of potting mix with either Mineral Dust (MD) or Mineral Mulch (MM) and subsequent inoculation with Ci and Pc, Experiment 3. 2 2 3 Experiment 3 2.2.3. Experiment 3 Amendment to Potting Media a Seedling Growth (cm) Leaf and Stem Biomass b (g) Root Biomass (g) % Necrotic Roots FW DW FW DW MD (2 g) 5.97 a 28.7 ab 9.31 ab 27.3 a 2.73 ab 1.25 c MD (2 g) + Ci 1.54 b 25.9 ab 9.27 ab 19.3 b 2.11 b 27.1 b MD (2 g) + Pc 0.83 b 19.9 b 6.78 b 7.98 c 0.73 c 99.2 a MM (2 g) 6.13 a 33.1 a 10.9 a 28.7 a 3.03 a 0.50 c MM (2 g) + Ci 1.29 b 26.5 ab 9.59 ab 21.9 ab 2.45 ab 29.6 b MM (2 g) + Pc 0.79 b 21.0 b 7.35 ab 8.00 c 0.94 c 99.1 a Control 4.92 ab 27.3 ab 8.74 ab 24.1 ab 2.72 ab 0.92 c Control + Ci 2.29 ab 24.0 ab 8.18 ab 18.6 b 1.97 b 35.4 b Control + Pc 0.79 b 21.4 b 7.44 ab 8.83 c 0.86 c 99.3 a p <0.001 <0.01 <0.05 <0.001 <0.001 <0.001 Mean values followed by the same letter are not significantly different at the p values indicated (n = 12). a Two separate doses of MD/MM were applied. First application was 30 days before Ci/Pc inoculation and second application three days after inoculation. Control received neither MD nor Table 10. Plant growth and severity of root rot after amendment of potting mix with either Mineral Dust (MD) or Mineral Mulch (MM) and subsequent inoculation with Ci and Pc, Experiment 3. 2.2.2. Experiment 2 2.2.2. Experiment 2 Amendment to Potting Media a Seedling Growth (cm) Leaves and Stem Biomass b (g) Root Biomass (g) % Necrotic Roots FW DW FW DW MD (2 g) + Ci 5.67 cd 32.6 bcd 10.8 16.6 b 2.01 abc 41.7 b MD (2 g) 10.50 a 41.2 a 13.5 27.4 a 2.63 a 0.64 c MM (2 g) + Ci 3.79 d 28.9 cd 10.0 15.4 b 1.82 bc 47.1 b MM (2 g) 9.33 ab 38.0 ab 11.7 27.2 a 2.57 ab 1.08 c Control + Ci 3.13 d 25.8 d 8.78 12.9 b 1.54 c 68.3 a Control 7.25 bc 35.9 abc 11.3 26.1 a 2.55 ab 1.10 c p <0.001 <0.01 NS <0.001 <0.05 <0.001 Mean values followed by the same letter are not significantly different at the p values indicated (n = 12). a Two separate doses of MD/MM were applied. First application was 30 days before Ci inoculation and second application three days after inoculation. Control received neither MD nor MM. b FW, Fresh weight; DW, Dry weight. p <0.001 <0.01 NS <0.001 <0.05 <0.001 Mean values followed by the same letter are not significantly different at the p values indicated (n = 12). a Two separate doses of MD/MM were applied. First application was 30 days before Ci l d d l h d f l l d h Mean values followed by the same letter are not significantly different at the p values indicated (n = 12). a Two separate doses of MD/MM were applied. First application was 30 days before Ci inoculation and second application three days after inoculation. Control received neither MD nor MM. b FW, Fresh weight; DW, Dry weight. 8 of 15 8 of 15 Plants 2017, 6, 51 Pla t 2017 6 51 Figure 2. Silicon content (mg/kg) in dried root and leaf tissue four weeks after treatment with Mineral dust (MD) and Mineral mulch (MM), Experiment 2. Control plants received neither MD nor MM; MD (2 g), plants received 2 g MD in total; MM (2 g), plants received 2 g MM in total; control + Ci, MD (2 g) + Ci, MM (2 g) + Ci, plants inoculated with Ci; Root, No significant differences among treatments, (p > 0.05, n = 6); Leaf, Bars labeled with the same letter are not significantly different (p < 0.05, n = 6). 2.2.2. Experiment 2 2.2.2. Experiment 2 bcd a cd ab d abc 0 20 40 60 80 100 ROOT LEAF Silicon content (mg/kg) Control + Ci Control MD (2g) + Ci MD (2g) MM (2g) + Ci MM (2g) Figure 2. Silicon content (mg/kg) in dried root and leaf tissue four weeks after treatment with Mineral dust (MD) and Mineral mulch (MM), Experiment 2. Control plants received neither MD nor MM; MD (2 g), plants received 2 g MD in total; MM (2 g), plants received 2 g MM in total; control + Ci, MD (2 g) + Ci, MM (2 g) + Ci, plants inoculated with Ci; Root, No significant differences among treatments, (p > 0.05, n = 6); Leaf, Bars labeled with the same letter are not significantly different (p < 0.05, n = 6). bcd a cd ab d abc LEAF Figure 2. Silicon content (mg/kg) in dried root and leaf tissue four weeks after treatment with Mineral dust (MD) and Mineral mulch (MM), Experiment 2. Control plants received neither MD nor MM; MD (2 g), plants received 2 g MD in total; MM (2 g), plants received 2 g MM in total; control + Ci, MD (2 g) + Ci, MM (2 g) + Ci, plants inoculated with Ci; Root, No significant differences among treatments, (p > 0.05, n = 6); Leaf, Bars labeled with the same letter are not significantly different (p < 0.05, n = 6). Figure 2. Silicon content (mg/kg) in dried root and leaf tissue four weeks after treatment with Mineral dust (MD) and Mineral mulch (MM), Experiment 2. Control plants received neither MD nor MM; MD (2 g), plants received 2 g MD in total; MM (2 g), plants received 2 g MM in total; control + Ci, MD (2 g) + Ci, MM (2 g) + Ci, plants inoculated with Ci; Root, No significant differences among treatments, (p > 0.05, n = 6); Leaf, Bars labeled with the same letter are not significantly different (p < 0.05, n = 6). 2 2 3 Experiment 3 2.2.3. Experiment 3 Amendment to Potting Media a Seedling Growth (cm) Leaf and Stem Biomass b (g) Root Biomass (g) % Necrotic Roots FW DW FW DW MD (2 g) 5.97 a 28.7 ab 9.31 ab 27.3 a 2.73 ab 1.25 c MD (2 g) + Ci 1.54 b 25.9 ab 9.27 ab 19.3 b 2.11 b 27.1 b MD (2 g) + Pc 0.83 b 19.9 b 6.78 b 7.98 c 0.73 c 99.2 a MM (2 g) 6.13 a 33.1 a 10.9 a 28.7 a 3.03 a 0.50 c MM (2 g) + Ci 1.29 b 26.5 ab 9.59 ab 21.9 ab 2.45 ab 29.6 b MM (2 g) + Pc 0.79 b 21.0 b 7.35 ab 8.00 c 0.94 c 99.1 a Control 4.92 ab 27.3 ab 8.74 ab 24.1 ab 2.72 ab 0.92 c Control + Ci 2.29 ab 24.0 ab 8.18 ab 18.6 b 1.97 b 35.4 b Control + Pc 0.79 b 21.4 b 7.44 ab 8.83 c 0.86 c 99.3 a p <0.001 <0.01 <0.05 <0.001 <0.001 <0.001 Mean values followed by the same letter are not significantly different at the p values indicated (n = 12). a Two separate doses of MD/MM were applied. First application was 30 days before Ci/Pc inoculation and second application three days after inoculation. Control received neither MD nor MM. b FW, Fresh weight; DW, Dry weight. p Table 10. Plant growth and severity of root rot after amendment of potting mix with either Mineral D t (MD) Mi l M l h (MM) d b t i l ti ith Ci d P E i t 3 Table 10. Plant growth and severity of root rot after amendment of potting mix with either Mineral Dust (MD) or Mineral Mulch (MM) and subsequent inoculation with Ci and Pc, Experiment 3. p <0.001 <0.01 <0.05 <0.001 <0.001 <0.001 Mean values followed by the same letter are not significantly different at the p values indicated (n = 12). a Two separate doses of MD/MM were applied. First application was 30 days before Ci/Pc inoculation and second application three days after inoculation. Control received neither MD nor Mean values followed by the same letter are not significantly different at the p values indicated (n = 12). a Two separate doses of MD/MM were applied. First application was 30 days before Ci/Pc inoculation and second application three days after inoculation. p <0.001 <0.01 <0.05 <0.001 <0.001 <0.001 Mean values followed by the same letter are not significantly different at the p values indicated (n = 12). a Two separate doses of MD/MM were applied. First application was 30 days before Ci/Pc i l i d d li i h d f i l i C l i d i h MD Mean values followed by the same letter are not significantly different at the p values indicated (n = 12). a Two separate doses of MD/MM were applied. First application was 30 days before Ci/Pc inoculation and second application three days after inoculation. Control received neither MD nor MM. b FW, Fresh weight; DW, Dry weight. 2.3. Preliminary Field Trials with Mineral Mulch 2.3. Preliminary Field Trials with Mineral Mulch Table 11 shows the average tree (canopy) health for treated and untreated trees at each site at the date of MM application (July 2016) and at the first assessment (May 2017). At the Kumbia site, all trees improved in health, irrespective of treatment, and, in May 2017, there was no significant difference in canopy health between treatments (Table 11). At Kumbia, there were no differences in heights and diameters between trees treated with MM or those which received no treatments (data not shown). At the Goodwood orchard, canopy health in 2016 was significantly worse in the trees selected for MM application than those in adjacent rows selected as the untreated controls (Table 11). However, 10 months after MM application, the tree health had improved considerably, to be the same as that in untreated trees. A similar trend was also observed in the Childers orchard, where the improvement in tree health over time was more rapid for the MM treated trees than untreated controls (Table 11). Fruit were collected in 2017 for postharvest disease assessments and nutrient analyses. There were no differences amongst orchards or treatments in the interval between harvest and “eating ripe” stage. There were significant differences among treatments (and orchards) in severity of anthracnose disease, where fruit from Kumbia or Childers treated with MM, or Childers untreated, had significantly more disease than from Goodwood, or Kumbia untreated (Table 11). A similar trend was observed in stem end rot. These results must be viewed with extreme caution, as most trees from Kumbia and Childers had no fruit, so a composite sample from only a few trees was available for testing. There were also limited numbers (replicates) from each treatment at each site. The usual practice for fruit assessments is to harvest a full tray of fruit (18–20 pieces) from a replicate tree, ensuring at least six replicate trees per treatment. Future trials should include more treated trees and incorporate more robust sampling and replication. Si concentration in leaves and fruit peel was determined, however, it must be noted that statistical analyses were not performed on the data, as there was only one replicate sample for each tissue type and treatment at each site. 2 2 3 Experiment 3 2.2.3. Experiment 3 Control received neither MD nor MM. b FW, Fresh weight; DW, Dry weight. Plants 2017, 6, 51 9 of 15 2.3. Preliminary Field Trials with Mineral Mulch 3. Discussion 3. Discussion It is possible that more frequent applications of Agsil targeted more closely to autumn and spring root flushes, may improve uptake and translocation of available Si and hence efficacy. While the disease reducing beneficial effects of Si applications are most frequently attributed Fruit postharvest evaluations from one trial showed that stem end rot was significantly reduced in the first year of Agsil treatment, resulting in a greater proportion of marketable fruit. Anthracnose was not reduced by potassium silicate drench treatments. Previous studies showed the reverse with potassium silicate injection treatments, i.e., reducing anthracnose but having no effect on stem end rot [14,15]. Lack of effect of spray applications on disease severity was, however, consistent with a previous study [16]. It is possible that more frequent applications of Agsil targeted more closely to autumn and spring root flushes, may improve uptake and translocation of available Si and hence efficacy. While the disease-reducing beneficial effects of Si applications are most frequently attributed to enhancement of physical barriers to pathogen ingress and activation of plant defense mechanisms, a direct fungicidal effect is also possible, depending of course on the formulation of Si and its applied concentration. Soluble potassium silicate (20.7% SiO2) was tested for its effects on in vitro growth of several phytopathogenic fungi including Sclerotinia sclerotiorum, Fusarium oxysporum, F. solani, Colletotrichum coccodes, A. solani and oomycetes, P. cinnamomi and Pythium sp. [30]. Concentrations of 5 mL and 10 mL potassium silicate/L agar completely inhibited growth of P. cinnamomi and S. sclerotiorum, however, mycelial growth of F. oxysporum, F. solani and Verticillium theobromae was increased compared with controls at these concentrations. Growth was inhibited at the higher concentrations tested, 20, 40 and 80 mL/L agar [30]. Potassium silicate amendment increased pH of media, however, mycelial growth of all fungi tested occurred at corresponding high pH (adjusted with KOH), demonstrating the fungicidal effect of the potassium silicate solution was not simply due to alkalization of the agar media [30]. This information is important when considering field applications It would be counterproductive to apply a treatment which enhanced While the disease-reducing beneficial effects of Si applications are most frequently attributed to enhancement of physical barriers to pathogen ingress and activation of plant defense mechanisms, a direct fungicidal effect is also possible, depending of course on the formulation of Si and its applied concentration. 2.3. Preliminary Field Trials with Mineral Mulch Si concentration in leaves was higher in MM treated trees at two sites, Kumbia and Childers, and higher in fruit peel at each site, compared with leaves and peel from untreated control trees (Figure 3). This provides strong indication that Si is taken up by roots in mature orchard trees and translocated and deposited in leaves and fruit peel, within a relatively short time frame (10 months). Table 11. Fruit quality and canopy health of avocado 10 months after Mineral Mulch application to trees in three orchards in Queensland. Table 11. Fruit quality and canopy health of avocado 10 months after Mineral Mulch application to trees in three orchards in Queensland. Site and Treatment n No. Days to Ripe Anthracnose Severity (%) Stem End Rot Severity (%) n Canopy Health 2016 Canopy Health 2017 Kumbia Untreated 16 14.2 4.06 b 14.1 ab 8 4.13 d 3.5 b Kumbia Mineral Mulch 16 12.6 35.9 a 25.3 a 8 4.25 cd 3.6 b Goodwood Untreated 15 13.2 3.00 b 1.67 b 20 4.55 bcd 4.6 a Goodwood Mineral Mulch 12 13.3 1.25 b 17.1 a 10 5.20 a 4.6 a Childers Untreated 31 12.6 22.9 a 2.74 b 29 4.75 abc 4.5 a Childers Mineral Mulch 18 11.7 26.4 a 1.67 b 10 4.90 ab 4.4 a p NS <0.001 <0.001 <0.01 <0.01 Mean values followed by the same letter are not significantly different at p values indicated. 10 of 15 10 of 15 Plants 2017, 6, 51 Plants 2017 6 51 Figure 3. Silicon content (mg/kg) in dried avocado leaf and peel tissue 10 months after treatment of avocado trees at three locations in Queensland with Mineral Mulch (MM). n = 1 and thus no statistical analysis was undertaken. 0 50 100 150 200 250 300 LEAF PEEL Silicon content (mg/kg) Kumbia control Kumbia MM Goodwoood control Goodwoood MM Childers control Childers MM Figure 3. Silicon content (mg/kg) in dried avocado leaf and peel tissue 10 months after treatment of avocado trees at three locations in Queensland with Mineral Mulch (MM). n = 1 and thus no statistical analysis was undertaken. Figure 3. Silicon content (mg/kg) in dried avocado leaf and peel tissue 10 months after treatment of avocado trees at three locations in Queensland with Mineral Mulch (MM). n = 1 and thus no statistical analysis was undertaken. 3. Discussion 3. Discussion The four trials with soluble potassium silicate (AgSil®32) conducted on growers’ orchards highlight the inconsistent effects of Agsil under field conditions. Tree health was generally improved in Agsil treated trees, but not significantly compared with untreated trees. In two of the three trials where yields were measured, average yields per tree were 13–39% greater from Agsil- drenched trees compared with untreated trees. Fruit quality and packout data demonstrate that, after two years of application, fruit from Agsil treated trees had higher proportions of fruit in 2nd grades (but not Premium grade), superior quality with reduced stem end rot and defects attributed to pepper spot (superficial necrotic lesions <0.5 mm, caused by Colletotrichum spp.), than fruit from non-drenched trees. One packingshed estimated the improved quality represented a 20% increase in net return to the grower, based on prices received at the time of packing. Fruit postharvest evaluations from one trial showed that stem end rot was significantly The four trials with soluble potassium silicate (AgSil®32) conducted on growers’ orchards highlight the inconsistent effects of Agsil under field conditions. Tree health was generally improved in Agsil treated trees, but not significantly compared with untreated trees. In two of the three trials where yields were measured, average yields per tree were 13–39% greater from Agsil-drenched trees compared with untreated trees. Fruit quality and packout data demonstrate that, after two years of application, fruit from Agsil treated trees had higher proportions of fruit in 2nd grades (but not Premium grade), superior quality with reduced stem end rot and defects attributed to pepper spot (superficial necrotic lesions <0.5 mm, caused by Colletotrichum spp.), than fruit from non-drenched trees. One packingshed estimated the improved quality represented a 20% increase in net return to the grower, based on prices received at the time of packing. Fruit postharvest evaluations from one trial showed that stem end rot was significantly reduced in the first year of Agsil treatment, resulting in a greater proportion of marketable fruit. Anthracnose was not reduced by potassium silicate drench treatments. Previous studies showed the reverse with potassium silicate injection treatments, i.e., reducing anthracnose but having no effect on stem end rot [14,15]. Lack of effect of spray applications on disease severity was, however, consistent with a previous study [16]. 2.3. Preliminary Field Trials with Mineral Mulch 0 50 100 150 200 250 300 LEAF PEEL Silicon content (mg/kg) Kumbia control Kumbia MM Goodwoood control Goodwoood MM Childers control Childers MM Figure 3. Silicon content (mg/kg) in dried avocado leaf and peel tissue 10 months after treatment of avocado trees at three locations in Queensland with Mineral Mulch (MM). n = 1 and thus no statistical analysis was undertaken. Figure 3. Silicon content (mg/kg) in dried avocado leaf and peel tissue 10 months after treatment of avocado trees at three locations in Queensland with Mineral Mulch (MM). n = 1 and thus no statistical analysis was undertaken. Figure 3. Silicon content (mg/kg) in dried avocado leaf and peel tissue 10 months after treatment of avocado trees at three locations in Queensland with Mineral Mulch (MM). n = 1 and thus no statistical analysis was undertaken. 3. Discussion 3. Discussion Soluble potassium silicate (20.7% SiO2) was tested for its effects on in vitro growth of several phytopathogenic fungi including Sclerotinia sclerotiorum, Fusarium oxysporum, F. solani, Colletotrichum coccodes, A. solani and oomycetes, P. cinnamomi and Pythium sp. [30]. Concentrations of 5 mL and 10 mL potassium silicate/L agar completely inhibited growth of P. cinnamomi and S. sclerotiorum, however, mycelial growth of F. oxysporum, F. solani and Verticillium theobromae was increased compared with controls at these concentrations. Growth was inhibited at the higher concentrations tested, 20, 40 and 80 mL/L agar [30]. Potassium silicate amendment increased pH of media, however, mycelial growth of all fungi tested occurred at corresponding high pH (adjusted with KOH), demonstrating the fungicidal effect of the potassium silicate solution was not simply due to alkalization of the agar media [30]. This information is important when considering field Plants 2017, 6, 51 11 of 15 applications. It would be counterproductive to apply a treatment which enhanced growth of an economically important pathogen rather than suppressing it! An effect of Si on pathogen or other microbial populations (including beneficial or suppressive microbes) and diversity in soil has not been reported, but requires further investigation. There are strong indications that Mineral Mulch and/or Mineral Dust amendment to potting mix of greenhouse avocado plants reduces the root necrosis caused by C. ilicicola, the nectriaceous fungus causing black root rot of avocado. g In greenhouse Experiment 3, it is clear that the inoculation with P. cinnamomi and subsequent conditions favouring disease were too severe, and there was little chance of observing treatment effects when seedlings were overwhelmed by the pathogen. In a fourth greenhouse experiment, there was an indication that treatment with MD and MM reduced root necrosis after inoculation with either Ci or Pc (data not shown). MD reduced necrosis by approximately 30% and 18%, while MM reduced necrosis by approximately 19% and 9%, compared with Ci or Pc inoculated controls, respectively. This was also observed for MD and MM in Ci inoculated plants, in Experiments 2 and 3, where MD appeared to be more effective at reducing necrosis than MM. However, the higher rate, 6 g, of MD applied in greenhouse Experiment 1, was likely to have been too high, and resulted in less healthy (lower biomass and more necrosis) than those treated with 2 g. 3. Discussion 3. Discussion Perhaps 6 g MD was too much in a short timeframe and overwhelmed plant defense responses. It is possible that the smaller particle size of MD contributed to faster nutrient uptake and defense responses than MM, but this requires further investigation, and supports the idea of a combined MD + MM treatment compared with MD and MM alone, in planned field trial investigations. Si accumulation in roots and leaves were measured in dried tissue from two greenhouse experiments. Amendment with MD or MM enhanced accumulation by around 30% in roots compared with untreated controls (Figures 1 and 2). However, smaller increases (7–18%) in leaf Si accumulation were observed in Experiment 1, while concentration actually decreased compared with untreated controls in Experiment 2. In preliminary field trials with MM, application at approximately 2 t/Ha had a beneficial effect on tree health at two sites, and Si accumulation in leaves and fruit peel, after only 10 months. Some larger particles of MM were still clearly visible underneath trees at each site, and may provide a consistent and sustained delivery of Si over a period of time. These preliminary results are extremely promising and warrant further replicated field trials, with suggested treatments of MM at 2 and 4 t/Ha and a combined MM and MD application. MD may provide a rapid delivery of Si, while the larger particle size of MM ensures release over several months. Canopy health as well as fruit yields and quality, and nutrient analyses will be necessary to evaluate the efficacy and cost benefit of MM applications in commercial avocado orchards. Comprehensive soil testing, including microbial and chemical (nutrient, pH, EC) analyses would also be desirable. Future trials should evaluate treatments applied to whole rows (e.g., 30 trees), compared with grower standard treatment, at different orchards across Australia (for replication), and across at least three years. Whole-row treatments can be assessed for productivity (yield) as well as commercial packout and quality. This approach would give more meaningful and realistic outputs. 4.1. Field Trials with AgSil32 AgSil®32 is a soluble potassium silicate product from PQ Australia Pty. Ltd., and contains 32% w/v SiO2 and 21% w/v K2O. AgSil®32 is a soluble potassium silicate product from PQ Australia Pty. Ltd., and contains 32% w/v SiO2 and 21% w/v K2O. Field trial sites were conducted at 4 locations between Central Queensland and the mid New South Wales coast to assess the effect of potassium silicate on tree health and productivity by reducing PRR. Each site was visited between April and June 2014 to establish trial sites, establish baseline tree health and establish communication links with growers for potassium silicate application 12 of 15 Plants 2017, 6, 51 and ongoing management. Grower collaborators were responsible for Agsil applications at three sites (all drench applications), and the pathology team independently conducted the 4th trial on an orchard at Beechmont, QLD. The selected trial sites enabled evaluation of the potential for potassium silicate application to manage PRR across 3 growing regions, with unique orchard management activities at each site. Agsil was applied as a drench at 300 mL/tree delivered in 20 L (5000 ppm solution of Si delivered in 20 L water per tree), to the active root zone. Application was planned to coincide with periods of active root flush (autumn and summer), however, frequency and timing varied across sites for a number of reasons. Tree canopy health was determined at each site visit by visual assessment according to a widely-used rating scale [29]. Fruit yields were collected from Sites 1 and 2 in 2015, and Site 4 in 2016. Additionally, commercial packout data were obtained for fruit from Site 1 in 2015 and Site 4 in 2016. Fruit was harvested from trees in the fully replicated trial at Site 3 in 2015 and 2016, ripened and assessed for development of postharvest anthracnose and stem end rot. p p p Site 1 (Childers, QLD): The entire orchard is currently treated with a low dose of potassium silicate on a monthly basis by fertigation. Sick/declining trees were treated at a high “treatment” rate of 300 mL Agsil per tree (5000 ppm solution of Si delivered in 20 L water per tree) on 3 occasions annually. Given the entire orchard currently receives Agsil, this trial was set up to examine the effects of withdrawing Agsil treatment from selected trees with obvious signs of decline due to PRR. 4.1. Field Trials with AgSil32 A total of 22 trees were selected for monitoring over the duration of the trial, with 11 of these clearly identified for Agsil treatment withdrawal. At the trial initiation, the average health ratings of trees subject to Agsil withdrawal and continuing treatment was 5.36 and 5.67 respectively. The tree health rating system used across all trial sites is based on the established system, where 1 = totally healthy and 10 = dead tree [29]. Root windows to facilitate measurements of root growth were installed under a selection of Agsil drench and non-drenched trees. Trees received 300 mL Agsil per tree, delivered in 20 L of water at each treatment date throughout the trial (February, May, July and October 2014; February, May, August and December 2015; and May 2016). Site 2 (Goodwood, QLD): This trial site was established on a well-managed orchard with no history of potassium silicate treatment. The trial consisted of two treatment rows in which potassium silicate soil drenches were applied, one in a block of relatively “healthy” trees, and the other row within a block of “sick” declining trees. Rows either side of the Agsil treated rows were assessed for canopy health as the untreated controls. Trees received 300 mL Agsil per tree, delivered in 20 L of water (i.e., approximately 5000 ppm) at each treatment date throughout the trial (July, October and December 2014; October and November 2015; and March 2016). Yield data was collected in May 2015. y Site 3 (Beechmont, QLD): This trial site was established on a small orchard with no history of potassium silicate treatment. Trees currently receive a phosphorous acid injection annually, however an area of the orchard in mid-decline was selected to evaluate a range of potassium silicate application methods. Treatments were undertaken by project staff, (i.e., not the grower), and included AgSil soil drenches as per trial sites 1 and 2 (300 mL Agsil per tree, delivered in 20 L of water, i.e., 5000 ppm solution), foliar spray (50 mL Agsil per tree delivered in 5 L water, i.e., 3300 ppm solution) and trunk injection (approximately 1 mL Agsil per tree in 100 mL water, i.e., 3000 ppm solution delivered in 5 × 20 mL injection into vascular tissue per tree). 4.2. Greenhouse Trials with Mineral Mulch and Mineral Dust MM and MD are sustainable soil amendment by-products from the manufacture of cement building boards. The major components are sand (approximately 50%), pine pulp, cement and water, and contains 51% total silica (as SiO2), with 5% as plant-available silica (monosilic acid) and calcium silicate. The waste boards are milled so that 80% of MM is composed of particles 1000 µm in size, while 88% of MD has particles in the 75–300 µm size range. The effects of Mineral Mulch in avocado have not previously been reported. Four greenhouse trials with avocado seedlings were undertaken between 2015 and 2017, and consisted of three steps; treatment of avocado seedlings with MD or MM, inoculation of the plants with Ci or Pc and assessment of seedling growth, root necrosis and nutrient uptake. There were 12 replicate seedlings per treatment, and MD or MM was applied twice, approximately 4–6 weeks apart. Treatments are outlined in result Tables 8–11 The dust/mulch was gently incorporated into the top 1–2 cm of potting mix. Approximately 30 days later, all plants were re-potted into 140 mm diam. round pots and inoculated with Pc or Ci. Pathogen colonized grain, 10–20 mL (or uncolonized grain as controls) was sprinkled on a thin layer of potting mix sitting at the bottom of each pot. The seedling was placed on top of the inoculum, and the pot back-filled with potting mix. Pc inoculated pots were watered until damp, placed on saucers to simulate waterlogging for 5 days before saucers were removed and all plants watered as required. Plant height was measured from the base of the stem to the apex and recoded just after inoculation and at the conclusion of the experiment. At the conclusion of the trial, plants were uprooted, roots were washed to remove potting mix, and percentage of necrotic roots was assessed for each plant. Above-ground parts (leaves and stems excised above the seed) and root systems of each plant were placed into paper bags, weighed and dried for three days at 55 ◦C. Dried roots and leaves from selected treatments of Experiments 1 and 2 were ground to a fine powder using a domestic coffee and spice grinder, then sent to the Analytical Services Unit, School of Agriculture and Food Science, University of Queensland. 4.1. Field Trials with AgSil32 In 2014 trees were treated in June (all treatments) and December (spray and drench), 2015 trees treated in May (all treatments) and December (spray and drench), 2016 treated in May (spray and drench) and June (all treatments). Tree health assessments have been obtained at least twice each year, and fruit were harvested for quality assessments in 2015 and 2016. Site 4 (Comboyne, NSW): This trial site was established on an orchard with no history of potassium silicate treatment. Trees have been receiving recent spray applications of phosphorous acid to restore tree health, however a row of trees in mid-decline was identified as a treatment site. Nine and ten trees, respectively, were selected for untreated controls or Agsil drench applications (300 mL Agsil per tree, delivered in 20 L of water, i.e., 5000 ppm). Agsil was applied by farm staff in August 2014; 13 of 15 Plants 2017, 6, 51 February, June, November and December 2015; and May and June 2016. Trees were either pruned heavily or staghorned in March 2015. This trial was visited twice only, prior to treatment application and at the end of the trial in 2016. All trees in the trial were harvested at the time of final tree health assessment (August 2016) and one bin of fruit from each treatment were transported to a commercial packingshed to obtain packout data. 4.2. Greenhouse Trials with Mineral Mulch and Mineral Dust The samples were prepared by closed-vessel microwave assisted acid digestion, (to solubilize elements in the presence of organic molecules), and then analyzed by ICP-OES (inductively coupled plasma optical emission spectrometry). 4.3. Field Trials with Mineral Mulch Canopy heath was again assessed approximately 10 months later, and leaves and fruit were collected for quality and nutrient analyses. rot. Ten trees in mid-decline were selected and tagged. MM was applied by manual broadcast to the drip zone of 10 trees in the centre row at the rate of 4.5 kg/tree. Canopy heath was again assessed approximately 10 months later, and leaves and fruit were collected for quality and nutrient analyses. Site 3 (Childers, QLD): A section of 5-year-old trees near a farm dam was selected, as trees were showing decline due to Phytophthora root rot. Trees were also showing signs of moderate salt (chloride) leaf burn. Canopy health was recorded, and MM applied by manual broadcast to the d i f 10 t i th t t th t f 1 9 k /t C h th i d pp y q y y Site 3 (Childers, QLD): A section of 5-year-old trees near a farm dam was selected, as trees were showing decline due to Phytophthora root rot. Trees were also showing signs of moderate salt (chloride) leaf burn. Canopy health was recorded, and MM applied by manual broadcast to the drip zone of 10 trees in the centre row at the rate of 1.9 kg/tree. Canopy heath was again assessed approximately 10 months later, and leaves and fruit were collected for quality and nutrient analyses, as described previously. There were limited numbers of fruit on trees. Acknowledgments: Project AV13021 “Exploring alternatives for managing Phytophthora root rot in avocados” was funded by Hort Innovation using contributions from the Australian Government and PQ Australia Pty Ltd. Hort Innovation is the grower owned, not-for-profit research and development corporation for Australian horticulture. Trials with Mineral Mulch and Mineral Dust were funded by Shiftwaste P/L and Rob and Wendy Howe are acknowledged for their support. Several avocado growers, orchard managers and farm staff collaborated in the field trials and their assistance is greatly appreciated. Casual employees assisted with field trial treatments, fruit harvests and quality assessments. Author Contributions: E.K.D. conceived and designed the experiments, obtained research funding, analy most of the data and wrote the paper. D.P.L. performed the greenhouse experiments. Conflicts of Interest: The authors declare no conflict of interest. 4.3. Field Trials with Mineral Mulch Site 1 (Kumbia, QLD): Trees in this orchard were approximately 5 years old at the start of the trial. A poorer section of the orchard was selected, where trees had not established well, or had died. Eight pairs of trees which were stunted, yellow and not thriving were tagged. Each tagged tree was rated for canopy health (0 = completely healthy and 10 = dead), and height and canopy diameter measured. MM was spread under one of the trees in each pair, in an area corresponding to the canopy drip zone, at the rate of 255 g MM/m2. Tree height varied considerably throughout the trial, so MM applications ranged from a minimum of 0.5 kg applied to the smallest tree up to 2.7 kg applied to the largest tree. The same canopy heath, height and diameter measurements were recorded approximately 10 months after the MM application. Leaves and fruit were collected for quality and nutrient analyses, as described previously, but many of the trees had either no fruit or very little fruit, potentially skewing results. Site 2 (Goodwood, QLD): Trees in this orchard were approximately 11 years old at the start of the trial, and sections of the orchard are showing fairly uniform decline due to Phytophthora root 14 of 15 Plants 2017, 6, 51 rot. Ten trees in mid-decline were selected and tagged. MM was applied by manual broadcast to the drip zone of 10 trees in the centre row at the rate of 4.5 kg/tree. Canopy heath was again assessed approximately 10 months later, and leaves and fruit were collected for quality and nutrient analyses. Site 3 (Childers, QLD): A section of 5-year-old trees near a farm dam was selected, as trees were showing decline due to Phytophthora root rot. Trees were also showing signs of moderate salt (chloride) leaf burn. Canopy health was recorded, and MM applied by manual broadcast to the drip zone of 10 trees in the centre row at the rate of 1.9 kg/tree. Canopy heath was again assessed approximately 10 months later, and leaves and fruit were collected for quality and nutrient analyses, as described previously. There were limited numbers of fruit on trees. rot. Ten trees in mid-decline were selected and tagged. MM was applied by manual broadcast to the drip zone of 10 trees in the centre row at the rate of 4.5 kg/tree. References 1. Pozza, E.A.; Pozza, A.A.A.; dos Santos Botelho, D.M. Silicon in plant disease control. Rev. Ceres 2015, 62, 323–331. [CrossRef] 2. Tubana, B.S.; Babu, T.; Datnoff, L.E. A review of silicon in soils and plants and its role in us agriculture: History and future persectives. Soil Sci. 2016, 181, 393–411. [CrossRef] 3. Liang, Y.; Sun, W.; Zhu, Y.-G.; Christie, P. Mechanisms of silicon-mediated alleviation of abiotic stresses in higher plants: A review. Environ. Pollut. 2007, 147, 422–428. [CrossRef] [PubMed] 4. Cazorla, F.M.; Arrebola, E.; Olea, F.; Velasco, L.; Hermoso, J.M.; Perez-Garcia, A.; Tores, J.A.; Farre, J.M.; de Vicente, A. Field evaluation of treatments for the control of the bacterial apical necrosis of mango (Mangifera indica) caused by Pseudomonas syringae pv. syringae. Eur. J. Plant Pathol. 2006, 116, 279–288. [CrossRef] 5. Gutierrez-Barranquero, J.A.; Arrebola, E.; Bonilla, N.; Sarmiento, D.; Cazorla, F.M.; de Vicente, A. Environmentally friendly treatment alternatives to bordeaux mixture for controlling bacterial apical necrosis (ban) of mango. Plant Pathol. 2012, 61, 665–676. [CrossRef] 6. Asanzi, N.M.; Taylor, N.J.; Vahrmeijer, J.T. Can silicon be used to prevent Alternaria alternata in citrus trees? SA Fruit J. 2014, 13, 48–51. 7. Helaly, M.N.; El-Hoseiny, H.; El-Sheery, N.I.; Rastogi, A.; Kalaji, H.M. Regulation and physiological role of silicon in alleviating drought stress of mango. Plant Physiol. Biochem. 2017, 118, 31–44. [CrossRef] [PubMed] 8. Marais, L.J. Efficacy of water soluble silicon in managing Fusarium dry root rot of citrus. Acta Hortic. 2015, 7. Helaly, M.N.; El-Hoseiny, H.; El-Sheery, N.I.; Rastogi, A.; Kalaji, H.M. Regulation and physiological role of silicon in alleviating drought stress of mango. Plant Physiol. Biochem. 2017, 118, 31–44. [CrossRef] [PubMed] silicon in alleviating drought stress of mango. Plant Physiol. Biochem. 2017, 118, 31–44. [CrossRef] [PubMed] 8. Marais, L.J. Efficacy of water soluble silicon in managing Fusarium dry root rot of citrus. Acta Hortic. 2015, 1065, 993–999. [CrossRef] 8. Marais, L.J. Efficacy of water soluble silicon in managing Fusarium dry root rot of citrus. Acta Hortic. 2015, 1065, 993–999. [CrossRef] 9. Jamar, L.; Lefrancq, B.; Fassotte, C.; Lateur, M. A during-infection spray strategy using sulphur compounds, copper, silicon and a new formulation of potassium bicarbonate for primary scab control in organic apple production. Eur. J. Plant Pathol. 2008, 122, 481–493. [CrossRef] 10. Vieira, D.L.; Barbosa, V.D.O.; Souza, W.C.O.D.; Silva, J.G.D.; Malaquias, J.B.; Batista, J.D.L. Potassium silicate-induced resistance against blackfly in seedlings of Citrus reticulata. Fruits 2016, 71, 49–55. References [CrossRef] Edgerton, L.J.; Veinbrants, N.; Hutchinson, J.F. Foliar sprays of silicon dioxide-containing compound reduce russeting in ‘Golden Delicious’ apple fruits. HortScience 1976, 11, 508–509. 12. Costa, I.J.S.; Pereira, M.C.T.; Mizobutsi, G.P.; Maia, V.M.; Silva, J.F.; Oliveira, J.A.A.; Oliveira, M.B.; Souza, V.N.R.; Nietsche, S.; Santos, E.F.; et al. Influence of silicon fertilization on ‘Palmer‘ mango tree cultivation. Acta Hortic. 2015, 1075, 229–234. [CrossRef] Plants 2017, 6, 51 15 of 15 15 of 15 13. Matichenkov, V.; Bocharnikova, E.; Calvert, D. Response of citrus to silicon soil amendments. Proc. Fla. State Hortic. Soc. 2001, 114, 94–97. 14. Anderson, J.M.; Pegg, K.G.; Coates, L.M.; Dann, E.K.; Cooke, A.W.; Smith, L.; Dean, J.R. Silicon and disease management in avocados. Talk. Avocados 2004, 15, 23–25. 15. Anderson, J.M.; Pegg, K.G.; Dann, E.K.; Cooke, A.W.; Smith, L.A.; Willingham, S.L.; Giblin, F.R.; Dean, J.R.; Coates, L.M. New Strategies for the Integrated Control of Avocado Fruit Diseases. In Proceedings of the New Zealand and Australia Avocado Growers Conference, Tauranga, New Zealand, 20–22 September 2005; p. 6. 16. Smith, L.A.; Dann, E.K.; Leonardi, J.; Dean, J.R.; Cooke, A.W. Exploring Non-Traditional Products for Management of Postharvest Anthracnose and Stem End Rot in Avocado. In Proceedings of the VII World Avocado Congress 2011 (Actas VII Congreso Mundial del Aguacate 2011), Cairns, Australia, 5–9 September 2011. 17. Kaluwa, K.; Bertling, I.; Bower, J.P.; Tesfay, S.Z. Silicon application effects on ‘Hass’ avocado fruit physiology. S. Afr. Avocado Grower Assoc. Yearb. 2010, 33, 44–47. [CrossRef] 18. Bosse, R.J.; Bower, J.P.; Bertling, I. Pre- and post-harvest treatments on ‘Fuerte’ avocados to control anthracnose (Colletotrichum gloeosporioides) during ripening. S. Afr. Avocado Grower Assoc. Yearb. 2011, 34, 65–69. 19. Tesfay, S.Z.; Bertling, I.; Bower, J.P. Effects of postharvest potassium silicate application on phenolics and other anti-oxidant systems aligned to avocado fruit quality. Postharvest Biol. Technol. 2011, 60, 92–99. [CrossRef] 20. Giblin, F. Silicon and the control of Phytophthora root rot in avocado seedlings. Talk. Avocados 2006, 1 21. Bekker, T.F.; Labuschagne, N.; Aveling, T.; Kaiser, C. The inhibition of Phytophthora root rot of avocado with potassium silicate application under greenhouse conditions. S. Afr. Avocado Grower Assoc. Yearb. 2007, 30, 49–56. 22. 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Evaluation of alternatives to phosphorous acid contro Phytophthora root rot. N. Z. Avocado Grower Assoc. Annu. Res. Rep. 2008, 8, 71–76. 25. Dann, E.K.; Ploetz, R.C.; Coates, L.M.; Pegg, K.G. Foliar, fruit and soilborne diseases. In The Avocado: Botany, Production and Uses, 2nd ed.; Schaffer, B., Wolstenholme, N., Whiley, A., Eds.; CABI Publishing: Wallingford, UK, 2013; pp. 380–422. 26. Dann, E.K.; Cooke, A.W.; Forsberg, L.I.; Pegg, K.G.; Tan, Y.P.; Shivas, R.G. Pathogenicity studies in avocado with three nectriaceous fungi, Calonectria ilicicola, Gliocladiopsis sp. and Ilyonectria liriodendri. Plant Pathol. 2012, 61, 896–902. [CrossRef] 27. Parkinson, L.E.; Shivas, R.G.; Dann, E.K. Pathogenicity of nectriaceous fungi on avocado in Australia. Phytopathology 2017. [CrossRef] [PubMed] 28. Berthelsen, S.; Hurney, A.; Kingston, G.; Rudd, A.; Garside, A.L.; Noble, A.D. Plant cane responses to silicated products in the Mossman, Innisfail and Bundaberg districts. Proc. Aust. Soc. Sugar Cane Technol. 2001, 23, 297–303. 29. Darvas, J.M.; Toerien, J.C.; Milne, D.L. Control of avocado root rot by trunk injection with phosethyl-Al. Plant Dis. 1984, 68, 691–693. [CrossRef] 30. Bekker, T.F.; Kaiser, C.; Merwe, R.V.D.; Labuschagne, N. In-Vitro inhibition of mycelial growth of several phytopathogenic fungi by soluble potassium silicate. S. Afr. J. Plant Soil 2006, 23, 169–172. [CrossRef] © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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The two-year impact of first generation protease inhibitor based antiretroviral therapy (PI-ART) on health-related quality of life.
Health and quality of life outcomes
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City Research Online City Research Online City, University of London Institutional Repository Citation: Eriksson, L. E., Bratt, G. A., Sandström, E. & Nordström, G. (2005). The two-year impact of first generation protease inhibitor based antiretroviral therapy (PI-ART) on health- related quality of life. Health and Quality of Life Outcomes, 3(1), 32. doi: 10.1186/1477- 7525-3-32 Citation: Eriksson, L. E., Bratt, G. A., Sandström, E. & Nordström, G. (2005). The two-year impact of first generation protease inhibitor based antiretroviral therapy (PI-ART) on health- related quality of life. Health and Quality of Life Outcomes, 3(1), 32. doi: 10.1186/1477- 7525-3-32 This is the unspecified version of the paper. Received: 16 December 2004 Accepted: 04 May 2005 Health and Quality of Life Outcomes 2005, 3:32 doi:10.1186/1477-7525-3- 32 This article is available from: http://www.hqlo.com/content/3/1/32 © 2005 Eriksson et al; licensee BioMed Central Ltd. © 2005 Eriksson 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. ticle distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0 d use, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit Link to published version: https://doi.org/10.1186/1477-7525-3-32 Link to published version: https://doi.org/10.1186/1477-7525-3-32 Copyright: City Research Online aims to make research outputs of City, University of London available to a wider audience. Copyright and Moral Rights remain with the author(s) and/or copyright holders. URLs from City Research Online may be freely distributed and linked to. Reuse: Copies of full items can be used for personal research or study, educational, or not-for-profit purposes without prior permission or charge. Provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. Open Ac Research The two-year impact of first generation protease inhibitor based antiretroviral therapy (PI-ART) on health-related quality of life Lars E Eriksson*1,2,3, Göran A Bratt2, Eric Sandström2 and Gun Nordström1 Open Access Open Ac Research The two-year impact of first generation protease inhibitor based antiretroviral therapy (PI-ART) on health-related quality of life Lars E Eriksson*1,2,3, Göran A Bratt2, Eric Sandström2 and Gun Nordström1 Address: 1Department of Nursing, Karolinska Institutet, 23300, SE-141 83 Huddinge, Sweden, 2Department of Venhälsan, South Stockholm General Hospital, SE-118 83 Stockholm, Sweden and 3Department of Virology, Swedish Institute for Infectious Disease Control, SE-171 82 Solna Address: 1Department of Nursing, Karolinska Institutet, 23300, SE-141 83 Huddinge, Sweden, 2Department of Venhälsan, South Stockholm General Hospital, SE-118 83 Stockholm, Sweden and 3Department of Virology, Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden Email: Lars E Eriksson* - Lars.Eriksson@omv.ki.se; Göran A Bratt - Goran.Bratt@karolinska.se; Eric Sandström - Eric.Sandstrom@karolinska.se; Gun Nordström - Gun.Nordstrom@kau.se * Corresponding author Received: 16 December 2004 Accepted: 04 May 2005 Health and Quality of Life Outcomes BioMed Central BioMed Central Background HRQL instrument described below before the start of PI- ART (pre PI-ART). HIV infection was documented by at least two laboratory tests (two repeated ELISA tests or one ELISA test and one Western Blot). The patients were treated according to best clinical practice and the partici- pant's physician chose the drug combination of the PI- ART (at least 2 nucleoside analogue reverse transcriptase inhibitors and either indinavir (n = 55) or ritonavir (n = 17) at start). Approximately two years after the introduc- tion of PI-ART (follow-up; mean 25.1 months, standard deviation (SD) 2.8 months, post initiation), 54 of the 72 subjects (75 %) completed the follow-up measure. Thirteen patients had died, four had changed clinic and one did not receive the follow-up questionnaire. g Protease inhibitor based antiretroviral therapy (PI-ART), defined as the combination of at least two nucleoside ana- logues with at least one protease inhibitor (PI) [1], was introduced in 1996 and has greatly reduced the incidence of HIV-related morbidity and mortality in the industrial- ised world [2,3]. PI-ART would thus be expected to have a positive effect on health-related quality of life (HRQL). On the other hand, HRQL might be negatively affected by strict adherence requirements as well as by short and long- term adverse effects [4,5]. Several studies (cross-sectional or longitudinal) have focused on the HRQL of HIV-positive individuals in dif- ferent stages of the HIV infection and under different treatment regimes. The results have varied but in general HIV infection affects several physical, psychological and social dimensions of HRQL and patients with sympto- matic disease and/or an AIDS-defining complication are more severely affected than those with other comparable chronic diseases [6-8]. HRQL has been shown to be related to the CD4 value, viral load and symptoms, so that patients with a more advanced state of HIV infection reported poorer HRQL [9-15]. Moreover, symptoms, physical function, role function and sexual function dete- riorated over time, while emotional domains were unchanged or improved [9,10,16]. Only a few studies on the influence of long-term (>1 year) PI-ART on the quality of life have yet been published. Nieuwkerk et al. [17] com- pared the development of HRQL during three different PI based regimes and concluded that in terms of HRQL, patients with higher CD4 values at start experienced less benefit from the treatment. Burgoyne et al. Abstract Background: Protease inhibitor based antiretroviral therapy (PI-ART) was introduced in 1996 and has greatly reduced the incidence of HIV-related morbidity and mortality in the industrialised world. PI-ART would thus be expected to have a positive effect on health-related quality of life (HRQL). On the other hand, HRQL might be negatively affected by strict adherence requirements as well as by short and long- term adverse effects. The aim of this study was to assess the influence of two years of first generation PI- ART on HRQL in patients with a relatively advanced state of HIV-infection. Furthermore, we wanted to investigate the relation between developments in HRQL and viral response, self-reported adherence and subjective experience of adverse effects in patients with PI-ART. Methods: HRQL was measured by the Swedish Health-Related Quality of Life Questionnaire (SWED- QUAL). Sixty-three items from the SWED-QUAL forms two single-item and 11 multi-item dimension scales. For this study, two summary SWED-QUAL scores (physical HRQL composite score and emotional HRQL composite score) were created through a data reduction procedure. At the 2-year follow-up measurement (see below), items were added to measure adherence and subjective experience of adverse effects. Demographic and medical data were obtained from specific items in the questionnaires and from the medical files. Seventy-two patients who were among the first to receive PI-ART (indinavir or ritonavir based) responded to the questionnaire before the start of PI-ART. Of these, 54 responded to the same instrument after two years of treatment (13 had died, four had changed clinic and one did not receive the questionnaire). Results: The main findings were that the emotional HRQL deteriorated during two years of PI-ART, while the physical HRQL remained stable. Multiple linear regression analyses showed that experience of adverse effects contributed most to the deterioration of emotional HRQL. Conclusion: In this sample of patients with relatively advanced state of HIV-infection, our data suggested that a negative development of physical HRQL had been interrupted by the treatment and that the emotional dimension of HRQL deteriorated during two years after start of PI-ART. Subjective experience of adverse effects made a major contribution to the decrease in emotional HRQL. The results underline the importance of including HRQL measures in the evaluation of new life prolonging therapies. Page 1 of 8 (page number not for citation purposes) (page number not for citation purposes) Health and Quality of Life Outcomes 2005, 3:32 http://www.hqlo.com/content/3/1/32 Background [18,19] fol- lowed HRQL in 41 patients with different treatment status over a period of four years and found no overall change of HRQL and that HRQL was less sensitive to CD4 changes than to symptom changes as well as that change in HRQL was somewhat related to change in social support. The Swedish Health-Related Quality of Life Questionnaire (SWED- QUAL) The Swedish Health-Related Quality of Life Questionnaire (SWED- QUAL) Q ) The patients completed the SWED-QUAL at the pre PI-ART and two-year follow-up visits. SWED-QUAL was developed by Brorsson et al. [20] from the measures used in the US Medical Outcomes Study (MOS) [21-24]. The question- naire, which is designed to measure HRQL, consists of 70 items, of which 63 forms two single-item and 11 multi- item dimension scales of Likert type: physical functioning (7 items), mobility (1 item), satisfaction with physical ability (1 item), role limitations due to physical health (3 items), pain (6 items), emotional well-being: positive affect (i.e. positive feelings; 6 items), emotional well- being: negative affect (i.e. negative feelings; 6 items), role limitations due to emotional health (3 items), sleep prob- lems (7 items), satisfaction with family life (relations with parents, siblings, children etc.; 4 items), relation to part- ner (6 items), sexual functioning (4 items) and general health perception (9 items). In the present study, the rela- tion to partner section was slightly modified to make it suitable for the investigated group (i.e. the word "spouse" was replaced by "partner"). Each scale is transformed into a 0–100 index; the higher the score, the better the per- ceived HRQL. In a general population sample, the inter- nal-consistency reliability coefficients (Cronbach's α) ranged from 0.79 to 0.89 [20]. Brorsson et al. have also reportedpreliminary support for the construct validity [20]. The instrument has been used in our previous study of HIV-positive subjects in Sweden [25]. The main aim of the present study was to investigate HRQL before and after two years of first generation PI- ART. The following research questions were addressed: (a) Does HRQL change during two years of PI-ART? (b) Do viral response, adherence, subjective experience of adverse effects and initial CD4 count predict changes in HRQL during two years of PI-ART? A factor analysis (see below) was performed on a merged material from three groups of patients: (a) the group described above at pre PI-ART (n = 72), (b) a previously described group of protease inhibitor naïve HIV patients (n = 73) [25] and (c) an unpublished material of 164 HIV- negative men who have sex with men visiting our infor- mation and screening clinic for sexually transmitted dis- eases (own unpublished data). Page 2 of 8 (page number not for citation purposes) Viral outcome On the basis of the participants' long-term virologic out- come to PI-ART, they were subdivided into viral respond- ers and viral non-responders. Viral responders were defined as participants who, after the first 3 months of PI- ART, had either HIV RNA less than 500 copies ml-1 in more than 75% of the analysed samples or a continuous decrease in HIV RNA to below 500 copies ml-1 before 18 months. All other participants were regarded as viral non- responders [28]. Demographic and medical data Demographic and medical data were obtained from spe- cific items in the questionnaires and from the medical files. A set of hierarchical multiple linear regression analyses [31] were performed to investigate whether the initial CD4 value, viral response, adherence and subjective expe- rience of adverse effects predicted the change in HRQL. The two analyses (one for PCS and one for ECS) were con- ducted in two forced steps with the follow-up PCS and ECS as dependent variables. The pre PI-ART PCS and ECS, respectively, were entered in the first step, while the three variables viral response, adherence and subjective experi- Health and Quality of Life Outcomes 2005, 3:32 Table 1: Rotated factor loading (varimax) of the principal component analysis (n = 309) Rotated factor loadings PCS ECS SWED-QUAL Scale Physical functioning 0.92 0.07 Mobility 0.83 0.04 Satisfaction with physical ability 0.79 0.24 Pain 0.56 0.45 Role limitations due to physical health 0.66 0.46 Sexual functioning 0.63 0.32 Role limitations due to emotional health 0.33 0.75 Emotional well-being, positive affect 0.08 0.81 Emotional well-being, negative affect 0.08 0.85 Sleep problems 0.32 0.70 PCSPhysical HRQL composite score; ECSEmotional HRQL composite score Table 1: Rotated factor loading (varimax) of the principal component analysis (n = 309) Adherence/adverse effects ff At the follow-up, the patients' adherence and drug-related adverse effects were examined. Adherence was assessed by asking the following question, modified from Morisky et al. [26]: "How many times during the last month did you skip doses of your HIV medication for the following rea- sons: because you felt so good that it did not matter, because you felt worse due to the medication or because you forgot?" For each of the three given reasons the patients were asked to rate the number of missed doses on a scale with five alternatives (1 = 0 doses, 2 = 1–2 doses, 3 = 3–5 doses, 4 = 6–9 doses or 5 = >9 doses). Patients were classified as non-adherent if they indicated >9 missed doses in at least one of the three mentioned categories (i.e. indicating 90% adherence or less) or if they indicated missed doses in all three categories. The subjective experience of medication-related adverse effects was addressed in one question, which asked the patients to rate their global experience of adverse effects during the past month on a 10 cm visual analogue scale (VAS) ranging from "none at all" to "severe". test [29] was used to compare differences between two related groups (i.e. pre PI-ART versus follow-up). Laboratory measures h b Lymphocyte subsets were determined, using routine flow cytometry [27], at least every 3–4 months. The level of HIV-1 RNA in plasma was quantified at the Swedish Insti- tute for Infectious Disease Control by a commercially available reverse transcriptase polymerase chain reaction (PCR)-based assay (HIV Monitor; Roche Diagnostic Sys- tems, Branchburg New Jersey, USA). The level of quantifi- cation at the time of the study was 500 viral copies ml-1. In order to achieve data reduction to create composite scores for use in multiple regression analyses of the mate- rial, a rotated component matrix analysis [30] was per- formed on the scores from ten of the 13 SWED-QUAL scales (i.e. those measuring physical and emotional health). The following scales were used in the factor anal- ysis: physical functioning, mobility, satisfaction with physical ability, role limitations due to physical health, pain, emotional well-being: positive affect, emotional well-being: negative affect, role limitations due to emo- tional health, sleep problems and sexual functioning. The analysis resulted in two factors: (a) physical HRQL com- posite score, i.e. PCS, comprising the averaged scores of the SWED-QUAL scales physical functioning, mobility, satisfaction with physical ability, pain, role limitations due to physical health and sexual functioning, and (b) emotional HRQL composite score, i.e. ECS, comprising the averaged scores of the SWED-QUAL scales role limita- tions due to emotional health, positive affect, negative affect and sleep problems (Table 1). These two factors explained 65 % of the total variance. Data collection Subjects The study was performed at the Department of Venhälsan at South Stockholm General Hospital, Sweden. A conven- ient sample of 72 subjects (70 men and 2 women) in an advanced state of HIV infection and who were among the first patients to receive PI-ART in Sweden responded to the Page 2 of 8 (page number not for citation purposes) Health and Quality of Life Outcomes 2005, 3:32 Health and Quality of Life Outcomes 2005, 3:32 http://www.hqlo.com/content/3/1/32 Page 3 of 8 (page number not for citation purposes) Statistical methods A comparison of the sin- gle SWED-QUAL scales at pre PI-ART with the same scales at follow-up revealed statistically significant decreases in the SWED-QUAL scales role limitations due to emotional health (pre PI-ART Md 88.9, IQR 66.7 – 100 versus follow- up Md 77.8, IQR 50.0 – 100; p < 0.05) and emotional well-being: negative affect (pre PI-ART Md 62.5, IQR 37.5 – 94.8 versus follow-up Md 50.0, IQR 25.0 – 75.0; p < 0.01; Table 3). The effect sizes of the change were 0.34 for the PCS, 0.36 for role limitations due to emotional health and 0.37 for emotional well-being: negative affect. Ethical approval Ethical approval The study was approved by the Local Ethical Committee at Huddinge University Hospital. Information about the study was given to the subjects in connection with an ordi- nary, scheduled visit to the clinic. Demographic and medical data At pre PI-ART, the mean age of the 72 patients was 41 (SD 9, range 23 – 65) years, 59 were of Swedish origin, 61 had prior antiretroviral treatment with only nucleoside ana- logue reverse transcriptase inhibitors, 32 had a CD4 count of <200 × 106 cells l-1 and 28 had an AIDS diagnosis. Fur- ther demographic and medical data are shown in Table 2. Statistical methods of subjects Percentage Mode of transmission Male to male 69 96 Female to male/male to female 3 4 Working status Full or part time work/studies 36 50 Sick leave or disability pension 31 43 Not stated 5 7 Education Compulsory school 13 18 Upper secondary school/high school 27 38 University studies 27 38 Not stated 5 7 Having a partner: Yes/no 30/41 42/57 not stated 1 1 CD4 count, cells × 106 l-1: median 127 range 1–660 HIV RNA, log10 copies ml-1: median 4.87 range 2.70–6.18 Time since first start of antiretroviral treatment, months: mean 33 SD 31.6 PI-ARTProtease inhibitor based antiretroviral therapy; SDStandard deviation Table 2: Demographic and medical data regarding a Swedish sample of HIV-infected persons before the start of PI-ART (n = 72) N f bj t P t Table 2: Demographic and medical data regarding a Swedish sample of HIV-infected persons before the start of PI-ART (n = 72) No. of subjects Percentage Table 2: Demographic and medical data regarding a Swedish sample of HIV-infected persons before the start of PI-ART (n = 72) No. of subjects Percentage Mode of transmission Male to male 69 96 Female to male/male to female 3 4 Working status Full or part time work/studies 36 50 Sick leave or disability pension 31 43 Not stated 5 7 Education Compulsory school 13 18 Upper secondary school/high school 27 38 University studies 27 38 Not stated 5 7 Having a partner: Yes/no 30/41 42/57 not stated 1 1 CD4 count, cells × 106 l-1: median 127 range 1–660 HIV RNA, log10 copies ml-1: median 4.87 range 2.70–6.18 Time since first start of antiretroviral treatment, months: mean 33 SD 31.6 PI-ARTProtease inhibitor based antiretroviral therapy; SDStandard deviation ence of adverse effects were entered in the second step together with the initial CD4 value. The patients' emotional well-being deteriorated during the first two years of PI-ART years of PI-ART The pre PI-ART and follow-up results of the composite scores and the single SWED-QUAL scales are shown in Table 3. The ECS decreased from pre PI-ART to follow-up (Md 71.2, interquartile range (IQR) 47.1 – 86.9 versus Md 63.6, IQR 41.0 – 77.3; p < 0.01;). Statistical methods Statistical calculations were performed with the assistance of the personal-computer program SPSS for Windows, version 11.0.0. The criterion for statistical significance was p < 0.05. Since some parameters did not fulfil the assump- tions of a normal distribution, the Wilcoxon signed ranks Page 3 of 8 (page number not for citation purposes) Page 3 of 8 (page number not for citation purposes) Health and Quality of Life Outcomes 2005, 3:32 http://www.hqlo.com/content/3/1/32 ence of adverse effects were entered in the second step together with the initial CD4 value. Ethical approval The study was approved by the Local Ethical Committee at Huddinge University Hospital. Information about the study was given to the subjects in connection with an ordi- nary, scheduled visit to the clinic. Results Demographic and medical data At pre PI-ART, the mean age of the 72 patients was 41 (SD 9, range 23 – 65) years, 59 were of Swedish origin, 61 had prior antiretroviral treatment with only nucleoside ana- logue reverse transcriptase inhibitors, 32 had a CD4 count of <200 × 106 cells l-1 and 28 had an AIDS diagnosis. Fur- ther demographic and medical data are shown in Table 2. Changes from pre PI-ART to follow-up The patients' emotional well-being deteriorated during the first two years of PI-ART The pre PI-ART and follow-up results of the composite scores and the single SWED-QUAL scales are shown in Table 3. The ECS decreased from pre PI-ART to follow-up (Md 71.2, interquartile range (IQR) 47.1 – 86.9 versus Md 63.6, IQR 41.0 – 77.3; p < 0.01;). A comparison of the sin- gle SWED-QUAL scales at pre PI-ART with the same scales at follow-up revealed statistically significant decreases in the SWED-QUAL scales role limitations due to emotional health (pre PI-ART Md 88.9, IQR 66.7 – 100 versus follow- up Md 77.8, IQR 50.0 – 100; p < 0.05) and emotional well-being: negative affect (pre PI-ART Md 62.5, IQR 37.5 – 94.8 versus follow-up Md 50.0, IQR 25.0 – 75.0; p < 0.01; Table 3). The effect sizes of the change were 0.34 for the PCS, 0.36 for role limitations due to emotional health and 0.37 for emotional well-being: negative affect. Subjective experience of adverse effects contributed to deteriorated Table 2: Demographic and medical data regarding a Swedish sample of HIV-infected persons before the start of PI-ART (n = 72) No. Subjective experience of adverse effects contributed to deteriorated emotional HRQL at follow-up The higher the score the better the health-related quality of life (n = 54) Score Pre PI-ART Median (IQR) Follow-up Median (IQR) P-valuea Physical HRQL composite score (PCS) 79.3 (64.5–92.5) 77.2 (58.4–92.9) NS Emotional HRQL composite score (ECS) 71.2 (47.1–86.9) 63.6 (41.0–77.3) <0.01 Physical functioning 95.2 (81.0–95.2) 95.2 (84.5–100) NS Mobility 100 (66.7–100) 100 (66.7–100) NS Satisfaction with physical ability 66.7 (33.3–83.3) 66.7 (33.3–100) NS Pain 100 (53.8–100) 100 (84.7–100) NS Role limitations due to physical health 66.7 (55.6–100) 88.9 (50.0–100) NS Sexual functioning 75.0 (41.7–91.7) 58.3 (41.7–91.7) NS Role limitations due to emotional health 88.9 (66.7–100) 77.8 (50.0–100) <0.05 Emotional well-being, positive affect 72.9 (47.9–83.3) 62.5 (37.5–83.3) NS Emotional well-being, negative affect 62.5 (37.5–94.8) 50.0 (25.0–75.0) <0.01 Sleep problems 67.9 (39.3–87.5) 57.1 (36.6–75.0) NS Satisfaction with family life 72.9 (49.5–90.6) 72.9 (55.7–85.4) NS Relation to partner 87.5 (75.0–100) 79.2 (52.1–95.8) NS General health perception 55.6 (36.1–75.0) 63.9 (38.9–80.6) NS aWilcoxon signed ranks test, change from pre PI-ART to follow-up; NSNon significant; IQRInterquartile range; HRQLHealth-related quality of life Table 4: Hierarchical multiple linear analyses of the influence of initial CD4 value and the variables viral response, subjective experience of adverse effects and adherence on the change in PCS and ECS from before start (pre PI-ART) to after two year of treatment (follow-up) Follow-up PCS Follow-up ECS Step 1 Partial correlationa Partial correlationa Pre PI-ART PCS 0.722*** - Pre PI-ART ECS - 0.681*** R2 0.605*** 0.456*** F 78.26 42.72 d.f. (regression;residual) 1;51 1;51 Step 2 Partial correlation Partial correlation Viral response 0.202 0.076 Adherence 0.167 0.235 Adverse effects 0.091 -0.366* Pre PI-ART CD4 value -0.043 -0.024 R2-change 0.028 0.128* F-change 0.91 3.63 d.f. (regression;residual) 4;47 4;47 PCSPhysical HRQL composite score; ECSEmotional HRQL composite score; PI-ARTProtease inhibitor based antiretroviral therapy; apartial correlation of the final model (step 2); *p < 0.05; ***p < 0.001 Table 3: The Swedish Health-Related Quality of Life Questionnaire (SWED-QUAL) results before and after 2 years of protease inhibitor based antiretroviral therapy (pre PI-ART and follow-up respectively). The higher the score the better the health-related quality of life (n = 54) Table 3: The Swedish Health-Related Quality of Life Questionnaire (SWED-QUAL) results before and after 2 years of protease inhibitor based antiretroviral therapy (pre PI-ART and follow-up respectively). Subjective experience of adverse effects contributed to deteriorated emotional HRQL at follow-up Subjective experience of adverse effects contributed to deteriorated emotional HRQL at follow-up From the pre PI-ART to the follow-up, the CD4 count of the surviving patients increased from a median (Md) of 150 (range 1 – 660) × 106 cells l-1 to 325 (range 5 – 840) × 106 cells l-1 (p < 0.001) and the HIV-1 RNA level decreased from 4.90 (range 2.70 – 6.00) log10 copies ml-1 to 2.70 (range 2.70 – 6.31) log10 copies ml-1 (p < 0.001). Thirty patients had switched PI during the first two years of PI- ART. A total of 36 subjects were classified as viral respond- ers and 18 as viral non-responders. Forty-seven (87 %) of the 54 patients were classified as adherent and seven (13 %) as non-adherent. The mean VAS score for subjective experience of adverse effects at fol- low-up was 3.39 (SD 2.95, Md 2.57, IQR 0.79 – 5.84) cm on a 10 cm scale. Two hierarchical multiple regression analyses were per- formed to investigate whether the change in physical and Page 4 of 8 (page number not for citation purposes) Page 4 of 8 (page number not for citation purposes) http://www.hqlo.com/content/3/1/32 Health and Quality of Life Outcomes 2005, 3:32 Table 3: The Swedish Health-Related Quality of Life Questionnaire (SWED-QUAL) results before and after 2 years of protease inhibitor based antiretroviral therapy (pre PI-ART and follow-up respectively). Page 5 of 8 (page number not for citation purposes) Discussion In this study we used the SWED-QUAL instrument to investigate the HRQL of 54 relatively advanced HIV patients before and after two years of first generation PI- ART in a setting where protease inhibitors were intro- duced. We also studied the patients' viral outcome, self reported adherence and subjective experience of adverse effects and the relationship between these variables and the development in HRQL. To minimise multiple com- parisons, a set of linear regression models were used. To increase the power to detect relations as a result of these models, we created two SWED-QUAL composite scores (PCS and ECS) through a data reduction procedure. When we further investigated the relation between the change in HRQL and the variables viral response, adher- ence, subjective experience of adverse effects and baseline CD4 value, our study showed that it was the subjective experience of adverse effects that contributed most to the deterioration in the emotional HRQL. It should be stressed that our measure of adverse effects was a global single item, where the patients were asked to rate their total perception of adverse effects during the previous month and that we did not sub-analyse this experience further into different symptoms. However, the negative impact of perceived side effects/symptoms on HRQL, is confirmed in a great number of quantitative investiga- tions [12,18,37-42]. Also, the results from our quantita- tive study agreed with those of a qualitative study that aimed to elicit the patients' real-life descriptions of their experience of combination therapy. Erlen & Mellors [43] found that side effects were one of the major problems associated with the therapy. Furthermore, symptoms or adverse events have been shown to be related to medica- tion adherence [44,45]. In order to improve HRQL and adherence, it is therefore crucial to find treatment combi- nations and strategies that minimise these negative effects and to individualize treatment. The simplified and less toxic treatment regimes available today, with antiretrovi- ral therapy based on three nucleoside analogue reverse transcriptase inhibitors or nucleoside analogues com- bined with non-nucleoside analogue reverse transcriptase inhibitors or ritonavirboosted PIs once or twice daily [1], may be less liable to have a negative impact on HRQL. Subjective experience of adverse effects contributed to deteriorated emotional HRQL at follow-up The higher the score the better the health-related quality of life (n = 54) Score Pre PI-ART Median (IQR) Follow-up Median (IQR) P-valuea Physical HRQL composite score (PCS) 79.3 (64.5–92.5) 77.2 (58.4–92.9) NS Emotional HRQL composite score (ECS) 71.2 (47.1–86.9) 63.6 (41.0–77.3) <0.01 Physical functioning 95.2 (81.0–95.2) 95.2 (84.5–100) NS Mobility 100 (66.7–100) 100 (66.7–100) NS Satisfaction with physical ability 66.7 (33.3–83.3) 66.7 (33.3–100) NS Pain 100 (53.8–100) 100 (84.7–100) NS Role limitations due to physical health 66.7 (55.6–100) 88.9 (50.0–100) NS Sexual functioning 75.0 (41.7–91.7) 58.3 (41.7–91.7) NS Role limitations due to emotional health 88.9 (66.7–100) 77.8 (50.0–100) <0.05 Emotional well-being, positive affect 72.9 (47.9–83.3) 62.5 (37.5–83.3) NS Emotional well-being, negative affect 62.5 (37.5–94.8) 50.0 (25.0–75.0) <0.01 Sleep problems 67.9 (39.3–87.5) 57.1 (36.6–75.0) NS Satisfaction with family life 72.9 (49.5–90.6) 72.9 (55.7–85.4) NS Relation to partner 87.5 (75.0–100) 79.2 (52.1–95.8) NS General health perception 55.6 (36.1–75.0) 63.9 (38.9–80.6) NS aWilcoxon signed ranks test, change from pre PI-ART to follow-up; NSNon significant; IQRInterquartile range; HRQLHealth-related quality of life Table 4: Hierarchical multiple linear analyses of the influence of initial CD4 value and the variables viral response, subjective experience of adverse effects and adherence on the change in PCS and ECS from before start (pre PI-ART) to after two year of treatment (follow-up) Follow-up PCS Follow-up ECS Step 1 Partial correlationa Partial correlationa Pre PI-ART PCS 0.722*** - Pre PI-ART ECS - 0.681*** R2 0.605*** 0.456*** F 78.26 42.72 d.f. (regression;residual) 1;51 1;51 Step 2 Partial correlation Partial correlation Viral response 0.202 0.076 Adherence 0.167 0.235 Adverse effects 0.091 -0.366* Pre PI-ART CD4 value -0.043 -0.024 R2-change 0.028 0.128* F-change 0.91 3.63 d.f. (regression;residual) 4;47 4;47 PCSPhysical HRQL composite score; ECSEmotional HRQL composite score; PI-ARTProtease inhibitor based antiretroviral therapy; apartial correlation of the final model (step 2); *p < 0.05; ***p < 0.001 PCSPhysical HRQL composite score; ECSEmotional HRQL composite score; PI-ARTProtease inhibitor based antiretroviral therapy; apartial correlation of the final model (step 2); *p < 0.05; ***p < 0.001 Page 5 of 8 (page number not for citation purposes) (page number not for citation purposes) Health and Quality of Life Outcomes 2005, 3:32 http://www.hqlo.com/content/3/1/32 emotional HRQL composite scores could be predicted by initial CD4 count, viral response, adherence and subjec- tive experience of adverse effects (Table 4). Subjective experience of adverse effects contributed to deteriorated emotional HRQL at follow-up Only the ECS model showed a statistically significant R2 change; that is, the subjective experience of adverse effects predicted a decrease in the emotional HRQL. only single drug antiretroviral therapy were followed for two years (own unpublished data). However, a study investigating the HRQL of patients receiving didanosine monotherapy or in combination with delavirdine (not approved in Europe due to side effects) showed slightly declining mental health scores for up to two years after the start of the trial [33]. After the introduction of PI-ART, improvements in depressive symptoms and mental health was reported in a shorter time period, i.e. after up to one year following the addition of a PI to existing antiretrovi- ral therapy [36,37]. Similarly, Rabkin et al. [38] found a reduction in psychological distress and clinical depression during a two-year period when PI-ART became widely available. In the latter study, however, PI-ART was intro- duced to the cohort continuously during the follow-up, resulting in a mean time with PI-ART shorter than two years (i.e. the analysis did not evaluate patients before and after start of PI-ART in a consistent manner). From a longer term perspective, however, emotional HRQL was found to be stable over a four-year period [18]. Internal consistency of the SWED-QUAL scales Cronbach's α reliability estimates for the 11 SWED-QUAL multi-item scales for the pre PI-ART and follow-up ranged between 0.74 and 0.92 indicating good internal consist- ency. The internal consistency for the two HRQL compos- ite scores was for the PCS α 0.88 and 0.85 (pre PI-ART and follow-up, respectively) and for the ECS α 0.91 and 0.89 (pre PI-ART and follow-up, respectively). Page 6 of 8 (page number not for citation purposes) Authors' contributions influence on HRQL than the combination with the PI [46]. Similarly, Carrieri et al. [37] found a positive devel- opment of HRQL in patients switching to a non-PI-con- taining therapy, as compared to patients continuing stable PI-ART. However, these aspects need to be further investi- gated and there is a need for further study of the effect of different treatment regimes on HRQL, in experienced as well as naïve patients. influence on HRQL than the combination with the PI [46]. Similarly, Carrieri et al. [37] found a positive devel- opment of HRQL in patients switching to a non-PI-con- taining therapy, as compared to patients continuing stable PI-ART. However, these aspects need to be further investi- gated and there is a need for further study of the effect of different treatment regimes on HRQL, in experienced as well as naïve patients. LEE, ES and GN conceived of the study. All authors made substantial contributions to conception, planning and design. LEE participated in the coordination, carried out the statistical analysis and interpretation of data and drafted the manuscript. GAB participated the coordina- tion and acquisition of the study and helped draft the manuscript. ES participated in the acquisition of the study and helped draft the manuscript. GN participated in the interpretation of data and helped draft the manuscript. All authors read and approved the final manuscript. Certain study limitations should be emphasized when interpreting the present results. Firstly, the small sample may mean that the study failed to detect further relations between the investigated variables. Secondly, a large pro- portion of the investigated patients were in an advanced state of their HIV infection and many had previous expe- rience of antiretroviral monotherapy. The inferences drawn from this study may not be applicable to ART naïve patients starting antiretroviral therapy in a less advanced state of the disease. Today treatment is started when cer- tain laboratory criteria are fulfilled and before symptoms, including AIDS-defining disease, are at a high risk of appearing. Thirdly, our study population may not be totally representative of all patients receiving PI-ART. The majority of our patients were well-educated males with a homosexual route of transmission. The influence on HRQL may be different for female patients or patients with other educational status or routes of transmission. It should be noted that we chose to consider viral outcome of the treatment in a longitudinal context, i.e. Acknowledgements g We would like to thank the nurses and physicians at the Department of Venhälsan, South Stockholm General Hospital for their help in administer- ing the instruments to the participants, Eva-Lena Fredriksson, Department of Venhälsan, South Stockholm General Hospital for her great assistance in the co-ordination of the study and J. Petter Gustavsson, Department of Nursing, Karolinska Institutet for helpful discussions. We would also like to acknowledge the assistance of the participants, who gave time and effort to respond to the questionnaires. This study was supported by grants from the Board of Research for Health and Caring Sciences at Karolinska Institutet. Discussion This was indicated in a randomised study comparing two nucleoside analogue reverse transcriptase inhibitors com- bined with either a PI or efavirenz, over a one year period and where the combination with efavirenz had a better The main findings from the present study were that the physical HRQL remained stable while the emotional HRQL deteriorated for two years of first generation PI-ART and that subjective experience of adverse effects was the strongest predictor of the deterioration in emotional HRQL ratings. The present study thus suggests that first generation PI- ART interrupted the progressive negative development of the physical domain of HRQL that has been reported in studies performed before the introduction of PI-ART [9,10,16,32,33]. Our findings are consistent with those of Goujard et al. [34] and Burgoyne et al. [18] who also failed to detect any changes in HRQL after one and a half and four years, respectively, when monitoring patients in a period after PI-ART had become available. The somewhat surprising decrease in emotional health found in the present study has, to our knowledge, not been reported in other longitudinal studies of PI-ART. Longitudinal studies performed before the introduction of PI-ART have, in general, showed stable or improved mental/emotional health over time [9,10,16,35]. We also found stable scores in the emotional domain of HRQL in a previous pre PI-ART era study, where patients with no or Page 6 of 8 (page number not for citation purposes) Health and Quality of Life Outcomes 2005, 3:32 http://www.hqlo.com/content/3/1/32 Authors' contributions viral response defined by repeated HIV RNA measures over up to 18 months. The impact on the development of HRQL might have been different if only one single measure of viral load at follow-up had been taken into account. p 5. Carr A, Cooper DA: Adverse effects of antiretroviral therapy. Lancet 2000, 356:1423-1430. p 5. Carr A, Cooper DA: Adverse effects of antiretroviral therapy. Lancet 2000, 356:1423-1430. In this sample of mainly advanced patients, the emotional dimension of HRQL had deteriorated for two years after the start of first generation PI-ART, and the subjective per- ception of adverse effects made a major contribution to this decrease. 6. Ragsdale D, Morrow JR: Quality of life as a function of HIV classification. Nurs Res 1990, 39:355-359. 7. Wu AW, Rubin HR, Mathews WC, Ware JE, Brysk LT, Hardy WD, Bozzette SA, Spector SA, Richman DD: A health status question- naire using 30 items from the Medical Outcomes Study. Med Care 1991, 29:786-798. 8. Wachtel T, Piette J, Mor V, Stein M, Fleishman J, Carpenter C: Qual- ity of life in persons with human immunodeficiency virus infection: Measurement by the Medical Outcomes Study Instrument. Ann Intern Med 1992, 116:129-137. The results of the present study show the importance of studying HRQL in a situation where there is a desperate need for life-saving new therapies. Therefore, consistently taking HRQL into account when treating HIV patients is of the utmost importance. Finding treatment combina- tions and strategies with the least negative long-term influence on HRQL is essential at a time when those hav- ing access to antiretroviral combination therapy have a dramatically increased life span. Furthermore, consider- ing the current numerous treatment options together with the fluctuations in HRQL over time, further short and long-term investigations of HRQL in patients receiving antiretroviral therapy are of the utmost importance. 9. Holzemer WL, Wilson HS: Quality of life and the spectrum of HIV infection. Annu Rev Nurs Res 1995, 13:3-29. 10. Wu AW, Rubin HR: Approaches to health status assessment in HIV disease overview of the conference. Psychol Health 1994, 9:1-18. 11. Call SA, Klapow JC, Stewart KE, Westfall AO, Mallinger AP, DeMasi RA, Centor R, Saag MS: Health-related quality of life and viro- logic outcomes in an HIV clinic. Qual Life Res 2000, 9:977-985. g 12. Lorenz KA, Shapiro MF, Asch SM, Bozzette SA, Hays RD: Associa- tions of symptoms and health-related quality of life: findings from a national study of persons with HIV infection. Ann Intern Med 2001, 134:854-860. 13. Murri R, Fantoni M, Borgo CD, Visona R, Barracco A, Zambelli A, Testa L, Orchi N, Tozzi V, Bosco O, Wu AW: Determinants of health-related quality of life in HIV-infected patients. AIDS Care 2003, 15:581-590. References 1. British HIV Association Writing Committee: British HIV Associa- tion (BHIVA) guidelines for the treatment of HIV-infected adults with antiretroviral therapy. HIV Med 2003, 4(Suppl 1):1-41. 2. ) 2. Palella FJ Jr, Delaney KM, Moorman AC, Loveless MO, Fuhrer J, Sat- ten GA, Aschman DJ, Holmberg SD: Declining morbidity and mortality among patients with advanced human immunode- ficiency virus infection. HIV Outpatient Study Investigators. N Engl J Med 1998, 338:853-860. 3. Mocroft A, Katlama C, Johnson AM, Pradier C, Antunes F, Mulcahy F, Chiesi A, Phillips AN, Kirk O, Lundgren JD: AIDS across Europe, 1994 98: the EuroSIDA study Lancet 2000 356:291 296 Chiesi A, Phillips AN, Kirk O, Lundgren JD: AIDS across Europe, 1994–98: the EuroSIDA study. Lancet 2000, 356:291-296. 4. Safrin S, Grunfeld C: Fat distribution and metabolic changes in patients with HIV infection. AIDS 1999, 13:2493-2505. 5 Carr A Cooper DA: Adverse effects of antiretroviral therapy 1994–98: the EuroSIDA study. Lancet 2000, 356:291-296. 4. Safrin S, Grunfeld C: Fat distribution and metabolic changes in patients with HIV infection. AIDS 1999, 13:2493-2505. p 5. Carr A, Cooper DA: Adverse effects of antiretroviral therapy. Lancet 2000, 356:1423-1430. Page 7 of 8 (page number not for citation purposes) Page 7 of 8 (page number not for citation purposes) Health and Quality of Life Outcomes 2005, 3:32 http://www.hqlo.com/content/3/1/32 36. Low-Beer S, Chan K, Yip B, Wood W, Montaner JSG, O'Shaughnessy MV, Hogg RS: Depressive symptoms decline among persons on HIV protease inhibitors. J Acquir Immune Defic Syndr 2000, 23:295-301. 14. Ichikawa M, Natpratan C: Quality of life among people living with HIV/AIDS in northern Thailand: MOS-HIV Health Survey. Qual Life Res 2004, 13:601-610. y 15. Kemmler G, Schmied B, Shetty-Lee A, Zangerle R, Hinterhuber H, Schüssler G, Mumelter B: Quality of life of HIV-infected patients: psychometric properties and validation of the Ger- man version of the MQOL-HIV. Qual Life Res 2003, 12:1037-1050. 37. Carrieri P, Spire B, Duran S, Katlama C, Peyramond D, Francois C, Chene G, Lang JM, Moatti JP, Leport C: Health-related quality of life after 1 year of highly active antiretroviral therapy. J Acquir Immune Defic Syndr 2003, 32:38-47. f y 38. Rabkin J, Ferrando S, Lin S, Sewell M, McElhiney M: Psychological effect of HAART: a 2-year study. Psychosom Med 2000, 62:413-422. 16. de Boer JB, van Dam FSAM, Sprangers MAG: Health-related qual- ity-of-life evaluation in HIV-infected patients. A Review of the literature. Pharmacoeconomics 1995, 8:291-304. 17. Nieuwkerk PT, Gisolf EH, Reijers MHE, Lange JMA, Danner SA, Sprangers MAG: Long-term quality of life outcomes in three antiretroviral treatment strategies for HIV-1 infection. AIDS 2001, 15:1985-1991. 39. Blanch J, Rousaud A, Martinez E, Lazzari ED, Milinkovic A, Peri J-M, Blanco J-L, Jaen J, Navarro V, Massana G, Gatell J-M: Factors asso- ciated with severe impact of lipodystrophy on the quality of life of patients infected with HIV-1. Clin Infect Dis 2004, 38:1464-1470. 18. Burgoyne RW, Rourke SB, Behrens DM, Salit IE: Long-term qual- ity-of-life outcomes among adults living with HIV in the HAART era: the interplay of changes in clinical factors and symptom profile. AIDS Behav 2004, 8:151-163. 40. Hudson A, Kirksey K, Holzemer W: The influence of symptoms on quality of life among HIV-infected women. West J Nurs Res 2004, 26:9-23. y p p 19. Burgoyne R, Renwick R: Social support and quality of life over time among adults living with HIV in the HAART era. Soc Sci Med 2004, 58:1353-1366. 41. p 5. Carr A, Cooper DA: Adverse effects of antiretroviral therapy. Lancet 2000, 356:1423-1430. Morisky DE, Green LW, Levine DM: Concurrent and predictive validity of a self-reported measure of medication adherence. Med Care 1986, 24:67-74. 46. Fumaz CR, Tuldrà A, Ferrer MJ, Paredes R, Bonjoch A, Jou J, Negredo E, Romeu J, Sirera G, Tural C, Clotet B: Quality of life, emotional status, and adherence of HIV-1-infected patients treated with efavirenz versus protease inhibitor-containing regimens. J Acquir Immune Defic Syndr 2002, 29:244-253. 27. 1994 revised guidelines for the performance of CD4+ T-cell determinations in persons with human immunodeficiency virus (HIV) infections. Centers for Disease Control and Prevention. MMWR Recom Rep 1994, 43:1-21. 28. Hejdeman B, Lenkei R, Leandersson A-C, Hultström AL, Wahren B, Sandström E, Bratt G: Clinical and immunological benefits from highly active antiretroviral therapy in spite of limited viral load reduction in HIV type 1 infection. AIDS Res Hum Retroviruses 2001, 17:277-286. 29. Shott S: Statistics for health professionals Philadelphia: W.B. Saunders Company; 1990. 30. Fayers PM, Machin D: Quality of life: assessment, analysis, and interpretation West Sussex, England: John Wiley & Sons Ltd; 2000. 31. Cohen J, Cohen P: Applied multiple regression/correlation analysis for the behavioral sciences Second edition. Hillsdale, NJ: Lawrence Erlbaum Associates, Inc; 1983. 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 8 of 8 (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 32. p 5. Carr A, Cooper DA: Adverse effects of antiretroviral therapy. Lancet 2000, 356:1423-1430. Préau M, Leport C, Salmon-Ceron D, Carrieri P, Portier H, Chene G, Spire B, Choutet P, Raffi F, Morin M, APROCO Study Group: Health-related quality of life and patient-provider relation- ships in HIV-infected patients during the first three years after starting PI-containing antiretroviral treatment. AIDS Care 2004, 16:649-661. , 20. Brorsson B, Ifver J, Hays RD: The Swedish Health-Related Qual- ity of Life Survey (SWED-QUAL). Qual Life Res 1993, 2:33-45. 21. Stewart AL, Hays RD, Ware JE: The MOS Short-form General Health Survey: Reliability and validity in a patient population. Med Care 1988, 26:724-735. 42. Tramarin A, Parise N, Campostrini S, Yin DD, Postma MJ, Lyu R, Gri- setti R, Capetti A, Cattelan AM, Di Toro MT, Mastroianni A, Pignat- tari E, Mondardini V, Calleri G, Raise E, Starace F, Palladio Study Group: Association between diarrhea and quality of life in HIV-infected patients receiving highly active antiretroviral therapy. Qual Life Res 2004, 13:243-250. p p 22. Tarlov AR, Ware JE, Greenfield S, Nelson EC, Perrin E, Zubkoff M: The Medical Outcomes Study. An application of methods for monitoring the results of medical care. JAMA 1989, 262:925-930. py Q f 43. Erlen JA, Mellors MP: Adherence to combination therapy in persons living with HIV: balancing the hardships and the blessings. J Assoc Nurses AIDS Care 1999, 10:75-84. 23. 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A label-free aptamer-based nanogap capacitive biosensor with greatly diminished electrode polarization effects
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View Article Online View Journal View Article Online View Journal View Article Online View Journal PCCP Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the author guidelines. You can find more information about Accepted Manuscripts in the author guidelines. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the ethical guidelines, outlined in our author and reviewer resource centre, still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. Accepted Manuscript Physical Chemistry Chemical P Accepted Manuscript Physical Chemistry Chemical P This article can be cited before page numbers have been issued, to do this please use: Z. Ghobaei Namhil, C. Kemp, E. Verrelli, A. Iles, N. Pamme, A. Adawi and N. T. Kemp, Phys. Chem. Chem. Phys., 2018, DOI: 10 1039/C8CP05510F This article can be cited before page numbers have been issued, to do this please use: Z. Ghobaei Namhil, C. Kemp, E. Verrelli, A. Iles, N. Pamme, A. Adawi and N. T. Kemp, Phys. Chem. Chem. Phys., 2018, DOI: 10.1039/C8CP05510F. This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. PCCP Physical Chemistry Chemical Physics www.rsc.org/pccp ISSN 1463-9076 PERSPECTIVE Darya Radziuk and Helmuth Möhwald Ultrasonically treated liquid interfaces for progress in cleaning and separation processes Volume 18 Number 1 7 January 2016 Pages 1–636 A Label-Free Aptamer-Based Nanogap Capacitive Biosensor with Greatly Diminished Electrode Polarization Effects View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript Zahra Ghobaei Namhil,a Cordula Kemp,b Emanuele Verrelli,a,c Alex Iles,a Nicole Pamme,a Ali M. Adawi,a,c Neil T. Kempa,c,* Zahra Ghobaei Namhil,a Cordula Kemp,b Emanuele Verrelli,a,c Alex Iles,a Nicole Pamme,a Ali M. Adawi,a,c Neil T. Kempa,c,* emistry Chemical Physics Accepted Ma a School of Mathematics and Physical Sciences, University of Hull, Hull, HU6 7RX, UK b School of Lifesciences, University of Hull, Hull, HU6 7RX, UK c George W Gray Centre for Advanced Materials, University of Hull, Hull, HU6 7RX, UK * Author to whom correspondence should be addressed. Electronic mail: N.Kemp@hull.ac.uk Physical Chemistry Chemical Physics Ac rsc.li/pccp Page 1 of 24 Physical Chemistry Chemical Physics Abstract A significant impediment to the use of impedance spectroscopy in bio-sensing is the electrode polarization effect that arises from the movement of free ions to the electrode-solution interface, forming an electrical double layer (EDL). The EDL screens the dielectric response of the bulk and its large capacitance dominates the signal response at low frequency, masking information particularly relevant for biological samples, such as molecular conformation changes and DNA hybridization. The fabrication of nanogap capacitors with electrode separation less than the EDL thickness can significantly reduce electrode polarization effects and provide enormous improvement in sensitivity due to better matching of the sensing volume with the size of the target entities. We report on the fabrication of a horizontal thin-film nanogap capacitive sensor with electrode separation of 40 nm that shows almost no electrode polarization effects when measured with water and ionic buffer solutions, thereby allowing direct quantification of their relative permittivity at low frequencies. Surface modification of the electrodes with thiol-functionalized single strand DNA aptamers transforms the device into a label-free biosensor with high sensitivity and selectivity towards the detection of a specific protein. Using this approach, we have developed a biosensor for the detection of human alpha thrombin. In addition, we also examine frequency dependent permittivity measurements on high ionic strength solutions contained within the nanogap and discuss how these support recent experimental observations of large Debye lengths. A large shift in the Debye relaxation frequency to lower frequency is also found, which is consistent with water molecules being in a rigid-like state, possibly indicating the formation of an ordered “ice-like” phase. Altogether, this work highlights the need for better understanding of fluids in confined, nanoscale geometries, from which important new applications in sensing may arise. Physical Chemistry Chemical 1 Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Page 2 of 24 Page 2 of 24 Introduction l Chemistry Chemical Physics Accepted Physical Chemistry Chemical Physics Ac Physical Chemistry Chemical Ph 2 2 Page 3 of 24 Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Page 3 of 24 Figure 1. a) Effect of electrode polarization on measurements of the electrical relative permittivity and b) overlapping electrical double layers leads to a constant electrical potential across a nanogap device, eliminating electrode polarization effects. View Article Online DOI: 10.1039/C8CP05510F Figure 1. a) Effect of electrode polarization on measurements of the electrical relative permittivity and b) overlapping electrical double layers leads to a constant electrical potential across a nanogap device, eliminating electrode polarization effects. View Article Online DOI: 10.1039/C8CP05510F Physical Chemistry Chemical Physics Accepted Manuscript cal Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript Nanogap electrodes having electrode separation less than the electrical double layer (EDL) thickness, typically <100 nm, can significantly reduce electrode polarization effects since the confined region causes the electrical double layers from opposite electrodes to overlap, leading to a constant electrical potential across the nanogap region as shown in Figure 1b). This has been shown theoretically by treating the distribution of ions in the EDL in terms of the Gouy-Chapman model16,17 and solving the Poisson-Boltzmann equation for the electrical potential. In this case, when the electrode separation, L, is less than the electrical double layer thickness, such that L~0.1 κ-1 (where κ is the Debye-Hückel parameter and κ-1 is the Debye length) the electrical potential is almost constant across the nanogap region,18 rather than dropping to zero in the middle of the gap. The small potential difference between the electrode interface and the middle region of the gap indicates an almost negligible contribution to the measured impedance from the electrical double layer and subsequently almost complete elimination of the electrode polarization effect. This has recently been demonstrated19 in a vertical electrode nanogap structure whereby the relative permittivity of water at 10 Hz was measured to be εr ~ 75 in contrast to macroscopic spaced electrodes where significant electrode polarization effects instead give a value many orders of magnitude larger, εr ~ 1.5×106. Introduction View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript Label-free electrochemical1 and field effect transistor2 bio-sensors have gained enormous attention in the last decade because of their low cost, portability and high potential for impact in point-of-care applications. In combination with recent advances in nanoscale engineering and surface functionalization for immobilization and detection,3 devices have attained extraordinary levels of sensitivity with new memristor-based Si nanowire sensors recently achieving detection levels as low as 23 attomole.4 Nowadays, a wide range of nanogap electrodes for bio-sensing applications have been investigated (see reviews5,6). However, the vast majority of these are resistive based sensors.7,8,9 Capacitive based biosensors, such as those made in this study, have been investigated much less and although they have not yet achieved the levels of sensitivity of resistive based sensors, they can provide important additional knowledge (via the frequency dependence and phase information) as well as have the same advantages of small footprint, simple instrumentation, low- cost fabrication and real-time detection. Although macroscopic sized capacitive biosensors have been used for quite some time10 their detection capabilities have been limited because of the large electrode polarization effect, which arises from the movement of free ions to the electrode-solution interface, forming an electrical double layer. The capacitance of this layer dominates the signal at low frequency, masking relaxation effects in the bulk11 and leading to incorrect measurement of the relative permittivity as shown in Figure 1a). True quantification of the dielectric properties of the relevant part of the material or solution has therefore relied on methods of either correcting the measurement via analytical means,12,13 which involves the fitting of various empirical functions or modelling of the electrode polarization via long-range many-body theory, or by physical compensation of the electrode double layer itself. The latter involving the use of reference electrodes or the insertion of thin electrically insulating blocking electrodes.14,15 However, because the electrode polarization effect is heavily dependent on the surface chemistry, electrode topography, and the involvement of both short and long-range interactions of ions at the electrode/solution interface, no simple correction techniques exists and all of these approaches pose significant flaws in the correct determination of the relative permittivity. Published on 06 December 2018. Downloaded by University of Hull on 12/6/2018 2:12:05 PM. Introduction The possibility to use low frequency electrical measurements, as shown in this work, may also prove to be particularly important since it could be used to detect large-scale changes in molecular structures, such as binding events and conformation changes in DNA. We should also note that the use of electrical detection methods in contrast to optical methods, which tend to require a larger footprint due to the need of both optical sources and detectors, mitigates the potential development of small, low cost, electronic handheld sensors. View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript Published on 06 December 2018. Downloaded by University of Hull on 12/6/2018 2:12:05 PM. In this work we demonstrate a new, simpler and low cost approach for making a nanogap device with a planar geometry and with electrodes that are metallic. This work contrasts with two previously reported nanogap biosensor devices that have significant differences in their design, use and experimental results. The first (earlier) device had a horizontal oriented nanogap18 but with electrodes made from silicon. This work also did not use aptamer functionalized electrodes and therefore was non-specific against the target species. The second nanogap device had a vertical oriented nanogap19 with gold electrodes and used aptamer functionalized electrodes but had a more complicated fabrication process and a more difficult geometry for connecting the nanogap capacitors in parallel. Our results importantly discriminates between the two sets of different experimental data obtained in these earlier reports, showing the importance of using metallic electrodes rather than silicon, whilst also confirming that electrode polarization effects can indeed be greatly diminished with the use of nanogap electrodes. Physical Chemistry Chemical Physics Ac Physical Chemistry Chemical To demonstrate a working prototype biosensor we have chosen human alpha thrombin as a target protein as this is a commonly used standard in the field. Thrombin is a Na+ activated protease enzyme that is central to the formation of blood coagulants during injury. It is not present in blood under normally circumstances but is instead generated from an inactive form, prothrombin (coagulation factor II), following an injury. During the clotting process prothrombin is cleaved to form thrombin, which clots blood through the conversion of fibrinogen into fibrin. As well as many blood related illnesses involving the improper coagulation of blood the detection and monitoring of thrombin and prothrombin is important in understanding many illnesses such as Alzheimer’s disease and cancer. Introduction The detection range of thrombin in blood varies widely from pM to μM depending on the injury or disease under investigation.23 Introduction emistry Chemical Physics Accepted Man ysical Chemistry Chemical Physics Acce Physical Chemistry Chemical Reduction of the gap size to nanoscale lengths in impedance spectroscopy also provides enormous advantages for the detection of surface bound species, since in comparison to macroscopic sized devices the size of the immobilized surface species makes up a significant proportion of the dielectric volume, thereby minimizing unwanted signal contribution from other material in the bulk. The use of short-chain single stranded nucleic acid aptamers for surface functionalization is particularly useful in this respect, since these molecules possess very high selectivity towards only the desired target species. In recent years20,21,22 the use of aptamers in biological sensing and the early detection of diseases has rapidly increased due to increased availability of aptamer types, better understanding of surface-immobilization techniques and advances in nanotechnology that have greatly improved device sensitivity. Aptamers are single-stranded nucleic acids (RNA, DNA) that possess unique binding capabilities in contrast to more traditional recognition elements, such as antibodies and enzymes. Aptamers have much greater stability and can be used for a wide variety of target molecules, including proteins, peptides, small organic molecules, metal ions, and even large supramolecular complexes such as cells and viruses. Aptamers are also readily produced in high concentration with specific sequence structures and are easily modified with linkers and functional groups. Thus, the combination of a nanogap impedance spectroscopy with aptamer functionalized 3 Page 4 o Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Page 4 of 24 Page 4 of 24 electrodes provides a powerful but simple label-free approach for the detection of target analytes with very high specificity and selectivity. The possibility to use low frequency electrical measurements, as shown in this work, may also prove to be particularly important since it could be used to detect large-scale changes in molecular structures, such as binding events and conformation changes in DNA. We should also note that the use of electrical detection methods in contrast to optical methods, which tend to require a larger footprint due to the need of both optical sources and detectors, mitigates the potential development of small, low cost, electronic handheld sensors. View Article Online DOI: 10.1039/C8CP05510F electrodes provides a powerful but simple label-free approach for the detection of target analytes with very high specificity and selectivity. Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics thiol functionalized aptamers, which would give the device both very high specificity and immobilization of the target protein directly within the active sensing region. Immobilization of the aptamer and target protein allows the device to be rinsed so as to remove unbound proteins and aptamers that affect both the specificity and sensitivity of the device. We particularly note here that aptamers were not used in the first nanogap biosensor study. Instead, the bio-sensor detected only whether DNA material was present in the sensor, meaning there was no specificity. The DNA was either measured in the solution or after evaporation of the buffer solution, the latter potentially problematic since this would leave behind ionic salts from the buffer in the process. Another potential problem of this study is that silicon and polysilicon electrodes were used that is likely to have influenced the dielectric response of both the water and ionic solutions measured as their response was not flat in the low frequency region and the relative permittivity value found for water was very different to the known theoretical value. In addition to a contribution to the overall permittivity from the native silicon oxide layer, the different value measured for water may also have resulted from strong van der Waals interactions with the underlying crystalline Si atoms, which influences ordering of molecules within the first 4 nm of the liquid layer.24 View Article Onlin DOI: 10.1039/C8CP05510 thiol functionalized aptamers, which would give the device both very high specificity and immobilization of the target protein directly within the active sensing region. Immobilization of the aptamer and target protein allows the device to be rinsed so as to remove unbound proteins and aptamers that affect both the specificity and sensitivity of the device. We particularly note here that aptamers were not used in the first nanogap biosensor study. Instead, the bio-sensor detected only whether DNA material was present in the sensor, meaning there was no specificity. The DNA was either measured in the solution or after evaporation of the buffer solution, the latter potentially problematic since this would leave behind ionic salts from the buffer in the process. Physical Chemistry Chemical Physics Another potential problem of this study is that silicon and polysilicon electrodes were used that is likely to have influenced the dielectric response of both the water and ionic solutions measured as their response was not flat in the low frequency region and the relative permittivity value found for water was very different to the known theoretical value. In addition to a contribution to the overall permittivity from the native silicon oxide layer, the different value measured for water may also have resulted from strong van der Waals interactions with the underlying crystalline Si atoms, which influences ordering of molecules within the first 4 nm of the liquid layer.24 View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript emistry Chemical Physics Accepted Man ysical Chemistry Chemical Physics Acce Our sensor design also focussed on simplification of the fabrication process so that it was more suitable to commercialization. A horizontal nanogap architecture utilizing metallic crossbar electrodes was chosen for simplicity in fabrication, since standard photolithography techniques can be used. This contrasts with the second reported nanogap study19 in the literature, which instead used a vertical nanogap architecture. However, this involved a complicated device fabrication process that included additionally depositing a silicon nitride layer and a convoluted final lift-off procedure involving planarization of the Au electrodes by chemical-mechanical polishing. Instead, our methodology uses only a simple two-step photolithography method and a single HF etching step. Furthermore, low cost glass substrates can be used and only small quantities of gold are evaporated during the process, i.e. two 50 nm thick gold evaporation in contrast to two 1 micron thick gold evaporations. Physical Chemistry Chemical Ph Sensor Design 4 A focus of the sensor design was to improve upon the two earlier reported nanogap sensors, utilizing the advantages of both but also making the device fabrication more suited for commercialization. In contrast to the first nanogap study,18 which utilized silicon electrodes, it was important to use a fabrication methodology that permitted the use of metallic electrodes, since this allows the use of 4 Page 5 of 24 Page 5 of 24 Physical Chemistry Chemical Physics 1. Nanofabrication The schematic of the nanogap bio-sensor device is shown in Figure 2 and consists of top and bottom gold electrodes separated by a SiO2 support layer. Device fabrication involved standard photolithography processes for the metal electrodes and an etching step to form the nanogap cavity. The simple techniques involved in the device fabrication are key advantages of this method and should enable low cost fabrication of devices in relatively large quantities. Figure 3 demonstrates the main sequence of steps used in the fabrication. A highly polished flat quartz substrate was first 5 Physical Chemistry Chemical Physics Page 6 of 24 Page 6 of 24 cleaned using acetone, isopropanol and de-ionized water in an ultrasonic bath. The substrate was then coated with the adhesion promoter TI prime followed by the image reversal photoresist AZ5214E (MicroChemicals) for the first photolithography step. An image reversal photoresist was used to give inclined side-walls for improved removal of the metal layer during lift-off. Metallization of the first layer electrodes consisted of 5 nm (chromium) and 50 nm (gold), deposited using an HHV Auto 500 e-beam evaporator. Following this, a 40 nm SiO2 dielectric layer, acting as a sacrificial/support layer, was deposited by RF magnetron sputtering deposition (at 100 W). The process gas consisted of an argon:oxygen ratio of 3:1 at 1.2x10-2 mbar with the substrate held at 300°C, to improve stoichiometry and minimize leakage current. The second photolithography step was used to form the top metal electrode. A similar method to the first photolithography step was used for this procedure. To make the nanogap, wet etching of the sacrificial SiO2 layer was carried out using a weak solution of DI water:HF in a ratio of 10:1. Figure 4a) shows an SEM image of a typical etched nanogap with 40 nm separation between the top and bottom electrodes. The final device is shown in Figure 4b) with each device consisting of 100 capacitors in parallel. This is used to increase the overall device capacitance so that is in an acceptable range (~ pF) for ease of measurement. A protective resist, observable in the optical photo as the outer region around the square window, was also used so that only the active sensing region came into contact with the solution. View Article Onlin DOI: 10.1039/C8CP05510 cleaned using acetone, isopropanol and de-ionized water in an ultrasonic bath. 1. Nanofabrication The substrate was then coated with the adhesion promoter TI prime followed by the image reversal photoresist AZ5214E (MicroChemicals) for the first photolithography step. An image reversal photoresist was used to give inclined side-walls for improved removal of the metal layer during lift-off. Metallization of the first layer electrodes consisted of 5 nm (chromium) and 50 nm (gold), deposited using an HHV Auto 500 e-beam evaporator. Following this, a 40 nm SiO2 dielectric layer, acting as a sacrificial/support layer, was deposited by RF magnetron sputtering deposition (at 100 W). The process gas consisted of an argon:oxygen ratio of 3:1 at 1.2x10-2 mbar with the substrate held at 300°C, to improve stoichiometry and minimize leakage current. The second photolithography step was used to form the top metal electrode. A similar method to the first photolithography step was used for this procedure. To make the nanogap, wet etching of the sacrificial SiO2 layer was carried out using a weak solution of DI water:HF in a ratio of 10:1. Figure 4a) shows an SEM image of a typical etched nanogap with 40 nm separation between the top and bottom electrodes. The final device is shown in Figure 4b) with each device consisting of 100 capacitors in parallel. This is used to increase the overall device capacitance so that is in an acceptable range (~ pF) for ease of measurement. A protective resist, observable in the optical photo as the outer region around the square window, was also used so that only the active sensing region came into contact with the solution. View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript mistry Chemical Physics Accepted Manus al Chemistry Chemical Physics Accepted Physical Chemistry Chemical Ph Figure 2. Schematic of the biosensor device structure consisting of top and bottom gold electrodes separated by an etched SiO2 support layer. Binding of biomolecules to the aptamer functionalized electrodes modifies the impedance between the top and bottom electrodes. Physical Chemistry Che Figure 2. Schematic of the biosensor device structure consisting of top and bottom gold electrodes separated by an etched SiO2 support layer. Binding of biomolecules to the aptamer functionalized electrodes modifies the impedance between the top and bottom electrodes. 6 6 Physical Chemistry Chemical Physics Page 7 of 24 Page 7 of 24 Figure 3. Fabrication steps used to construct the biosensor. 1. Nanofabrication a) Deposition of the bottom electrode using standard photolithography with a metallization and lift-off process, b) deposition of a 40 nm thick SiO2 layer by RF magnetron sputtering, c) deposition of the top electrode using photolithography, and d) HF etching of the SiO2 layer to form the nanogap space between the two electrodes. View Article Onlin DOI: 10.1039/C8CP05510 View Article Onlin DOI: 10.1039/C8CP05510 View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript mistry Chemical Physics Accepted Manus al Chemistry Chemical Physics Accepted Figure 3. Fabrication steps used to construct the biosensor. a) Deposition of the bottom electrode using standard photolithography with a metallization and lift-off process, b) deposition of a 40 nm thick SiO2 layer by RF magnetron sputtering, c) deposition of the top electrode using photolithography, and d) HF etching of the SiO2 layer to form the nanogap space between the two electrodes. Physical Chemistry Chemical Physics Ac a) b) Figure 4. a) SEM image of the etched region of the nanogap biosensor confirming a 40 nm etched gap seprates the top and bottom electrodes. b) Optical image of the final nanogap sensor consisting of 10 by 10 overlapping crossbar electrodes to form 100 active capactors in parallel. The outer region of the device (dotted) is a protective resist so that only the active sensing region comes into contact with the solution. b) a) Physical Chemistry Chemical a) b) a) Figure 4. a) SEM image of the etched region of the nanogap biosensor confirming a 40 nm etched gap seprates the top and bottom electrodes. b) Optical image of the final nanogap sensor consisting of 10 by 10 overlapping crossbar electrodes to form 100 active capactors in parallel. The outer region of the device (dotted) is a protective resist so that only the active sensing region comes into contact with the solution. 2. Surface Functionalization Surface functionalization by the DNA oligonucleotide was carried out using the following process. The thiol modified aptamer for thrombin with sequence25 oligo 5’-GGT TGG TTT GGT TGG TTT/3- 7 Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Page 8 of 24 Page 8 of 24 (CH2)3SH-3’ (Integrated DNA Technologies) was purchased in its protected S=S form. Reduction was carried out by treating the oligo (100 μM) with DTT (10 mM). The DTT was subsequently removed by passing the mixture through a Sephadex column. The aptamers were denatured at 90°C for 5 min and left to cool to room temperature to ensure correct folding. Functionalization involved a co-immobilization process with mercaptohexanol (MCH) acting as a surface blocking-agent, as well as a spacer between aptamers, providing sufficient room to enable hybridization with the target molecules.26 The co-immobilization process involved incubating the device with 1 μM of aptamer + 100μM MCH in a PBS buffer (0.1 M, pH 7.0) overnight in the presence of a moist atmosphere to prevent the solution from evaporating. Afterwards, repeated backfilling for 50 min in a 1 mM MCH solution was used to block any remaining sites and lift any aptamers bending down on the surface. View Article Online DOI: 10.1039/C8CP05510F (CH2)3SH-3’ (Integrated DNA Technologies) was purchased in its protected S=S form. Reduction was carried out by treating the oligo (100 μM) with DTT (10 mM). The DTT was subsequently removed by passing the mixture through a Sephadex column. The aptamers were denatured at 90°C for 5 min and left to cool to room temperature to ensure correct folding. Functionalization involved a co-immobilization process with mercaptohexanol (MCH) acting as a surface blocking-agent, as well as a spacer between aptamers, providing sufficient room to enable hybridization with the target molecules.26 The co-immobilization process involved incubating the device with 1 μM of aptamer + 100μM MCH in a PBS buffer (0.1 M, pH 7.0) overnight in the presence of a moist atmosphere to prevent the solution from evaporating. Afterwards, repeated backfilling for 50 min in a 1 mM MCH solution was used to block any remaining sites and lift any aptamers bending down on the surface. 2. Surface Functionalization View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript emistry Chemical Physics Accepted Man Detection of protein binding involved first incubating the functionalized devices with the protein mixture for two hours at room temperature, followed by washing multiple times with 0.5xSSC buffer solution, in order to remove any unbound protein. The protein solution consisted of 1 μM of human α-Thrombin (HCT-0020 purchased from Cambridge BioScience) in 0.5xSSC buffer. Capacitance measurements in the presence of the buffer solution were then used to detect changes in the capacitance of the device due to the presence of the protein. Physical Chemistry Chemical Physics Ac A non-binding control oligonucleotide was used to confirm if changes in the relative permittivity are due to hybridization of the thrombin with the oligonucleotide. The control oligo, having the structure 5’-GTG TGT GTG TGT GTG TTT-(CH2)3SH-3’, does not have the correct nucleotide sequence for forming the G quadruplex structure that is necessary for specific binding, and therefore cannot bind to the human alpha thrombin. Experiments were carried out in exactly the same manner as above except that the control oligonucleotide was used instead to functionalize the devices. Physical Chemistry Chemical Results The impedance spectroscopy results for air, water and buffer solutions ranging from 0.1 x SSC to 1 x SSC are shown in figure 5. The relative permittivity values are determined directly from the measured capacitance results and are uncorrected for any electrode polarization effects. The relative permittivity of the solution with the gap, εcalc, is determined from the following equation for the measured capacitance of the sensor, , where 100 is the number 𝐶𝑚𝑒𝑎𝑠= 100 × (𝜀𝑐𝑎𝑙𝑐𝜀0 𝐴 𝐷+ 𝜀𝑆𝑖𝑂2𝜀0 𝐴𝑆𝑖𝑂2 𝐷) of capacitors in parallel, A is the total area of the active sensing region of a single capacitor device (2  5 μm  0.8 μm), D is the nanogap separation (40nm) and and are the area (5 μm  𝐴𝑆𝑖𝑂2 𝜀𝑆𝑖𝑂2 3.4 μm) and relative permittivity of the SiO2 support layer respectively. The relative permittivity calculation assumes a 0.8 micron undercut etch on either side of each SiO2 support layer (determined by comparing the capacitance of the device before and after etching) and a relative permittivity of the SiO2 of 3.8 (measured independently using a metal oxide semiconductor approach). cal Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript emistry Chemical Physics Accepted Man Physical Chemistry Chemical Physics A The relative permittivity (’) results for air, deionized water and the buffer solutions are particularly interesting since they all exhibit a flat response at low frequency, indicating the absence of the electrode polarization effect. In the case of air, since there is no electrical double layer present, the relative permittivity is constant over the entire range from 10 Hz to 500 kHz. Deionized water instead shows a flat response at low frequency but undergoes a broad transition to lower relative permittivity from about 200 Hz onwards, reaching a value approaching of 1 at around 100 kHz. 3. AC Impedance Measurements To characterise the devices impedance spectroscopy measurements were taken over the range of 10 Hz to 600 kHz using 0 V DC bias and a 10 mV AC signal with a Solartron 1260 impedance analyser and Solartron 1296 dielectric interface. In some cases, a Stanford SR830 Lock-in amplifier was additionally used. The devices were tested in air and de-ionized water as well as a range of dilutions of saline-sodium citrate buffer (SSC) varying from 0.1SSC to 1.0SSC, whereby a stock 20SSC solution consisted of 3.0 M NaCl and 0.15 M sodium citrate buffered to pH 6.8. 8 8 Physical Chemistry Chemical Physics Page 9 of 24 View Article Online DOI: 10.1039/C8CP05510F Physical Chemistry Chemical Physics Page 10 of 24 Figure 5 also shows the results of the relative permittivity of different concentration SSC buffer solutions. These show many similarities to deionized water in the nanogap. The dielectric response is flat below a frequency of approximately 1000 Hz for low ionic concentration, indicating the absence of electrode polarization effects, and the transition to a lower relative permittivity occurs at a similarly low frequency and with a similar shape. The static relative permittivity of the SSC buffer is however higher than water and increases with increasing SSC concentration, rising from εr ≈167 in the case of 0.1SSC to εr≈262 for 1.0SSC. This behaviour is unexpected since the relative permittivity should instead decrease with increasing ion concentration since the presence of strong internal electric fields between ions inhibits polarization of the water molecules. However, interestingly these results are in perfect agreement with a previous report of the relative permittivity of SSC in a nanogap device.19 The possible reasons for this occurring is detailed in the discussion section. View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript stry Chemical Physics Accepted Manusc al Chemistry Chemical Physics Accepted Figure 5. Relative permittivity as a function of log (frequency) for various concentrations of SSC buffer solution ranging from 0.1xSSC (blue), 0.2xSSC, (magenta) 0.5xSSC (green) and 1xSSC (black). Also shown are curves for de-ionized water (red) and air (black). Physical Chemistry Chemical Ph Figure 5. Relative permittivity as a function of log (frequency) for various concentrations of SSC buffer solution ranging from 0.1xSSC (blue), 0.2xSSC, (magenta) 0.5xSSC (green) and 1xSSC (black). Also shown are curves for de-ionized water (red) and air (black). Figures 6 and 7 report on the use of the device as a biosensor. In this case, single strand nucleic acid aptamers with a sequence specific to the target protein, human α-thrombin, functionalizes the gold surfaces of the device. The device works by detecting a change in the capacitance of the device due to a change in the dielectric permittivity of the active sensing volume when the target protein molecules bind to the aptamers. The data is presented in terms of relative permittivity since capacitance is geometry specific and the contribution of the SiO2 support structure must be taken Figures 6 and 7 report on the use of the device as a biosensor. Results This result is very similar to another report of a nanogap device19 with similar electrode spacing but is very different to the accepted value of the relative permittivity of water in macroscopic systems, which almost matches the ideal Debye relaxation model and has instead the step to lower permittivity at much high frequency, typically in the vicinity of 2-20 GHz.27,28 The possible reasons behind these differences may be related to the confined environment of the water molecules within the nanogap and the potential formation of an ice-like phase.18,19 The discussion section examines this possibility in more detail. The measured value of the static relative permittivity of water in the nanogap, εr≈93, is larger than the accepted value of water,29 which ranges from εr=78.30 to εr=78.58 (at 25°C). The relative permittivity value is however very close to the measured static relative permittivity value of ice (Ih)30,31 with a hexagonal crystalline structure, s91.5, which lends further support to the possibility that water in the nanogap may be in a highly ordered state. This is debated further in the discussion section. We should also stress that the value measured is likely to be affected by uncertainty in the length of the undercut etch on either side of the SiO2 support, as well as changes in surface roughness and uniformity in the nanogap thickness across the device. The presence of air bubbles in the nanogap can also not be ruled out. Physical Chemistry Chemical 9 Page 10 o Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics In this case, single strand nucleic acid aptamers with a sequence specific to the target protein, human α-thrombin, functionalizes the gold surfaces of the device. The device works by detecting a change in the capacitance of the device due to a change in the dielectric permittivity of the active sensing volume when the target protein molecules bind to the aptamers. The data is presented in terms of relative permittivity since capacitance is geometry specific and the contribution of the SiO2 support structure must be taken 10 of 24 Physical Chemistry Chemical Physics Page 11 of 24 Physical Chemistry Chemical Physics into consideration. The black curve in Figure 6 is the relative permittivity as a function of frequency for a bare (non-functionalized) nanogap device in the presence of the 0.5xSSC buffer solution. The same device was measured (again in a 0.5xSSC buffer) but after the aptamer functionalization process (red curve). The curve indicates an overall shift to lower relative permittivity, which is likely due to a reduction in the content of the higher permittivity buffer solution within the nanogap volume, due to the presence of the aptamers on both surface electrodes. The same device was then measured (blue curve) after a 2 hour exposure of the device to a 0.5xSSC buffer solution containing the human α-thrombin (concentration of 1 μM) and following a wash cycle with just pure 0.5xSSC buffer to remove any excess unbound proteins. The results shown in Figure 6 indicate a substantial shift to lower relative permittivity across the frequency spectrum that is consistent with binding of the lower permittivity protein within device. View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript mistry Chemical Physics Accepted Manus al Chemistry Chemical Physics Accepted Figure 6. Relative permittivity vs log (frequency) for a bare nanogap device in the presence of a 0.5x SSC buffer (black curve), after surface functionalization with the single strand DNA aptamer (red curve), measured again in 0.5x SSC buffer, and after binding with human α-thrombin protein (concentration 1 μM in 0.5xSSC buffer solution). Physical Chemistry Chemical Ph Figure 6. Relative permittivity vs log (frequency) for a bare nanogap device in the presence of a 0.5x SSC buffer (black curve), after surface functionalization with the single strand DNA aptamer (red curve), measured again in 0.5x SSC buffer, and after binding with human α-thrombin protein (concentration 1 μM in 0.5xSSC buffer solution). Physical Chemistry Chemical Physics Figure 7 reports measurements of the relative permittivity of devices that are instead functionalized with a non-binding control oligonucleotide. The control oligo does not have the correct amino acid sequence for forming the G quadruplex structure that is necessary for binding, and therefore cannot bind to the human alpha thrombin. Experiments were carried out in exactly the same manner as above. The black curve in Figure 7 is the relative permittivity as a function of frequency for the case 11 Physical Chemistry Chemical Physics Page 12 of 24 Page 12 of 24 of a bare (non-functionalized) nanogap device in the presence of the 0.5xSSC buffer. The same device was measured, again in a 0.5xSSC buffer, but after the functionalization process with the control aptamer (red curve). The data demonstrates an overall shift to lower permittivity, similar to that observed in Figure 6, indicating successful surface functionalization. The same device was then measured (blue curve) after a 2 hour exposure of the device to a 0.5xSSC buffer solution containing the human α-thrombin (concentration of 1μM) and following a wash cycle with just pure 0.5xSSC buffer. In contrast to Figure 6, there is no shift to lower permittivity in this case, indicating the protein has not bound to the control aptamer. Interestingly there is a slight shift to higher permittivity in the higher frequency part of the spectrum. The cause of this is not yet fully understood but will be examined in future studies. View Article Online DOI: 10.1039/C8CP05510F of a bare (non-functionalized) nanogap device in the presence of the 0.5xSSC buffer. The same device was measured, again in a 0.5xSSC buffer, but after the functionalization process with the control aptamer (red curve). The data demonstrates an overall shift to lower permittivity, similar to that observed in Figure 6, indicating successful surface functionalization. The same device was then measured (blue curve) after a 2 hour exposure of the device to a 0.5xSSC buffer solution containing the human α-thrombin (concentration of 1μM) and following a wash cycle with just pure 0.5xSSC buffer. In contrast to Figure 6, there is no shift to lower permittivity in this case, indicating the protein has not bound to the control aptamer. Interestingly there is a slight shift to higher permittivity in the higher frequency part of the spectrum. The cause of this is not yet fully understood but will be examined in future studies. Physical Chemistry Chemical Physics The process involved determining the 12 Page 13 of 24 Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Page 13 of 24 Physical Chemistry Chemical Physics concentration of the ions present in the SSC buffer (Na+, Cl-, OH-, H+) as well as those in the following reactions (using the Henderson-Hasselbach equation). View Article Onlin DOI: 10.1039/C8CP05510 concentration of the ions present in the SSC buffer (Na+, Cl-, OH-, H+) as well as those in the following ti ( i th H d H lb h ti ) View Article Onlin ng the Henderson-Hasselbach equation). View Article Onl DOI: 10.1039/C8CP0551 View Article Online DOI: 10.1039/C8CP05510F 𝐻3𝑐𝑖𝑡↔𝐻2𝑐𝑖𝑡― + 𝐻+ 𝐻2𝑐𝑖𝑡― ↔𝐻𝑐𝑖𝑡2 ― + 𝐻+ 𝐻𝑐𝑖𝑡2 ― ↔𝑐𝑖𝑡3 ― + 𝐻+ 𝑁𝐻+ 3 𝑂𝐻↔𝑁𝐻2𝑂𝐻+ 𝐻+ 𝐻3𝑐𝑖𝑡↔𝐻2𝑐𝑖𝑡― + 𝐻+ 𝐻2𝑐𝑖𝑡― ↔𝐻𝑐𝑖𝑡2 ― + 𝐻+ 𝐻𝑐𝑖𝑡2 ― ↔𝑐𝑖𝑡3 ― + 𝐻+ 𝑁𝐻+ 3 𝑂𝐻↔𝑁𝐻2𝑂𝐻+ 𝐻+ 𝐻3𝑐𝑖𝑡↔𝐻2𝑐𝑖𝑡― + 𝐻+ 𝐻2𝑐𝑖𝑡― ↔𝐻𝑐𝑖𝑡2 ― + 𝐻+ 𝐻𝑐𝑖𝑡2 ― ↔𝑐𝑖𝑡3 ― + 𝐻+ 𝑁𝐻+ 3 𝑂𝐻↔𝑁𝐻2𝑂𝐻+ 𝐻+ cal Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript mistry Chemical Physics Accepted Manus The molar concentrations of the ions are used to determine the total ionic strength of the solution using: The molar concentrations of the ions are used to determine the total ionic strength of the solution using: (1) 𝐼= 1 2∑𝑖𝑧2𝑖𝑐𝑖 (1) 𝐼= 1 2∑𝑖𝑧2𝑖𝑐𝑖 (1) cal Chemistry Chemical Physics Accepte where I = total ionic strength of the solution and ci and zi are the molar concentration and charge of ion i. However, since the activity coefficients and equilibria constants are dependent upon the ionic strength, the molar concentrations of the ions must be re-calculated iteratively until convergence is reached. where I = total ionic strength of the solution and ci and zi are the molar concentration and charge of ion i. However, since the activity coefficients and equilibria constants are dependent upon the ionic strength, the molar concentrations of the ions must be re-calculated iteratively until convergence is reached. The EDL thickness (-1), or Debye length (or Debye-Huckel parameter), can then be determined using: Physical Chemistry Chemical Ph (2) 𝜅―1 = ε𝑟ε0𝑘𝐵𝑇 2𝑁𝐴𝑒2𝐼 (2) where r is the dielectric constant (or relative permittivity), 0 is the permittivity of free space, kB is the Boltzmann constant, T is the temperature, NA is the Avogadro number, e is the elementary charge and I is the ionic strength (note: mol/m3). Physical Chemistry Chemical Physics View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript stry Chemical Physics Accepted Manusc al Chemistry Chemical Physics Accepted Figure 7. Relative permittivity vs log (frequency) for a bare nanogap device in the presence of a 0.5x SSC buffer (black curve), after surface functionalization with a control (non-binding) single strand DNA aptamer (red curve), and after exposure to human α-thrombin protein (concentration 1 μM in 0.5xSSC buffer solution). In this case, the immobilized control oligomer is incapable of forming the active G-quadruplex structure with the protein so hybridization should not occur. Physical Chemistry Chemical Physics Ac Physical Chemistry Chemical Figure 7. Relative permittivity vs log (frequency) for a bare nanogap device in the presence of a 0.5x SSC buffer (black curve), after surface functionalization with a control (non-binding) single strand DNA aptamer (red curve), and after exposure to human α-thrombin protein (concentration 1 μM in 0.5xSSC buffer solution). In this case, the immobilized control oligomer is incapable of forming the active G-quadruplex structure with the protein so hybridization should not occur. Figure 7. Relative permittivity vs log (frequency) for a bare nanogap device in the presence of a 0.5x SSC buffer (black curve), after surface functionalization with a control (non-binding) single strand DNA aptamer (red curve), and after exposure to human α-thrombin protein (concentration 1 μM in 0.5xSSC buffer solution). In this case, the immobilized control oligomer is incapable of forming the active G-quadruplex structure with the protein so hybridization should not occur. Figure 8 gives the results of calculations of the electrical double layer thickness (EDL) of various dilutions of the 20xSSC stock solution buffer. The EDL values were determined using a self- consistent numerical approach involving the calculation of the ionic strength of the solution based on the Extended Debye Hückel model and Davies equations. The process involved determining the Figure 8 gives the results of calculations of the electrical double layer thickness (EDL) of various dilutions of the 20xSSC stock solution buffer. The EDL values were determined using a self- consistent numerical approach involving the calculation of the ionic strength of the solution based on the Extended Debye Hückel model and Davies equations. Physical Chemistry Chemical Physics In the case of water (r=78) at 25C this can be simplified to: to: (3) 𝜅―1(nm) = 0.304 𝐼 𝜅―1(nm) = 0.304 𝐼 (3) where -1 is expressed in nanometer and I is in mol/L. here -1 is expressed in nanometer and I is in mol/L. The graph shown in Figure 8 depicts the typical decay of the EDL thickness with increasing ionic strength and indicates that at low 0.001xSSC concentrations the EDL layers overlap since the EDL thickness on just one of the electrodes is  20 nm, whilst in the case of buffer solutions used in typical impedance spectroscopy measurements (and in this work i.e. 0.5xSSC concentration), the EDL is very small,  1 nm, and would importantly, appear to not overlap. Implications of this in relation to the effectiveness of the device and dielectric response, which surprisingly still appears flat at low frequency, are detailed in the discussion section. 13 Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Page 14 of 24 Page 14 of 24 Figure 8. Electrical double layer thickness vs concentration of the SSC buffer determined from numerical calculations based on the Extended Debye Hückel model and Davies equation. View Artic DOI: 10.1039/C8CP cal Physics Accepted Manuscript stry Chemical Physics Accepted Manusc al Chemistry Chemical Physics Accepted Physical Chemistry Chemical Physics Ac Figure 8. Electrical double layer thickness vs concentration of the SSC buffer determined from numerical calculations based on the Extended Debye Hückel model and Davies equation. Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics In this article, we have not investigated the detection limits and sensitivity of the device, which will be reported in future work. From a theoretical perspective, we expect the ultimate detection limit is in the vicinity of the attomolar range. This is based on a total sensor area of 1600 μm2 (active area of the 100 capacitors including top and bottom electrodes) with coating of the aptamer molecules32 at approximately 104 mol/μm2 to give the number of aptamer probes per device as 16106 mol. For the situation in which all of the aptamer probes have bound protein molecules the device sensitivity would be 16106 mol / 6.0221023 = 3010-18 mol. View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript The nanogap biosensors, once fabricated, have good repeatability (80 - 90 %) between different devices and also with repeated measurements. A greater challenge is the yield of successful devices during fabrication. Currently this is only about 15 %. A number of improvements can be made to greatly increase the yield of working devices, such as improving the adhesion of the electrodes to the underlying substrate and increasing the robustness of the top electrode. Optimization of these will allow more rigorous studies of the upper limit of the device sensitivity and investigations of different aptamer/protein types as well as their use in biological relevant fluids. These will be examined in future work. emistry Chemical Physics Accepted Man Physical Chemistry Chemical Physics Ac 1. Nanogap Bio-sensing Physical Chemistry Chemical Our results demonstrate successful operation of the device as a biosensor for the detection of small proteins with high specificity and sensitivity. Figure 6 shows that binding of human α-Thrombin to the aptamer functionalized surface leads to a 32% decrease in the measured relative permittivity at 1 Hz. In contrast, when a non-binding control aptamer was used, there was no decrease in the relative permittivity. Importantly, the figure indicates the device is most sensitive at low frequencies, which can be attributed to almost complete elimination of electrode polarization effects, which would otherwise normally swamp the device response and masking all important information in this frequency range. Low frequency detection measurement are particularly important since it can be used to better detect large-scale changes in molecules structures, such as binding events and conformation changes of DNA. In this study the reduction in the relative permittivity after binding of the protein is largely attributed to replacement of the high relative permittivity solution (0.5xSSC) in the nanogap region with the low relative permittivity protein. However, as discussed further below, the nature of the dielectric response of both water and high ionic strength solutions when contained in nanogaps is poorly understood, and this is made particularly more complex due to the presence of the aptamer and protein molecules at the electrode surfaces, whose additional effect on the electrical double layer and possible ordering of the solution molecules is unknown. 14 Page 15 of 24 Physical Chemistry Chemical Physics 2. Nature of the Dielectric Response and Electrical Double Layer in Nanogap Devices View Article Online DOI: 10.1039/C8CP05510F 100 kHz, is most similar to that of ice at a temperature of -20C to -0.1C. The most common phase transition is hexagonal crystalline ice (phase Ih), which occurs when liquid water is cooled to 0C at standard atmospheric pressure. The frequency response of the relative permittivity of ice Ih at 0C and it’s static relative permittivity,30,31 s91.5, are both in agreement33 with that of water in the nanogap device. View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript The question of whether water molecules can truly retain an ice-like structure over distances greater than a few atomic length scales is however still a topic of debate. Recent new experiments have given much insight to the adsorption of water monolayers on metals and the structure of the first few molecular layers. The work has challenged the commonly held theory of a bi-layer structure,39 consisting of a buckled hexagonal overlayer resembling the basal plane of ice. However, beyond the first few layers, and especially at ambient temperatures, there is still limited understanding on the structure and dynamics of both multilayer water adsorption and liquid-water thin-films on surfaces. Physical Chemistry Chemical Physics Ac In the case of the nanogap device the presence of strong electric fields within the electrical double layer must also be considered as these can easily reach up to 108 -109 V/m in the Helmholtz layer directly adjacent to the electrode. Electric fields orient the water molecules depending on its direction; a positively charged electrode orients water so that its hydrogen atoms are directed away from the surface whilst a negatively charge electrode orients the hydrogens to be instead directed toward the surface. Molecular dynamics simulations of water lamina embedded between two platinum electrodes40 have shown that without the presence of an electric field, the first adsorbed layer has a solid-like state whilst the second and third layers are liquid but with an orientation distribution favouring a hexagonal ice formation. However, with an electric field of 21010 V/m present, simulations have shown the water molecules reorient, aligning their dipoles along the field. A large change in the density profile is predicted and the water undergoes crystallisation process to give a cubic ice structure (phase IC). 2. Nature of the Dielectric Response and Electrical Double Layer in Nanogap Devices The nanogap capacitor device shows almost complete absence of electrode polarization, which indicates very good applicability of the device in bio-sensing. However, the permittivity results raise a number of interesting issues concerning the nature of the electrical double layer and the dielectric relaxation mechanisms of water and ionic solutions when confined in nanoscale dimensions. Physical Chemistry Chemical The first issue concerns the nature of the dielectric response in the case of when only de-ionized water is present within the nanogap. The real permittivity shows a flat response at low frequency but undergoes a broad transition to low values from about 200 Hz onwards, reaching a value approaching 1 at around 100 kHz. This is very different to the accepted relative permittivity values of water in macroscopic systems, which almost match the ideal Debye relaxation model and have instead a step to lower relative permittivity at much high frequency, typically in the vicinity of 2-20 GHz.27,28 The occurrence of the step at much lower frequency is possibly because of the inability of the water molecules’ dipole moment to sufficiently polarize in synchronization with the alternating electric field. It has been previously suggested18,19 that water in the nanogap may be in an ice-like phase, and indeed the dielectric response has many similarities with relative permittivity values of ice, which has almost the same real and imaginary permittivity features of liquid water,33 but are shifted to lower frequency by 106 Hz. Ice has as many as 17 known crystalline forms, each having different frequency dependent permittivity.34,35,36,37 A comparison of our data with frequency dependent relative permittivity measurements on pure ice38 over a range of temperature, -57C to - 0.1C, indicates that our data, although having a slightly broader transition ranging from 200 Hz to 15 Page 16 of 24 Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Page 16 of 24 Page 16 of 24 100 kHz, is most similar to that of ice at a temperature of -20C to -0.1C. The most common phase transition is hexagonal crystalline ice (phase Ih), which occurs when liquid water is cooled to 0C at standard atmospheric pressure. The frequency response of the relative permittivity of ice Ih at 0C and it’s static relative permittivity,30,31 s91.5, are both in agreement33 with that of water in the nanogap device. 2. Nature of the Dielectric Response and Electrical Double Layer in Nanogap Devices A similar point of view was also found in another study that examined the dielectric response,41 where it was shown the polarization underwent a non-linear transition starting from 0.1109 V/m, whilst at higher electric fields, above 30109 V/m, a phase transition occurred to a completely ordered ice-like structure. Experimental verification of results in this area is challenging however, and it is likely that other effects are also important, since impurities, defects and surface roughness may stimulate or inhibit crystallization and high electric fields are known to modify electrochemical reaction rates as well as promote the ionization42 of water. In this context, the nanogap devices could prove to be a useful probe to better understand such effects and improve the understanding of how molecules order and interact at liquid-metal interfaces. Physical Chemistry Chemical 16 ge 17 of 24 Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Page 17 of 24 Physical Chemistry Chemical Physics The second issue concerns the increase in the relative permittivity of the SSC buffer with increasing concentration. The higher relative permittivity indicates greater polarizability of the solution with increasing SSC concentration, which contrasts with that of macroscopic measurements on ionic solutions whereby the relative permittivity instead reduces with increasing salt concentration due to the effect of the electric field of the ions inhibiting the external electric field.43 For example, the accepted model in the case of a simple ionic solution, such as NaCl, is that the polar water molecules align themselves with the electric fields of the dissociated sodium and chloride ions, forming a hydration shell around individual ions. The water molecules are then less able to respond to the external electric field, which has the effect of lowering the dielectric constant. In a dilute solution with (<1.5 M) the relative permittivity decreases linearly with salt concentration with the relationship44 εr = εw -c, where c is the salt concentration,  is an ion specific constant and εw is the dielectric constant of pure water. The absence of this relationship in our data, which also supports results from other studies,18,19 is intriguing and requires further work to understand its cause. 2. Nature of the Dielectric Response and Electrical Double Layer in Nanogap Devices However, because of its peculiar properties imbibed from its molecular structure, which enables a strong dipole moment and weak van der Waals interaction, many of its properties are still not fully understood. The understanding of water in confined regions is hugely important for numerous fields, including biological systems (e.g. the interaction of proteins in cells), materials science (e.g. clays), chemistry (e.g. colloids) and the development of new technologies (e.g. electrolytic batteries and hydrogen fuel cells). Further studies on the origin of the dielectric relaxation in nanogap systems may help better understanding of the long running debate on the molecular origins of the Debye relaxation in water, which is a topic that is still under considerable debate.55 l Chemistry Chemical Physics Accepted Physical Chemistry Chemical Ph Conclusions We report on a horizontal thin-film nanogap capacitive sensor with electrode separation of 40 nm, fabricated using only standard photolithography techniques. The confined geometry of the active sensing region of the device causes overlap of the two electrical double layers when a low ionic strength solution is present within the 40 nm separated electrodes. Frequency dependent dielectric spectroscopy results indicates no electrode polarization effects are present when measured with either water or ionic buffer solutions, which greatly enhances the application of the devices since it enables low frequency measurements to be used for detection. By functionalizing the device electrodes with thiol-immobilized single strand DNA aptamers we have successfully fabricated a working nanogap biosensor targeted for the detection of human alpha thrombin. The wide range of aptamers variants available along with the simplicity of the device fabrication should enable use of the biosensor for the early detection of many diseases types. We as well discuss the interesting dielectric response of water and high ionic strength solutions contained within the nanogap device. 2. Nature of the Dielectric Response and Electrical Double Layer in Nanogap Devices This study has created much excitement in the field and a plethora of reports of long range screening lengths on a wide range of ionic liquids and salt solutions,46,47,48,49 as well as attempts to explain these puzzling observations.50,51,52,53 Although the 17 Page 18 Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Page 18 of 24 theory is not well understood, it is apparent from these recent studies that long range double layers can possibly be present in such systems with lengths of 10 nm or more. This effect could help to explain why increasing permittivity values were found with increasing ionic strength, and the lack of correlation between the large observed changes in the measured static relative permittivity (Figure 5) and the relatively small change in the EDL length for concentrations between 0.2SSC and 1.0SSC (Figure 8). View Article Online DOI: 10.1039/C8CP05510F theory is not well understood, it is apparent from these recent studies that long range double layers can possibly be present in such systems with lengths of 10 nm or more. This effect could help to explain why increasing permittivity values were found with increasing ionic strength, and the lack of correlation between the large observed changes in the measured static relative permittivity (Figure 5) and the relatively small change in the EDL length for concentrations between 0.2SSC and 1.0SSC (Figure 8). View Article Online DOI: 10.1039/C8CP05510F Physical Chemistry Chemical Physics Accepted Manuscript cal Physics Accepted Manuscript Lastly, we would like to emphasize the need for further work in the area so that a better understanding can be gained of one of the most important, yet unique and puzzling materials on this planet.54 Water plays a central role in life, biology, science and technology. It dominates a variety of solvation and charge transfer phenomena. However, because of its peculiar properties imbibed from its molecular structure, which enables a strong dipole moment and weak van der Waals interaction, Lastly, we would like to emphasize the need for further work in the area so that a better understanding can be gained of one of the most important, yet unique and puzzling materials on this planet.54 Water plays a central role in life, biology, science and technology. It dominates a variety of solvation and charge transfer phenomena. 2. Nature of the Dielectric Response and Electrical Double Layer in Nanogap Devices View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript mistry Chemical Physics Accepted Manus l Chemistry Chemical Physics Accepted The third area requiring further understanding is whether the electrical double layer (EDL) is really overlapping in the case of when the biosensor is used with the relatively high concentration SSC buffers. In the case of pure water it is straightforward to show the electrical double layers overlap. For weak ionic strength solutions, the Debye-Hückel regime is appropriate and the Debye screening (equation 2) is the governing length scale, giving a value of -1  1 m for pure water at 25°C, indicating a large overlap of the electrical double layers. However, the Debye-Huckel theory is only valid in this dilute regime, where the total ionic strength is I <0.005 M. At higher concentration, either the extended Debye-Hückel (I <0.1 M) or the Davies equation (I <0.5 M) should be used. The graph in Figure 8 give the results of calculations of the EDL thickness of various dilutions of the 20xSSC stock solution buffer. From this, it is transparent that the EDL thickness of the 0.5xSSC buffer solution has a relatively short electrical double layer thickness ( 1 nm), which is much less than the 40 nm gap separation of the electrodes. Thus, it is not clear in this model if the EDL’s are indeed overlapping. However, interestingly the electrode polarization effects still appear to be negligible because of the flat response in the frequency dependent permittivity. Physical Chemistry Chemical Physics Physical Chemistry Chemical Recent investigations45 involving the measurement of the screening length between two planar charged mica surfaces containing high ionic solutions indicates may shed some light on this dilemma. In contrast to classical electrolyte theories of dilute electrolytes, which indicates diminished screening at higher ionic concentrations, the measurements reported instead showed an increase in screening length with electrolyte concentration. Acknowledgements cal Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript We gratefully acknowledge the University of Hull PhD Scholarship scheme for funding of this research as well as the work of Tony Sinclair in carrying out the SEM investigations and help from Dr Chris Walton in microscopy work and thin-film investigations. Physical Chemistry Chemical Physics Accepted Manuscript stry Chemical Physics Accepted Manusc al Chemistry Chemical Physics Accepted Conflicts of Interest 18 age 19 of 24 Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Physical Chemistry Chemical Physics Page 19 of 24 There are no conflicts of interest to declare. 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View Article DOI: 10.1039/C8CP0 55 View Article Online DOI: 10.1039/C8CP05510F View Article Online DOI: 10.1039/C8CP05510F cal Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript stry Chemical Physics Accepted Manusc al Chemistry Chemical Physics Accepted Physical Chemistry Chemical 23 Physical Chemistry Chemical Physics Page 24 of 24 View Article Online DOI: 10.1039/C8CP05510F View Article Online DOI: 10.1039/C8CP05510F View Article Online DOI: 10.1039/C8CP05510F al Physics Accepted Manuscript Physical Chemistry Chemical Physics Accepted Manuscript 80x39mm (300 x 300 DPI) Physical Chemistry Chemical Physics Accepted Manuscript stry Chemical Physics Accepted Manusc l Chemistry Chemical Physics Accepted Physical Chemistry Chemical Physics Ac 80x39mm (300 x 300 DPI) 80x39mm (300 x 300 DPI) 80x39mm (300 x 300 DPI) Physical Chemistry Chemical
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Network meta-analysis reaches nutrition research Lukas Schwingshackl1,2 · Anette Buyken3 · Anna Chaimani4,5,6 Published online: 31 October 2018 © The Author(s) 2018 Published online: 31 October 2018 © The Author(s) 2018 Network meta-analysis (NMA) is increasingly recognized as a promising evidence synthesis method commonly allow- ing stronger conclusions on the comparative effectiveness of healthcare interventions than conventional pairwise meta- analysis [1]. Its strength arises from the fact that it allows to synthesize both direct and indirect evidence from ran- domized trials. It is hence timely that Hui et al. recently published an NMA in the European Journal of Nutrition [2], comparing the effects of different whole grains (oat, brown rice, barley, and wheat) and brans (oat bran and wheat bran) on blood lipids (total cholesterol, LDL-C, HDL-C, and tria- cylglycerols), using data from 55 trials. NMA allows infer- ence on every possible pairwise comparison of interventions within a connected network. For example, in the paper by Hui et al. [2], oat bran and barley have not been directly compared in a randomized trial, but each has been com- pared with wheat (Fig. 1). As such, an indirect comparison between oat bran and barley can be obtained. Sometimes, the relative effects estimated by the network may rely to a notable extent on indirect comparisons (i.e., for which no tri- als were ever conducted); the influence of direct and indirect evidence on the results can be seen using the contribution matrix [3, 4]. In fact, in the NMA by Hui et al., the contribu- tion of direct evidence to the relative effects estimated by the network was very low ranging from 0.3% (oat vs. wheat) to 15.9% (wheat vs. control).i Nutrition research can substantially profit from the poten- tial of NMA. However, it is crucial that authors meticulously plan, conduct, and report NMA [5, 6]; in particular, authors should follow a study protocol published a priori so as to improve transparency and perform a rigorous risk of bias assessment within and across studies as well as an evalua- tion of the quality of evidence. As Hui et al. are among the pioneers of applying NMA to the field of nutrition research, we draw on their article to highlight some methodological challenges that require specific attention when performing NMA.f Summary effects from NMA are usually presented in a league table including all comparisons: Hui et al. European Journal of Nutrition (2019) 58:1–3 https://doi.org/10.1007/s00394-018-1849-0 European Journal of Nutrition (2019) 58:1–3 https://doi.org/10.1007/s00394-018-1849-0 * Lukas Schwingshackl lukas.schwingshackl@dife.de 1 Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), Arthur‑Scheunert‑Allee 114‑116, 14558 Nuthetal, Germany 2 NutriAct-Competence Cluster Nutrition Research Berlin-Potsdam, Berlin, Germany 3 Public Health Nutrition, Institute of Nutrition, Consumption and Health, Faculty of Natural Sciences, University of Paderborn, 33098 Paderborn, Germany 4 Paris Descartes University, Paris, France 5 INSERM, UMR1153 Epidemiology and Statistics, Sorbonne Paris Cité Research Center (CRESS), METHODS Team, Paris, France 6 Cochrane France, Paris, France * Lukas Schwingshackl lukas.schwingshackl@dife.de Network meta-analysis reaches nutrition research Lukas Schwingshackl1,2 · Anette Buyken3 · Anna Chaimani4,5,6 They did, however, apply a number of approaches (loop- specific approach, node-splitting approach, and design-by- treatment interaction model) to examine statistical incoher- ence [2]. The coherence assumption suggests that direct and indirect evidence are in statistical agreement and its assess- ment is mandatory. Incoherence can be tested locally (i.e., in parts of the network) and globally (i.e., in the entire network) and the use of both types of tests is highly recommended [6]. If incoherence is identified, subgroup analyses and network meta-regression may be used to investigate potential sources. It should be noted that the absence of statistically significant incoherence—as in the case of Hui et al. [2]—is not neces- sarily evidence of the absence of incoherence. In particular in networks that built on scarce evidence—as in the NMA of Hui et al.—tests for incoherence have low power; thus, their results should be interpreted cautiously [10]. It is hence wel- come that the authors performed several sensitivity analyses confirming the findings of their main analyses.fi In spite of these risks, we expect that high-quality NMAs combining the results of dietary intervention tri- als will become the new evidence synthesis norm also in nutrition research. While widely applied in many medical fields [13], its use in the field of nutrition is at present sur- prisingly rare. A quick search in PubMed (September 10th, 2018) using search-terms network meta-analysis[tiab] OR multiple treatments meta-analysis[tiab] OR mixed-treatment comparison[tiab] AND (diet*[tiab] OR nutrition[tiab]) yielded only 38 hits. Out of these, only ~ 50% original NMA papers dealt with a nutrition-related topic and only two of them [14, 15] were published in a nutrition journal. Yet, NMAs have the potential to advance the knowledge in the field of nutrition as they provide insights that cannot be obtained by individual trials or pairwise meta-analysis: for example, the DASH dietary approach proved to be the most effective dietary approach to reduce blood pressure among hypertensive and pre-hypertensive patients [15], while the Mediterranean diet emerged as the most effective dietary ii Addressing publication bias in NMA is as difficult as in pairwise meta-analysis; thus, priority must be given to the exhaustive search for unpublished studies. Assessment of small-study effects is usually the first step in the for- mal investigation of reporting biases [11]. Hui el al used the ‘comparison adjusted funnel plot’, a modified funnel plot for application to a network of trials [4]. Network meta-analysis reaches nutrition research Lukas Schwingshackl1,2 · Anette Buyken3 · Anna Chaimani4,5,6 1   Example of indirect relative effects in a triangle comparing three interventions (wheat, oat bran, barley) estimation of the magnitude of small-study effects, while ‘selection models’ can investigate the potential for publica- tion bias [12].i Hui and colleagues [2] also assessed the confidence of evidence, using the Grading of Recommendations Assess- ment, Development and Evaluation (GRADE) framework that considers the following items: study limitations, imprecision, inconsistency, indirectness, and publication bias. While the confidence on evidence was low for most comparisons, for comparisons of oat bran vs. the control diet it was judged as high for TC and moderate for LDL-C and TG. Hence, further research will provide important evi- dence on the majority of the comparisons included in this NMA, yet confidence is appreciable for oat bran vs. control diets. As this is an important conclusion for future research, we believe that a confidence on evidence statement should always be incorporated in the conclusion section of any man- uscript. The use of the CINeMA (Confidence In Network Meta-Analysis: http://cinem​a.ispm.ch/) framework, which is an improvement of a previously suggested approach [3], can greatly facilitate judgements about the confidence that can be placed in results obtained by NMA. CINeMA modified and extended the five GRADE domains for use in NMA and is transparent and applicable to any network structure [3]. Combinaon of direct Wheat-Oat bran and direct Wheat-Barley -> indirect Oat Bran-Barley Fig. 1   Example of indirect relative effects in a triangle comparing three interventions (wheat, oat bran, barley) effect modifiers are comparable across the direct treatment comparisons. Transitivity would, for example, be violated if changes in body weight would differ strongly between the two direct comparisons, as depicted in Fig. 1. An important limitation of the NMA by Hui et al. is the lack of a formal investigation of the distributions of potential effect modifiers across the available direct comparisons. In view of its potentials, there is a risk that NMA is applied in cases, where the scarcity of available data pre- cludes the estimation of precise results and the evaluation of its assumptions. Specifically, in the absence of direct evi- dence for several comparisons transitivity and incoherence cannot be formally tested, although transitivity can always be evaluated clinically and epidemiologically. In that case, the resulting relative effects would possibly be estimated with large uncertainty and the relative rankings might be meaningless. Network meta-analysis reaches nutrition research Lukas Schwingshackl1,2 · Anette Buyken3 · Anna Chaimani4,5,6 [2] iden- tified oat bran as the most effective intervention strategy, revealing clinically relevant mean differences (MD) in comparison with the control diet [improvements in total cholesterol (TC) (MD: − 0.35 mmol/L, 95% CI − 0.47, − 0.23 mmol/L) and LDL-C (MD: − 0.32 mmol/L, 95% CI − 0.44, − 0.19 mmol/L)]. Another unique feature of NMA is its ability to rank interventions in relation with the stud- ied outcomes, using the distribution of the ranking prob- abilities and the surface under the cumulative ranking curve (SUCRA) [7]. SUCRA ranges from 0%, i.e., the treatment always ranks last without uncertainty, to 100%, i.e., the treat- ment always ranks first without uncertainty. In the NMA by Hui et al. [2] oat bran ranked as the best treatment for TC (SUCRA: 97%), LDL-C (SUCRA: 97%), and triacylglycer- ols (TG) (SUCRA: 78%), followed by oat (SUCRA: 79% for TC, 64% for LDL-C, 76% for TG). * Lukas Schwingshackl lukas.schwingshackl@dife.de 1 Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), Arthur‑Scheunert‑Allee 114‑116, 14558 Nuthetal, Germany 2 NutriAct-Competence Cluster Nutrition Research Berlin-Potsdam, Berlin, Germany 3 Public Health Nutrition, Institute of Nutrition, Consumption and Health, Faculty of Natural Sciences, University of Paderborn, 33098 Paderborn, Germany 4 Paris Descartes University, Paris, France 5 INSERM, UMR1153 Epidemiology and Statistics, Sorbonne Paris Cité Research Center (CRESS), METHODS Team, Paris, France 6 Cochrane France, Paris, France 1 Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), Arthur‑Scheunert‑Allee 114‑116, 14558 Nuthetal, Germany 1 Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), Arthur‑Scheunert‑Allee 114‑116, 14558 Nuthetal, Germany 2 NutriAct-Competence Cluster Nutrition Research Berlin-Potsdam, Berlin, Germany The extent to which NMA allows valid indirect inference depends on the extent to which the fundamental assump- tion of NMA usually called the ‘transitivity’, assumption is likely to be plausible. Transitivity requires that the trials comparing different sets of interventions are appreciably comparable in characteristics (other than the interventions being compared) which may affect the outcome [8, 9]. Tran- sitivity should be evaluated prior to conducting NMA [8, 9], e.g., by examining whether the distributions of potential 5 INSERM, UMR1153 Epidemiology and Statistics, Sorbonne Paris Cité Research Center (CRESS), METHODS Team, Paris, France 6 Cochrane France, Paris, France (0121 3456789) 3 European Journal of Nutrition (2019) 58:1–3 2 Combinaon of direct Wheat-Oat bran and direct Wheat-Barley -> indirect Oat Bran-Barley Wheat Oat bran Barley Fig. 3 References 1. Nikolakopoulou A, Mavridis D, Furukawa TA, Cipriani A, Tricco AC, Straus SE, Siontis GCM, Egger M, Salanti G (2018) Living network meta-analysis compared with pairwise meta-analysis in comparative effectiveness research: empirical study. BMJ 360:k585. https​://doi.org/10.1136/bmj.k585 16. Schwingshackl L, Chaimani A, Hoffmann G, Schwedhelm C, Boe- ing H (2018) A network meta-analysis on the comparative efficacy of different dietary approaches on glycaemic control in patients with type 2 diabetes mellitus. Eur J Epidemiol 33(2):157–170. https​://doi.org/10.1007/s1065​4-017-0352-x 2. Hui S, Liu K, Lang H, Liu Y, Wang X, Zhu X, Doucette S, Yi L, Mi M (2018) Comparative effects of different whole grains and brans on blood lipid: a network meta-analysis. Eur J Nutr. https​:// doi.org/10.1007/s0039​4-018-1827-6 2. Hui S, Liu K, Lang H, Liu Y, Wang X, Zhu X, Doucette S, Yi L, Mi M (2018) Comparative effects of different whole grains and brans on blood lipid: a network meta-analysis. Eur J Nutr. https​:// doi.org/10.1007/s0039​4-018-1827-6 17. Schwingshackl L, Bogensberger B, Bencic A, Knuppel S, Boe- ing H, Hoffmann G (2018) Effects of oils and solid fats on blood lipids: a systematic review and network meta-analysis. 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Using dietary exposures comparable to those exam- ined in observational studies on hard clinical endpoints in NMAs on intervention trials with intermediate disease mark- ers could ultimately strengthen the credibility of nutrition research findings [19, 20]. 9. Mavridis D, Giannatsi M, Cipriani A, Salanti G (2015) A primer on network meta-analysis with emphasis on mental health. Evid Based Mental Health 18(2):40–46. https​://doi.org/10.1136/ eb-2015-10208​8 10. Veroniki AA, Vasiliadis HS, Higgins JPT, Salanti G (2013) Evalu- ation of inconsistency in networks of interventions. Int J Epide- miol 42(3):919–919. https​://doi.org/10.1093/ije/dyt10​6 11. Chaimani A, Salanti G (2012) Using network meta-analysis to evaluate the existence of small-study effects in a network of interventions. Res Synth Methods 3(2):161–176. https​://doi. org/10.1002/jrsm.57 Compliance with ethical standards 12. Dimitris M, Alex S, Andrea C, Georgia S (2013) A fully Bayes- ian application of the Copas selection model for publication bias extended to network meta-analysis. Stat Med 32(1):51–66. https​ ://doi.org/10.1002/sim.5494 doi Conflict of interest  AEB is a member of the Editorial board of the European Journal of Nutrition (Section Epidemiology) and a member of the International Carbohydrate Quality Consortium (ICQC). LS and AC declare that they have no conflict of interest. 13. Petropoulou M, Nikolakopoulou A, Veroniki A-A, Rios P, Vafaei A, Zarin W, Giannatsi M, Sullivan S, Tricco AC, Chaimani A, Egger M, Salanti G (2017) Bibliographic study showed improv- ing statistical methodology of network meta-analyses published between 1999 and 2015. J Clin Epidemiol 82:20–28. https​://doi. org/10.1016/j.jclin​epi.2016.11.002 Open Access  This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat​iveco​ mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. g j j 14. Carter P, Achana F, Troughton J, Gray LJ, Khunti K, Davies MJ (2014) A Mediterranean diet improves HbA1c but not fasting blood glucose compared to alternative dietary strategies: a net- work meta-analysis. J Hum Nutr Diet 27(3):280–297. https​://doi. org/10.1111/jhn.12138​ 15. Schwingshackl L, Chaimani A, Schwedhelm C, Toledo E, Punsch M, Hoffmann G, Boeing H (2018) Comparative effects of different dietary approaches on blood pressure in hypertensive and pre- hypertensive patients: a systematic review and network meta-anal- ysis. Crit Rev Food Sci Nutr:1–14. https​://doi.org/10.1080/10408​ 398.2018.14639​67f References Hutton B, Salanti G, Caldwell DM, Chaimani A, Schmid CH, Cameron C, Ioannidis JP, Straus S, Thorlund K, Jansen JP (2015) The PRISMA extension statement for reporting of systematic reviews incorporating network meta-analyses of health care inter- ventions: checklist and explanations. Ann Intern Med 162. https​ ://doi.org/10.7326/m14-2385 20. Schwingshackl L, Hoffmann G, Iqbal K, Schwedhelm C, Boe- ing H (2018) Food groups and intermediate disease markers: a systematic review and network meta-analysis of randomized tri- als. Am J Clin Nutr 108(3):576–586. https​://doi.org/10.1093/ajcn/ nqy15​1 6. Chaimani A, Caldwell DM, Li T, Higgins JP, Salanti G (2017) Additional considerations are required when preparing a protocol for a systematic review with multiple interventions. J Clin Epide- miol 83:65–74. https​://doi.org/10.1016/j.jclin​epi.2016.11.015 1 3
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Spanish ATLAS Tier-2: facing up to LHC Run 2
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Spanish ATLAS Tier-2: facing up to LHC Run 2 Spanish ATLAS Tier-2: facing up to LHC Run 2 This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 J. Phys.: Conf. Ser. 664 052016 (http://iopscience.iop.org/1742-6596/664/5/052016) View the table of contents for this issue, or go to the journal homepage for more Download details: IP Address: 137.138.124.206 This content was downloaded on 24/02/2016 at 13:47 Please note that terms and conditions apply. 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 1 Santiago.gonzalez@ific.uv.es Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 E-mail: santiago.gonzalez@ific.uv.es Abstract. The goal of this work is to describe the way of addressing the main challenges of Run 2 by the Spanish ATLAS Tier-2. The considerable increase of energy and luminosity for the upcoming Run 2 with respect to Run 1 has led to a revision of the ATLAS computing model as well as some of the main ATLAS computing tools. In this paper, the adaptation to these changes will be described. The Spanish ATLAS Tier-2 is a R&D project which consists of a distributed infrastructure composed of three sites and its members are involved in ATLAS computing progress, namely the work in different tasks and the development of new tools (e.g. Event Index). Spanish ATLAS Tier-2: facing up to LHC Run 2 S González de la Hoz1, J Del Peso2, F Fassi1,4, A Fernández Casani1, M Kaci1, V Lacort Pellicer1, A Del Rocio Montiel2, E Oliver1, A Pacheco Pages3, J Sánchez1, V Sánchez Martínez1, J Salt1, M Villaplana1 (For the ATLAS Collaboration) S González de la Hoz1, J Del Peso2, F Fassi1,4, A Fernández Casani1, M Kaci1, V Lacort Pellicer1, A Del Rocio Montiel2, E Oliver1, A Pacheco Pages3, J Sánchez1, V Sánchez Martínez1, J Salt1, M Villaplana1 (For the ATLAS Collaboration) 2 Universidad Autónoma de Madrid, Spain 3 Institut de Física d’Altes Energies, Universitat Autònoma de Barcelona, Spain 3 Institut de Física d’Altes Energies, Universita 3 Institut de Física d’Altes Energies, Universitat Autònoma de Barcel 4 Mohammed V University, Rabat, Morocco 2. Updates of the ATLAS Computing Model WebDAV, XRootD, proxis and gridftp) and smarter and more automated data placement tools. Data access can be a bottleneck for data analysis. Some datasets can be very popular for short periods of time, with several analysis groups accessing them at the same time on the few sites where they are replicated. A way to ease the situation during peak request periods is to create a “data federation” in which data on disk at any site are directly accessible from jobs running at any other federated site. Evidently, the data access tools must be clever enough to choose the “best” data replica to access, depending on the bandwidth and latency between the destination and all possible data source sites. A data federation is needed also to allow remote access to data in case of unavailability of a given file in the local storage element, or sparse access to single events. FAX [8] is the ATLAS implementation of an XrootD based federation. It has two top-level redirectors, one in Europe and one in the US. It covers, so far, 56% of ATLAS sites, which contain 85% of data. The production and analysis workflows increased in number and complexity during Run 1, and are expected to increase and diversify further in the future. The system for defining and submitting tasks for the processing of event data had to be redesigned with a layered infrastructure; the new system, ProdSys2 [9], consists of four core components: the request interface allows production managers to define workflows; DEFT translates the user request into task definitions; JEDI generates the job definitions; and PanDA executes the jobs in the distributed infrastructure. From the point of view of the software, running one ATLAS job per core will be difficult to sustain without memory limits on the worker node being reached and so less jobs will have to be allocated per worker node unless memory pressure can be mitigated. The most significant innovation has been the development of software that is able to run in parallel on different CPU cores. As a result, substantial memory is saved: a parallel job running on an 8-core CPU uses 8 times less memory than eight jobs running on that CPU, with an equivalent processing time. Most, if not all, data centres of the ATLAS Collaboration are equipped with multi-core platforms and they already set up a new batch queue for this purpose [9]. 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 improvement of distributed analysis. Finally, in section 5, our participation in an important ATLAS R&D activity, the “Event Index project” in collaboration with other ATLAS groups is described. improvement of distributed analysis. Finally, in section 5, our participation in an important ATLAS R&D activity, the “Event Index project” in collaboration with other ATLAS groups is described. 2. Updates of the ATLAS Computing Model During 2013 and 2014, the LHC machine underwent a period of maintenance and upgrades, with the aim of restarting operation in spring/summer 2015 (Run 2) at higher centre-of-mass energies (13 TeV for p-p collisions), a reduced bunch spacing (25 instead of 50 ns) and higher luminosities, leading to an average number of collisions per bunch crossing around 40. The data-taking rate will also increase up to 1 kHz on average in ATLAS. p g The building blocks of the ATLAS Distributed Computing (ADC) architecture were designed and deployed before the start of LHC operations. The existing tools worked very well for ATLAS during Run 1 but at the same time showed some limitations: namely, the manpower required for operations was too high. The experience of Run 1 operations led to a redesign of workload management systems and to the addition of a few other services to help cope with increased data volumes and different kinds of computing resources [5]. ATLAS Computing Model changes are described in general in [6]. The Distributed Data Management (DDM) system was completely redesigned in 2013 and the new implementation Rucio [7] has been progressively deployed in 2014 and 2015. With respect to the previous DDM implementation, Rucio has data discovery based on name and metadata, no dependence on an external file catalogue, supports multiple data management protocols in addition to SRM (e.g. WebDAV, XRootD, proxis and gridftp) and smarter and more automated data placement tools. The building blocks of the ATLAS Distributed Computing (ADC) architecture were designed and deployed before the start of LHC operations. The existing tools worked very well for ATLAS during Run 1 but at the same time showed some limitations: namely, the manpower required for operations was too high. The experience of Run 1 operations led to a redesign of workload management systems and to the addition of a few other services to help cope with increased data volumes and different kinds of computing resources [5]. ATLAS Computing Model changes are described in general in [6]. The Distributed Data Management (DDM) system was completely redesigned in 2013 and the new implementation Rucio [7] has been progressively deployed in 2014 and 2015. With respect to the previous DDM implementation, Rucio has data discovery based on name and metadata, no dependence on an external file catalogue, supports multiple data management protocols in addition to SRM (e.g. 3. Maintenance and Operation of the Infrastructure 1. Introduction The Higgs boson discovery [1] acknowledged by the 2013 Nobel Prize in physics is a major Run 1 (data taking from 2010 to 2012) success for the experiments at LHC. Experiments like ATLAS [2] recorded 2 billion real events and 4 billion simulated events per year during 2011 and 2012. In addition, events were reprocessed and stored in several file formats, increasing numbers to 138 (109) billion real event instances, and 93 billion simulated events in year 2012 alone. The Spanish ATLAS Tier-2 (ES-ATLAS-T2) operations and performances during this period were reported in [3,4]. During Run 2 (event collisions starting in 2015), the LHC physics program will continue its unprecedented research, exploring collisions at even higher beam energy and luminosity, and taking data at up to five times the rate compared to Run 1. This change of “scenario” in the operation of the LHC in the coming years (2015-2017) introduces a large uncertainty in the estimation of computational resources and data storage needed; changes in the computing technologies and protocols used may be required. Section 2 of this paper presents an update of the ATLAS Computing Model for Run 2. In section 3 the current resources and the performance of the ES-ATLAS-T2 is reviewed. Section 4 discusses the changes needed to adapt our Tier-2 for the new Computing Model, such as to create multicore queues, and a Federated Data Storage System (FAX) to improve the use of computing resources and 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 2. Updates of the ATLAS Computing Model Even though this software has been tested successfully, the code must still be optimized to profit from additional features of modern compilers. p p p These upgrades have been tested on a large scale during an exercise called Data Challenge DC14 [5]. The sites of the ES-ATLAS-T2 performed its designated tasks and met the challenge successfully. 3. Maintenance and Operation of the Infrastructure 2 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 The ES-ATLAS-T2 has been built and is maintained to operate as an ATLAS Tier-2 Grid computing site. It can execute both data analysis and Monte Carlo event generation for the ATLAS experiment. According to the agreed commitment of Spain with the ATLAS experiment, the Tier-2 installations of the Spanish Groups must fulfil 5% of the total Tier-2 load. The infrastructure is integrated in the LHC Computing GRID following the Computing Model of the ATLAS Collaboration and will be ready to meet the requirements of Run 2. This Tier-2 has been developed in a distributed manner shared by the three Spanish institutes (all members of the ATLAS Collaboration) specifically: 50% IFIC (Valencia), 25% IFAE (Barcelona) and 25% UAM (Madrid). The fact that it is distributed leads to an increase of service redundancy, a faster response for problem solving, and optimises the use of existing computing expertise and human resources from the three institutes. The following subsections give an overview of the evolution of the resources and performance, respectively, of the ES-ATLAS-T2 during the Run 1 and just before the start of Run 2. 3.1. Evolution of the resources 3.1. Evolution of the resources In that period (during Run 1 and before the start of Run 2), the ES-ATLAS-T2 has provided the hardware resources fulfilling the ATLAS requirement of the Resource Review Board of the LHCC committee, as is shown in the Table 1: f In that period (during Run 1 and before the start of Run 2), the ES-ATLAS-T2 has provided the hardware resources fulfilling the ATLAS requirement of the Resource Review Board of the LHCC committee, as is shown in the Table 1: Table 1. Present resources (CPU and Disk) provided by the ES-ATLAS-T2 Table 1. Present resources (CPU and Disk) provided by the ES-ATLAS-T2 Site CPU installed / pledge 2014 (HEP-SPEC06) DISK installed / pledge 2014 (TB) IFIC 10478 / 10300 1400 / 1400 IFAE 6600 / 5150 857 / 700 UAM 5100 / 5150 721 / 700 A Tier-2 must run round-the-clock every day of the year, and personnel must serve it 12 hours a day during working days (12h/5d). Disk space is managed by two distributed storage systems, namely dCache at IFAE and UAM, and Lustre+StoRM at IFIC. The Worker Nodes have at least 2 GB of RAM per CPU core to be able to run highly demanding ATLAS production jobs. 3.2. Performance of the ES-ATLAS-T2 3.2. Performance of the ES-ATLAS-T2 ES-ATLAS-T2 availability and reliability metrics are calculated by the Service and Availability Monitoring system (SAM)[11], which runs a range of tests at regular intervals throughout the day. A site is considered to be available if a defined set of critical tests complete successfully. The availability and reliability metrics are defined as follows: availabiltiy = U TT −TU ;reliability = U TT −D −TU , (1) (1) where U means uptime (time the site is available), TT is the total time, TU is the time when the status was unknown and D is the scheduled downtime. where U means uptime (time the site is available), TT is the total time, TU is the time when the status was unknown and D is the scheduled downtime. The availability and reliability evolution for a four-year period (Run 1 and prior to Run 2) is shown in Figure 1. As can be seen, in both cases the average over all sites is always in the interval 90-100% since 2011. 3 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 Figure 1. Availability and Reliability of ES-ATLAS-T2 for a four-year period. Another useful metric is evaluated by ATLAS Computing operations. Sites are tested with typical analysis and Monte Carlo (MC) production jobs. Results from these Hammer Cloud (HC) tests are shown in Figure 2. It shows the average efficiency of the HC test is greater than 90% in the last four years for the ES-ATLAS-T2 sites. years for the ES ATLAS T2 sites. Figure 2. ATLAS availability for the ES-ATLAS-T2 analysis queues. ES-ATLAS-T2 sites have been and are T2D category showing they have good connectivity among other Tier-1s and Tier-2s. In terms of connectivity, the concept of so-called T2D was introduced: those that fulfil a certain performance are then classified as T2D. At the moment, the performance limit is set to an overall transfer performance of at least 5MB/s to at least 80% of Tier-1s in ATLAS, for very large files (>1GB). This implies direct transfer from/to Tier-0 and all ATLAS Tier-1s and Tier-2s. It also means the execution of simulation and analysis jobs are favoured in our Tier-2. 3.2. Performance of the ES-ATLAS-T2 4 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 Figure 4(b). Data transfer in the ES- ATLAS-T2 sites during Run 1 (January 2012) and before Run 2. Figure 4(a). Number of physical bytes processed in the ES-ATLAS-T2 sites during Run 1 and before Run 2. 4. Adaptation and involvement of the ATLAS Spanish Tier-2 in the new ATLAS computing model The necessary upgrades and changes performed to handle the challenges of the ATLAS Computing Model for Run 2 related to our Tier-2 can be classified into different areas. The most important ones are detailed below. 4.1. Progress in Multicore The ATLAS Software has been optimized to take advantage of the Multicore architectures so each site of ES-ATLAS-T2 has created a Multicore queue with several dedicated nodes. In some sites, static allocation for job resources has been configured based on the idea that the resources are only for multicore jobs; therefore no mix between Multicore or Monocore is possible in a CPU server. A usual approach is to assign 8 cores per job, with the same wall time restrictions as current ATLAS single core jobs. Currently, the number of Multicore jobs being processed is minimal, compared to Singlecore ones. g With time, the fraction of multicore-type jobs is expected to increase after technical problems (already explained in section 2) are resolved. Figure 5(a) compares the number of Multicore jobs completed in different sites since March 2014, having processed around 20k completed jobs. The average efficiency of the HC test is between 66 to 86% since March 2014 for the ES-ATLAS-T2 sites as is shown in Figure 5(b). g ( ) Figure 5(b). ATLAS availability for the ES- ATLAS-T2 multicore queues since March 2014. Figure 5(a). Completed multicore jobs in the ES-ATLAS-T2 sites since March 2014. Figure 5(b). ATLAS availability for the ES- ATLAS-T2 multicore queues since March 2014. Figure 5(a). Completed multicore jobs in the ES-ATLAS-T2 sites since March 2014. Figure 5(b). ATLAS availability for the ES- ATLAS-T2 multicore queues since March 2014. Figure 5(a). Completed multicore jobs in the ES-ATLAS-T2 sites since March 2014. 3.2. Performance of the ES-ATLAS-T2 A T2D site must be stable enough to guarantee data processing and analysis. Figure 3(a) compares the number of jobs completed by the different ES-ATLAS-T2 sites. During Run 1 and before Run 2, the ES-ATLAS-T2 processed around 19 million jobs (analysis + production). The number of events processed related to these completed jobs is shown in Figure 3(b): around 93 million events. In this period around 71 PB of data (both collision events and MC simulation jobs) have been Figure 2. ATLAS availability for the ES-ATLAS-T2 analysis queues. Figure 2. ATLAS availability for the ES-ATLAS-T2 analysis queues. Figure 2. ATLAS availability for the ES-ATLAS-T2 analysis queues. ES-ATLAS-T2 sites have been and are T2D category showing they have good connectivity among other Tier-1s and Tier-2s. In terms of connectivity, the concept of so-called T2D was introduced: those that fulfil a certain performance are then classified as T2D. At the moment, the performance limit is set to an overall transfer performance of at least 5MB/s to at least 80% of Tier-1s in ATLAS, for very large files (>1GB). This implies direct transfer from/to Tier-0 and all ATLAS Tier-1s and Tier-2s. It also means the execution of simulation and analysis jobs are favoured in our Tier-2. A T2D site must be stable enough to guarantee data processing and analysis. Figure 3(a) compares the number of jobs completed by the different ES-ATLAS-T2 sites. During Run 1 and before Run 2, the ES-ATLAS-T2 processed around 19 million jobs (analysis + production). The number of events processed related to these completed jobs is shown in Figure 3(b): around 93 million events. In this period around 71 PB of data (both collision events and MC simulation jobs) have been processed in our Tier-2 and read from our storage systems, as shown in Figure 4(a). The ES-ATLAS- T2 sites are getting more datasets and around 100 PB of data have been transferred as is shown in Figure 4(b) from January 2012 to before Run 2. Figure 3(b). Number of events processed belonging to simulation (same as production) plus analysis jobs. Figure 3(a). Completed jobs (50% analysis, 50% production) in the ES-ATLAS-T2 sites during Run 1 and before Run 2. Figure 3(b). Number of events processed belonging to simulation (same as production) plus analysis jobs. Figure 3(a). Completed jobs (50% analysis, 50% production) in the ES-ATLAS-T2 sites during Run 1 and before Run 2. 4.2. Towards a Federated Data Storage System 4.2. Towards a Federated Data Storage System Data federation using XRootD called FAX is a way to unify direct access to the diversity of storage services used by ATLAS. Since the start of the FAX initiative, several ATLAS sites belonging to different regions/clouds have joined the effort. Figure 6 shows the aggregated XRootD traffic at various Tier-2s. FAX implementation in ES-ATLAS-T2 is as follows: the IFAE and UAM sites are serving their disk space within the ATLAS FAX federation, whilst IFIC will join in the upcoming months. In any case, for all sites of ES-ATLAS-T2, PanDA is using FAX when a local copy of the input file fails; in the case stage-in fails due to a temporary problem, FAX will re-attempt the stage-in in a second time after a few minutes. 5 5 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 Figure 6. Aggregated XRootD Traffic at various Tiers of ATLAS. Figure 6. Aggregated XRootD Traffic at various Tiers of ATLAS. 4.3. Distributed Analysis in Run 2 Figure 7(a). Completed jobs in the ES- ATLAS-T2 by activity before the Run 2. g p j ATLAS-T2 by activity before the Run 2. g y Run 2 Derivation Framework. ES-ATLAS-T2 through the IFAE has contributed to the coordination of DA for two years and now the same person is the Grid Distributed Production (GDP) coordinator. ES-ATLAS-T2 through the IFAE has contributed to the coordination of DA for two years and now the same person is the Grid Distributed Production (GDP) coordinator. ES-ATLAS-T2 through the IFAE has contributed to the coordination of DA for two years and now the same person is the Grid Distributed Production (GDP) coordinator. 4.3. Distributed Analysis in Run 2 y Distributed Analysis (DA) has worked very well during Run 1 but several improvements are needed to speed-up the workflow and to profit from the more powerful computer architectures now available. Figure 7(a) shows the completed jobs in our Tier-2 by activity where analysis jobs comprise 55%. The Derivation Framework, see Figure 7(b), is the key to the success of the Run 2 Analysis Model. The framework performs bulk data processing for the majority of physicists, producing targeted samples, which physics groups and users will later process themselves. In Run 1 if one or more variables were missing from a particular private end-user collection of collision physics data, it had to be recreated from scratch, wasting time and resources. The new analysis model attempts to tackle this problem by producing automatically common data collections for all physics groups as soon as new data are produced or a new version of the data processing software is available. The Derivation Framewok is the chain of jobs that are executed regularly that produce either one or several common group data collections for a given input provided by the detector or a simulation. The data to be analysed is created with an initial calibration alignment which is the best known at the time of producing the files, but later can be recalibrated and realigned and will keep up-to-date all of the necessary variables. g p p y The computing resource constraints for Run 2 motivate a limit of the number of outputs of approximately ten. The target output size for a derived dataset is 1–10 TB, based on feedback on group workflows summing over the 2012 dataset and MC samples. This output volume fits well with the computing resource constraints. Data reduction can be achieved either by skimming (removing events) or slimming (removing per event information). Book-keeping tools will record the sum of the weights of events removed. Figure 7(b). Data Analysis Model for ATLAS Run 2 Derivation Framework. to the coordination of DA for two years and now the DP) coordinator. Figure 7(a). Completed jobs in the ES- ATLAS-T2 by activity before the Run 2. Figure 7(b). Data Analysis Model for ATLAS Run 2 Derivation Framework. ES-ATLAS-T2 through the IFAE has contributed to the coordination of DA for two years and now the same person is the Grid Distributed Production (GDP) coordinator. Figure 7(b). Data Analysis Model for ATLAS Run 2 Derivation Framework. 5. The Event Index Project j The Event Index Project [12] consists of the development and deployment of a complete catalogue of events for experiments with large amounts of data, such as the ATLAS experiment. Data to be stored 6 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 in the Event Index are produced by all production jobs that run at CERN or on the GRID; for every permanent output file, a snippet of information, containing the unique file identifier and the relevant attributes for each event, is sent to the central catalogue. The estimated insertion rate during the LHC Run 2 is about 80 Hz of file records containing ~15 kHz of event records. In Run 1, the ATLAS experiment had a catalogue of all real and simulated events in the database (TAGDB), implemented in Oracle, with the main purpose of the selection of events on the basis of physical variables, the identification of files that contain a list of events already selected by other means, and consistency checks on the completeness of event processing. Nevertheless the TAGDB is slow, complex, unreliable [13] and has scaling problems when the number of events exceeds 109. The new catalogue (Event Index Project) is based on the previous work on the TAGDB, but improving it to reach good performance. The most innovative part of this project is the adaptation and application of the NoSQL technologies for cataloguing data of a large experiment. They are cheaper, easier to use and faster for sparse searches over large amount of data, so the Event Index project is using them for the event cataloguing system. The contents of this new catalogue for each reconstruction campaign and for each data format are event identifiers, online trigger pattern and hit counts and references (pointers) to the events at each processing stage in all permanent files on the storage. 5. The Event Index Project Basically the use cases that Event Index is addressing are the following: hat Event Index is addressing are the following: - Event picking: give me the reference (pointer) to “this” event in “that” format for a given processing cycle. - Event picking: give me the reference (pointer) to “this” event in “that” format for a given processing cycle. - Production consistency checks: technical checks that processing cycles are complete (event counts match). - Event service: give me the references (pointers) for “this” list of events, or for the events satisfying given selection criteria, to be distributed individually to processes running on (for example) HPC or cloud clusters. - Event service: give me the references (pointers) for “this” list of events, or for the events satisfying given selection criteria, to be distributed individually to processes running on (for example) HPC or cloud clusters. The system design [12], as is shown in Figure 8, is highly modular, so that its components (data collection system, storage system based on Hadoop, and query web service) could be developed separately and in parallel. The Event Index is in operation for the start of LHC Run 2. Figure 8. Event Index architecture ES-ATLAS-T2 is participating actively in the Distributed Data Collection task [14] as a pure R&D task contained in the Tier-2 project. The Event Index contains records of all events processed by ATLAS; these records include the reference to the files containing each event (the GUID of the file) and the internal “pointer” to each event in the file. This information is collected by all jobs that run at Tier-0 or on the GRID and process ATLAS events. Each job produces a snippet of information for each permanent output file (producer architecture). This information is packed and transferred to a central broker at CERN using an ActiveMQ messaging system (consumer architecture), and then is unpacked, sorted and reformatted in order to be stored and catalogued into a central Hadoop server. The Event Index infrastructure at IFIC consists in a Hadoop cloudera cluster composed by 8 machines with 2 quad core Intel Xeon E5420@2.5GHz processors and 16 GB DDR2@667 MHz of RAM memory and a hardware by 2 SAS drives of 146 GB @ 10 Krpm. The purpose is to work on Event Figure 8. Event Index architecture Figure 8. Event Index architecture Figure 8. 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 Index developments (e.g. optimize queries from event service) and have another implementation for checking and comparison. Index developments (e.g. optimize queries from event service) and have another implementation for checking and comparison. 6. Conclusions and perspectives Run 2 data taking will begin in Spring/Summer 2015 and the ATLAS experiment is ready to address new challenges in Computing and Data Management. For this purpose the ATLAS computing model has been updated. The goals of every Tier-2 are to satisfy the revised computing requirements (disk and CPU) and the daily maintenance and operation. Moreover, the ES-ATLAS-T2 is adopting the necessary changes to address the new ATLAS Run 2 challenges. In particular: to apply several improvements in the Distributed Analysis and the Derivation Framework; to provide multicore queues to serve the multicore jobs; and to implement the Federated Data Storage System, ATLAS FAX. Moreover, the ES-ATLAS-T2 sites are leading the ATLAS Event Index Project, thus also embracing R&D. The tasks performed within this project, and ready for Run 2, are: automation of all the procedures; loading all Run 1 data; tuning of the system and providing a thorough monitoring system. Applying previous improvements will solve most of the new challenges mentioned at the introduction. 7. References [1] ATLAS & CMS Collaboration, 2012 First observation of a new particle in the search for the Standard Model Higgs boson at the LHC. Physics Letters B Volume 716, 1-29 & 30-61. [1] ATLAS & CMS Collaboration, 2012 First observation of a new particle in the search for the Standard Model Higgs boson at the LHC. Physics Letters B Volume 716, 1-29 & 30-61. [2] ATLAS Collaboration, 2008 The ATLAS Experiment at the CERN Large Hadron Collider. Journal of Instrumentation 3 (8) S08003. [2] ATLAS Collaboration, 2008 The ATLAS Experiment at the CERN Large Hadron Collider. Journal of Instrumentation 3 (8) S08003. [3] V. Sánchez et al., 2013 Lessons learned from ATLAS performance Studies of the Iberian Cloud for the first LHC running period, Journal of Physics: Conference Series 513 032082. [3] V. Sánchez et al., 2013 Lessons learned from ATLAS performance Studies of the Iberian Cloud for the first LHC running period, Journal of Physics: Conference Series 513 032082. [4] J. Salt et al., 2014 New Challenges of the Spanish ATLAS Tier-2 to address the Run-2 period of LHC. Proceeding of the IBERGRID 2014 conference, Aveiro (Portugal). [4] J. Salt et al., 2014 New Challenges of the Spanish ATLAS Tier-2 to address the Run-2 period of LHC. Proceeding of the IBERGRID 2014 conference, Aveiro (Portugal). [5] D. Barberis, 2014 ATLAS Computing challenges before the next LHC run. Proceedings of the ICHEP 2014 conference, Valencia (Spain). [5] D. Barberis, 2014 ATLAS Computing challenges before the next LHC run. Proceedings of the ICHEP 2014 conference, Valencia (Spain). [6] I. Bird et al., Update of the Computing Models of the WLCG and the LHC experiments, CERN- LHCC-2014-014/ LCG-TDR-002. [6] I. Bird et al., Update of the Computing Models of the WLCG and the LHC experiments, CERN- LHCC-2014-014/ LCG-TDR-002. [7] V. Garonne at al., 2014 Rucio – The next generation of large scale distributed sys ATLAS Data Management, Journal of Physics: Conference Series 513 042021. [7] V. Garonne at al., 2014 Rucio – The next generation of large scale distributed system for ATLAS Data Management, Journal of Physics: Conference Series 513 042021. R. Gardner et al., 2014 Data federation strategies for ATLAS using XRootD, Journal Physics: Conference Series 513 042049. [9] K. De, 2014 Task Management in the New ATLAS PS, JoP: Conference Series 513 032078. [9] K. De, 2014 Task Management in the New ATLAS PS, JoP: Conference Series 513 032078. 5. The Event Index Project Event Index architecture ES-ATLAS-T2 is participating actively in the Distributed Data Collection task [14] as a pure R&D task contained in the Tier-2 project. The Event Index contains records of all events processed by ATLAS; these records include the reference to the files containing each event (the GUID of the file) and the internal “pointer” to each event in the file. This information is collected by all jobs that run at Tier-0 or on the GRID and process ATLAS events. Each job produces a snippet of information for each permanent output file (producer architecture). This information is packed and transferred to a central broker at CERN using an ActiveMQ messaging system (consumer architecture), and then is unpacked, sorted and reformatted in order to be stored and catalogued into a central Hadoop server. The Event Index infrastructure at IFIC consists in a Hadoop cloudera cluster composed by 8 machines p g p The Event Index infrastructure at IFIC consists in a Hadoop cloudera cluster composed by 8 machines with 2 quad core Intel Xeon E5420@2.5GHz processors and 16 GB DDR2@667 MHz of RAM memory and a hardware by 2 SAS drives of 146 GB @ 10 Krpm. The purpose is to work on Event 7 21st International Conference on Computing in High Energy and Nuclear Physics (CHEP2015) IOP Publishing Journal of Physics: Conference Series 664 (2015) 052016 doi:10.1088/1742-6596/664/5/052016 7. References [10] CERN openlab Whitepaper on Future IT Challenges in Scientific Research, May2014, http://dx.doi.org/10.5281/zenodo.8765. p g 1] http://atlas.fis.utfsm.cl/atlas/adcos/AdcMonitoring.htm#Service_and_Availability_Monitor [12] D. Barberis et al., 2015 The ATLAS EventIndex: architecture, design choices, deployment and first operation experience. Proceedings of the CHEP2015 conference, Okinawa (Japan). [13] M. Mambelli et al., 2010 Job optimization in ATLAS TAG-based distributed analysis, Journal of Physics: Conference Series 219 072042. [14] J. Sánchez et al., 2015 Distributed Data Collection for the ATLAS EventIndex. Proceedings of the CHEP2015 conference, Okinawa (Japan). Acknowledgments This work has been supported by MINECO, Spain (Proj. Ref. FPA2010-21919-C03-01,02,03 & FPA2013-47424-C3,01,02,03), which include FEDER funds from the European Union 8
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A leishmaniose no município de Porto Ferreira
Revista Brasileira de Enfermagem
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I Resumo da Monografia apresentada no Curso de Especialização em Saúde Pública oferecido pelo Departamento de Enfermagem da UFSCar, 1 994. 2 Enfermeira Chefe da Vigilância Sanitária - Secretaria Municipal de Saúde - Porto Ferreira - SP. 3 Farmacêutico - Diretor da Vigilância Sanitária - ERSA 53 - São Carlos - SP. 4 Enfermeira - Professora Doutora do Departamento de Enfermagem da UFSCar. A LEISHMANIOSE NO MUNiCíPIO DE PORTO FERREIRA 1 LEJÇHMANIAÇIÇ AT PORTO FERREIRA DIITRICT Edelfrauf Nofhing Z6ia 2 Azumir E. Palombo 3 Elisefe Silva Pedrazzani 4 RESUMO: O Município de Porto Ferreira-SP., por suas características geográficas, hidrográficas, topológicas e climáticas, associadas ao tipo de vegetação existente, favorece a procriação da espécie de flebotomíneos e conseqüentemente um aumento na incidência da Leishmaniose a partir de 1 98 1 . Os dados foram obtidos junto à Ficha de Investigação Epidemiológica e também através de entrevista para os casos residentes na localidade. Foram registrados 40 casos até o primeiro semestre de 1 993 os quais ocorreram basicamente dentro de uma mesma área, que a denominamos de área crítica. O comportamento epidemiológico da doença no município difere do tradicional. citado na literatura, quando observamos que as mulheres estão sendo mais afetadas, com ocupação principal do lar. caracterizando uma transmissão autóctone, domiciliar e urbana. O fluxo de atendimento dos pacientes na rede básica de saúde também foi avaliado, identificando-se algumas questões a respeito do tempo decorrente entre os primeiros sintomas até o tratamento específico. Ações de controle epidemio-sanitário foram propostas no sentido de conter a incidência da doença no município. UNITERMOS: Leishmaniose - Epidemiologia A DOENÇA LEISHMANIOSE As Leishmanioses Tegumentares Americanas são parasitoses humanas e de outros vertebrados causadas por diversas espécies e subespécies do gênero Leishmania e sua transmissão se dá por meio da picada de insetos da família Phlebotominae, conhecido popularmente como "birigui", "mosquito palha" ou "cangalhinha" (1 , 9). O protozoário provoca uma infecção quase que exclusivamente na pele e mucosas. É uma zoonose própria de roedores silvestres e pode ser transmitida ao homem quando entra em contato com a mata, ou urbana, quando tem como reservatórios o próprio homem, os cães e gatos. 445 R. Bras: Enferm. Brasília, v. 49, n. 3, p. 445-458, jul./set. 1 996 A infecção no homem se estabelece pela inoculação da forma infectante (Iepfoma) nos elementos histiocitários da pele, desenvolvendo geralmente uma pápula ou eritema papuloso. Pode haver regressão das forr:nas primitivas ou progredirem para lesões ulcerosas ou vegetantes. Os parasitas da lesão primária, por metástase, invadem as mucosas da região naso-buco-faríngea produzindo alterações ulcerosas ou vegetantes de evolução crônica e raramente regressivas, se não receberem um tratamento apropriado. A Leishmaniose não chega a ser mortal mas contribui para a mortalidade geral como causa predisponente de outras enfermidades. É uma doença de notificação compulsória e encontra-se distribuída em populações localizadas em regiões ' ·de . . encostas montanhosas, com densa vegetação. Regiões quentes e úmidas favorecem mais a propagação da doença, sendo mais freqüente em altitudes compreendidas entre O a 800 metros acima do nível do mar, emborá encontram-se alguns tipos distribuídos em áreas de maior altitude (7, 1 0). No Brasil, praticamente todos os estados são atingidos, inclusive em regiões de vale e de matas no interior. A situação da Leishmaniose nó município de Porto Ferreira vem tornando-se preocupante Gevido ao aumento da incidência com o passar dos anos, chegando a serem notificados em média dois casos por mês (2). CARACTERIZAÇÃO DO MUNiCíPIO DE PORTO FERREIRA Porto Ferreira nasceu em conseqüência de três fatores distintos: a balsa que fazia a travessia do rio Mogi-Guaçu - 1 861 , a ferrovia implantada devido à expansão cafeeira do Estado -1 880 e a utilização da navegação fluvial - 1 884. Nesta época habitavam o va'le do ' Mogi-Guaçu.,'· indios Paing.uás, família Tupi­ Guarani, que com suas aldeias constituíram o atual município. Localiza-se a 47,30'de latitude oeste e a 21 ,51 'de longitude -sul, distando 21 7 km da capital de São Paulo, na Depressão Periférica Paulista (parte setentrional), no Vale do rio Mogi-Guaçu, a uma altitude de 51 9 m em relação ao nível do mar. Atualmente possui 246 Km2, sendo 21 ,46 urbano e ó restante 224,56, rural. A região , de · Porto Ferreira, de acordo com .0 Mapa Geomorfológico do Instituto de Pesquisas Tecnológicas de São Paulo' (1 981 ), caracteriza-se como uma área de transição, tanto nos aspectos litológicos (solo) pertencentes ao Grupo Tubarão, quanto aos de vegetação. A constituição do solo é predominantemente laterizada e arenosa, de caráter sedimentar, pertencente à subprovíncia geomorfológica do Rio Mogi-Guaçu, da Depressão Periférica Paulista. A vegetação natural é composta de mata tropical latifoliada, com a presença de cerrados e cerradões e vegetação típica de campo, como também campo cerrado. É marcante a presença de mata ciliar e de mata galeria, 'características do rio Mogi-Guaçu. . R. Bras. Enftrm. Brasília, v. 49, n. 3, p. 445-458, jul./set. 1996 446 A topografia do mumclplo caracteriza-se por baixa declividade em sua maioria, particularmente por localizar-se na planície · aluvial do rio Mogi-Guaçu. Ao norte é possível verificar a presença de morros e colinas e maior inclinação, de aspecto geomorfológico de transição às costas basálticas presentes no município circunvizinho. Na hidrografia do município, pertencente à Bacia do rio Mogi Pardo, é marcante e determinante a presença do rio Mogi-Guaçu, de largura média de 90 m, chegando em alguns pontos a 1 20 a 450m3/seg, conforme os períodos de cheia e de estiagem no município. Existem rios de pequeno porte que são de baixa declividade, pouco encachoeirados e que fazem uso da planície fluvial, constituindo-se por uma natureza favorável à exploração de areia e argila para uso industrial. Cortam a cidade também inúmeros córregos que se conservam naturais, inclusive com vegetação ciliar típica. CARACTERIZAÇÃO DO MUNiCíPIO DE PORTO FERREIRA O clima da região é caracterizado como do tipo mesotérmico, com inverno seco e verão úmido e quente, onde o índice pluviométrico anual oscila entre 1 . 1 00 e 1 .300 mm. A temperatura média anual varia entre 20 a 210 C. O ambiente natural da região vem sofrendo a ação antrópica, principalmente a vegetação, pois está sendo substituída por canaviais, que estão contribuindo para a alteração dos corpos d'água, provocando seu assoreamento e poluição . R. Bras. Enferm. Brasília, v . 49, n. 3, p. 445-458, jul./set. 1996 METODOLOGIA o presente estudo foi realizado junto ao Centro de Saúde de Porto Ferreira e foram considerados todos os casos notificados de Leishmaniose Cutâneo­ Mucosa do referido município desde 1 981 até o primeiro semestre de 1 993, num total de 40. Para coletar os dados da Ficha de Investigação Epidemiológica da Secretaria de Estado da Saúde de São Paulo, foi elaborado um instrumento contendo as variáveis: data da notificação (mês e ano), data dos primeiros sintomas, sexo, idade, residência, ocupação, locais das lesões, locais freqüentados pelos pacientes, exames laboratoriais realizados e locais procurados para o tratamento. Este instrumento foi testado preliminarmente procurando dessa forma garantir uma coleta de dados fidedigna e assim conhecer a população do presente estudo. As Fichas de Investigação Epidemiológica instituídas pela Secretaria de Estado da Saúde de São Paulo e os prontuários dos pacientes existentes no Centro de Saúde local serviram como fontes de dados. Também foram computados os dados existentes na pesquisa entomológica da SUCEN. Para levantamento do fluxo de atendimento dos pacientes, foram escolhidas informações contidas no próprio prontuário, como encaminhamentos e locais de atendimento. Foram realizadas entrevistas com os pacientes sobre esta temática. Serviram também como fonte de dados o mapa geográfico do município e dados cadastrais de ligações de água, para cálculo populacional aproximado. OS SERViÇOS DE SAÚDE EM PORTO FERREIRA O atendimento de saúde na rede básica é composto de um Centro de Saúde, que é responsável pelo desenvolvimento de ações de caráter preventivo, diagnóstico e /ou tratamento específico das doenças infecto-contagiosas e notificação compulsória; um Ambulatório Médico Central e 04 Postos de Saúde periféricos que realizam atendimento básico assistencial em pediatria e clínica geral. O atendimento com especialidade médica é feito basicamente pelo Ambulatório de Referência Regional do Escritório Regional de Saúde de São Carlos (ERSA-53), ficando no município um número reduzido de atendimentos. Existe também um pronto-socorro ligado ao Hospital local, que atende as urgências/emergências assim como o atendimento de clínica ambulatorial. O município conta com empresas médicas privadas que realizam os atendimentos através de convênios com as empresas locais. O objetivo deste estudo foi o de avaliar a situação local, identificando os principais aspectos epidemiológicos, determinando áreas potencialmente de risco e traçando diretrizes em saúde pública. A perspectiva é de aplicar ações intensivas de saúde à população exposta, num programa contínuo de orientação e busca ativa de casos Criovos, como também a reorganização do fluxo de atendimento junto à Rede Básica de Saúde 447 R. Bras. Enferm. Brasília, v . 49, n. 3, p. 445-458, jul./set. 1996 (3). Poderão ser desenvolvidas ações de controle sanitário em animais domésticos que possivelmente possam ser reservatórios assim como o controle de insetos transmissores (flebotomíneos). RESULTADOS E DISCUSSÃO Conforme apresentado anteriormente, a incidência da Leishmaniose vem aumentando no município de Porto Ferreira-SP, sendo que tanto a sua localização geográfica quanto os fatores topoclimáticos e a presença de Reserva Florestal e da Mata Ciliar às margens do Rio Mogi-Guaçu, favorecem a dispersão do mosquito transmissor. A expansão demográfica proporcionou a ocupação de dois novos bairros associados a outros bairros próximos, totalizando aproximadamente 6.500 habitantes. Desde que se tornou obrigatória a notificação da doença no ano de 1 976, o primeiro caso em Porto Ferreira foi registrado somente em 1 981 , sendo posteriormente notificados novos casos a partir de 1 986, atingindo níveis R. Bras. Enferm. Brasília, v. 49, n. 3, p. 445-458, jul./set. 1996 448 preocupantes, com excessão do ano de 1 990 . 9uando nãĠ se registrou nenhġm caso. Esses dados estão apresentados no Graflco 1 , totahzando 40 casos ate o primeiro semestre de 1 993. preocupantes, com excessão do ano de 1 990 . 9uando nãĠ se registrou nenhġm caso. Esses dados estão apresentados no Graflco 1 , totahzando 40 casos ate o primeiro semestre de 1 993. GRÁFICO 1 - DISTRIBUiÇÃO DOS CASOS DE LEISHMANIOSE CUT ÂNEO­ MUCOSA EM PORTO FERREIRA - 1 981 -93 CaOs 1nֱ--------------- 81 82 83 84 85 86 87 88 89 90 91 92 93 Anos É importante destacar que, nos anos de 1 987 e 1 988, se observa um registro superior aos anteriores, assim como nos anos de 1 991 a 1 993 que foram ainda maiores, tornando-se portanto necessário o acompanhamento epidemiológico no sentido de identificar e caracterizar os determinantes na ocorrência ou não de casos junto à população. Todos os casos de que se tem conhecimento receberam atendimento final no Centro de Saúde, onde foram preenchidas as fichas de investigação epidemiológica, realizada a notificação compulsória, a confirmação diagnóstica visto que os pacientes vêm encaminhados de outros serviços médicos e também iniciado o tratamento específico. A autoctonia foi caracterizada na própria ficha epidemiológica e confirmada por pesquisas entomológicas feitas pela SUCEN-Ribeirão Preto, em 1 988 e 1 992, de acordo com o relatório próprio (2,8). Nestas pesquisas detectou-se o mosquito transmissor em locais determinados da cidade, como no interior da Reserva Florestal (Mata do Procópio), Mata Ciliar do Rio Mogi-Guaçú e em domicílios próximos às margens do rio. Nos estudos realizados pela SUCEN, acima referidos, observa-se que a espécie L. R. Bras. Enferm. Brasília, v. 49, n. 3, p. 445-458, jul./set. 1996 R. Bras. Enferm. Brasília, v. 49, n. 3, p. 445-458, jul./set. 1996 RESULTADOS E DISCUSSÃO intermédia foi encontrada tanto no interior da Mata do Procópio e Mata Ciliar do Rio Mogi-Guaçú quanto no intra e peridomicílios próximos, mostrando as incursões que o mosquito transmissor vem fazendo na borda . da mata e com isso se aproximando da população e facilitando a propagação da doença. R. Bras. Enferm. Brasília, v. 49, n. 3, p. 445-458, jul./set. 1996 449 Todos os casos notificados no município foram confirmados clínica e/ou laboratorial mente como Leishmaniose Cutâneo-Mucosa. As lesões apareceram de forma única ou múltiplas, em vários locais do corpo, normalmente em áreas mais desprotegidas (desnudas), como nos braços, pernas, rosto e pescoço, em forma de úlceras, conforme dados do Gráfico 2. Observa-se que as pernas (37%) e os braços (35%) são as partes do corpo mais atingidas. GRÁFICO 2 - DISTRIBUiÇÃO DAS LESÕES SEGUNDO AS PARTES DO CORPO PERNAS DORSO 2% 14% VENTRAL 5% PESCOÇO BRAÇOS 35% Observando os locais das lesões, no momento da entrevista, conseguiu-se perceber que em dois pacientes as lesões apresentaram sinais de reativação ou de não completa cicatrização, os quais foram encaminhados à Unidade de Saúde local para esclarecimento clínico, sendo que um fez tratamento há dois anos e o outro há apenas sete meses, necessitando portanto de um acompanhamento pós alta. Um dos pacientes referiu que, no local do teste de Intradermorreação de Montenegro, após dois anos, se iniciou uma reação inflamatória; foi realizada uma biópsia no local, apresentando laudo negativo para Leishmaniose, tendo sido encaminhado para o otorrinolaringologista, para pesquisa de lesões de orofaringe e/ou nasal. O paciente será acompanhado pela Vigilância Epidemiológica local. R. Bras. Enferm. Brasília, v. 49, n. 3, p. 445-458, jul./set. 1996 450 GRÁFICO 3 - DISTRIBUiÇÃO DOS CASOS DE LEISHMANIOSE CUT ÂNEO­ MUCOSA SEGUNDO O SEXO Casos • MASCULINO • FEMININO 81 82 83 84 85 86 87 88 89 90 91 92 93 Anos o Gráfico 3 mostra que, apesar da maior incidência ocorrer no sexo masculino, se verifica que o registro de casos em mulheres ocorreu a partir de 1 987, não sendo observado nenhum caso feminino no ano de 1 989 e no último ano estudado (1 993) foi registrado o maior número de casos nesta categoria. R. Bras. Enferm. Brasília, v. 49, n. 3, p. 445-458, jul./set. 1996 RESULTADOS E DISCUSSÃO A observação sistematizada na análise desses dados é de extrema importância para a identificação do comportamento das mulheres, se elas estão saindo mais de seus domicílios e frequentando locais de maior risco ou se o mosquito transmissor continua se aproximando mais das áreas residenciais como já apontavam os estudos da SUCEN (2,5). Até então acreditava-se que os homens eram mais afetados devido à ocupação profissional e/ou atividades de lazer (pescaria). Os dados sobre ocupações estão apresentados no Gráfico 4. 45 1 GRÁFICO 4 - DISTRIBUiÇÃO DASA OCUPAÇÕES DE PACIENTES DE LEISHMANIOSE CUTANEO-MUCOSA GRÁFICO 4 DISTRIBUiÇÃO DASA OCUPAÇÕES DE PACIENTES DE LEISHMANIOSE CUTANEO-MUCOSA lavrador sem ocupação Casos o 1 2 3 4 5 6 7 8 9 10 Ç A Ç LEISHMANIOSE CUTANEO-MUCOSA lavrador sem ocupação Casos o 1 2 3 4 5 6 7 8 9 10 Observa-se que a maior ocorrência da doença está na ocupação "do lar", característica das mulheres, o que reforça a discordância dos dados na literatura, onde a doença apresenta-se freqüentemente em madeireiros, seringueiros, garimpeiros etc. O que também apresenta concordância com os dados obtidos pelo SUCEN sobre a localização de captura dos flebolomíneos. A distribuição geográfica da doença no município carateriza uma área em comum, compreendida pelos bairros Vila Sibila, Jardim Anibal, Jardim Porto Novo, Jardim Dornelles de Carvalho, Jardim Independência, população ribeirinha do Rio Mogi-Guaçú, associado a Reserva Florestal (Mata do Procópio) e Mata Ciliar ao longo do rio, onde a maioria dos pacientes ou residem nesta área ou a freqüentam regularmente, a qual optou-se por chamá-Ia de área crítica, conforme consta no Gráfico 5. R. Bras. Enferm. Brasília, v. 49, ll. 3, p. 445-458, jul./set. 1996 452 GRÁFICO 5 - DISTRIBl:JlÇÃO DA FREQÜÊNCIA DE CASOS NOTIFICADOS NA ÁREA (t;RíTICA Casos 81 >&2 83 84 85 86 87 88 89 90 91 92 93 Anos • NOTIFICADO El FREQÜÊNCIA • NOTIFICADO El FREQÜÊNCIA Com a confirmação da presença do flebótomo dos genêros Lutzomia intermedia, Lutzomia montigola, Lutzomia lanei, Lutzomia aragoi, Lutzomia migonei nesta área, caracteriza-se uma transmissão autóctone, domiciliar, diferente da epidemiologia tradicional da doença, quando os homens entravam em áreas enzoóticas de mata e se aproximavam de animais silvestres. A média de idade dos casos estudados é de 32 anos, caracterizando uma faixa etária da pt>pulação econômicamente ativa, mas que não apresenta uma ocupação profissional determinada. R. Bras. Enferm..Brasília, v. 49,'n. 3, p. 445-458, jul./set. 1996 RESULTADOS E DISCUSSÃO á Já para as :átᄚvidades de lazer, o município possui centenas de "Ranchos a beira-rio", além :de Clubes Recreativos e áreas de pequenos córregos que favorecem a pescaria , ou outras atividades afins, principalmente nos fins de semana, em períodos diuturnos. Não foi possível determinar com precisão o período de incubação da doença nos pacientes notificados, entretanto verificou-se a mesma variação entre semanas e meses, descrito na literatura (1 ,4,6,9). t Dentre os casos notificados, apenas três deles pertenciam à mesma família e os demais são membros isolados, confirmando uma suscétibilidade geral e uma resistência imunológica individual. Em relação ao vetor, a SUCEN-Ribeirão Preto, em seu Relatório Preliminar (2), aponta que pretende investigar mais alguns aspectos como a identificação das espécies de flebotomíneos, sua dispersão pelo espaço urbano, portas de entrada e sítios de repouso no intradomicílio e hábito alimentar. Quanto aos reservatórios, também se pretende levantar' a fauna silvestre da Reserva Florestal e doméstica, capazes de desempenhar tais papéis. O diagnóstico da doença, segundo os dados obtidos, foi feito basicamente pelo aspecto clínico das lesões, associado à biópsia na maioria dos casos, onde todos . acusaram a ausência do parasita, apresentando apenas um quadro R. Bras. Enferm..Brasília, v. 49,'n. 3, p. 445-458, jul./set. 1996 453 histológico compatível à doença. Não há informações, nos prontuários dos pacientes ou nas fichas de investigação epidemiológica, sobre a realização do Teste de Intradermorreação de Montenegro ou outro meio de diagnóstico laboratorial. histológico compatível à doença. Não há informações, nos prontuários dos pacientes ou nas fichas de investigação epidemiológica, sobre a realização do Teste de Intradermorreação de Montenegro ou outro meio de diagnóstico laboratorial. Identificou-se ainda que as biópsias foram realizadas em média após três meses do aparecimento dos primeiros sintomas, indicando serem lesões antigas, que portanto não apresentaram parasitas. Não se conseguiu detectar lesões recentes para realização de biópsias e dessa forma confirmar a presença dos parasitas (formas amastigotas). É importante confirmar, durante a realização dos exames, o estágio das lesões para não se esperar resultados positivos nas biópsias e, em conseqüência, um caso não ser confirmado. R. Bras. Enferm. Brasília, v. 49, n. 3, p. 445-458, jul.!set. 1 996 R. Bras. Enferm. Brasília, v. 49, n. 3, p. 445-458, jul./set. 1996 RESULTADOS E DISCUSSÃO Para avaliar o fluxo de atendimento dos pacientes de leishmaniose cutâneo­ mucosa, foi feita uma entrevista com os mesmos, onde se constatou o seguinte: dos 40 pacientes registrados no serviço, 07 mudaram de endereço, não sendo possível localizá-los, pois alguns foram para outro Estado e o endereço dos demais é ignorado; 02 pacientes faleceram e 06 residem em fazendas, portanto não foram entrevistados; 03 pacientes foram procurados em suas residências por várias vezes, mas não foram encontrados; o restante, ou seja, 22 pacientes foram entrevistados pelos pesquisadores em suas residências. Todos têm uma idéia do local onde adquiriram a doença, citando os lugares que freqüentaram, ou seja, locais onde foram picados por mosquitos, sejam eles em suas residências ou áreas de lazer que têm mosquitos que picam, próximos de rios e/ou de matas . Quatorze pacientes referiram ter demorado mais de dois meses para procurar atendimento, após o início das lesões; os demais procuraram com um mês ou menos. A tabela a seguir mostra a sequência de atendimento, onde observa-se que os pacientes procuraram para primeiro atendimento "Outros locais" (consultórios médicos particulares) e Farmácias, apontam ainda que a rede de Postos de Saúde do Bairro praticamente não foi utilizada pelos entrevistados, e por outro lado o Pronto-Socorro foi procurado por quatro indivíduos no seu primeiro atendimento. Esta situação se modifica a partir do terceiro atendimento onde ocorre um número maior pela procura do Centro de Saúde, onde é realizada a assistência específica para esta patologia. R. Bras. Enferm. Brasília, v. 49, n. 3, p. 445-458, jul.!set. 1 996 454 TABELA 1 - DISTRIBUiÇÃO DA FREQÜÊNCIA DE ATENDIMENTO DOS PACIENTES SEGUNDO A ORDEM DOS MESMOS. CONSIDERAÇÕES FINAIS Pelo presente estudo foi possível concluir que existe uma área crítica na cidade, compreendida pelos bairros Jardim Anibal, Vila Sibila', I?Orto Novo, Jardim I ndependência, Jardim Sérgio Dornelles de Carvalho, qߤe são próximos à Reserva Florestal (Mata do Procópio) e toda população, ribeirinha ao longo do Rio Mogi-Guaçu, margeados da Mata Ciliar, com presenߥ de flebotomíneos, segundo pesquisa entomológica da SUCEN . Salienta-se que está havendo uma expansão urbana em direção à área crítica, motivo pelo qual desperta uma preocupação epidemioJógica e também a necessidade de um planejamento tlrbano\ assim como uma: avaliação política sobre o assunto. No aspecto epidemiológico daߦ db,ença local, observa-se a necessidade de implantação de medidas de controle' epidemio-sanitárias como: No aspecto epidemiológico daߦ db,ença local, observa-se a necessidade de implantação de medidas de controle' epidemio-sanitárias como: orientação da população pertencente à área crítica quanto à epidemiologia da doença; s, orientação e adoção de medidas. preventivas quanto. as, Iilartes do corpo mais desprotegidas (desnudas); busca ativa e constante de casas novos, visando. diminttir o tempo entre a instalação dos primeiros. sintomas, o tratamento) e.sp.e:eifico. e: a: notificação compulsória; busca ativa e constante de casas novos, visando. diminttir o tempo entre a instalação dos primeiros. sintomas, o tratamento) e.sp.e:eifico. e: a: notificação compulsória; confirmação do período de maior incidência dQ/ mosquito transmissor (flebotomíneos) e conseqüente controle entomolágico> Jrof'parte da SUCEN; rofat investigar e acompanhar o próprio homem enquanto reservatório importante no ciclo de transmissão da doença; adequação da Rede Básica de atendimento à saúde para que encaminhe o paciente para tratamento específico o mais precocemente possível; adequação da Rede Básica de atendimento à saúde para que encaminhe o paciente para tratamento específico o mais precocemente possível; reciclagem médica sobre o assunto, incluindo a ação terapêutica do medicamento específico, período e técnica' adequada para a realização das biópsias, como também o atendimento, á Norma Tߧcnica da Secretaria de Estado da Saúde, referente à confirmação de casos de leishmaniose Tegumentar Americana; sistematizar as anotações nos prontuários dos pacientes. TABELA 1 - DISTRIBUiÇÃO DA FREQÜÊNCIA DE ATENDIMENTO DOS PACIENTES SEGUNDO A ORDEM DOS MESMOS. LOCAIS DE ATENDIMENTO ORDEM DE ATENDIMENTOS 12• 22• 32• 42. Farmácias 06 02 01 00 ! Posto de Saúde do Bairro 01 00 00 00 , Convênio Empresa 02 00 00 00 Pronto-Socorro 04 01 00 00 Ambulatório Central 01 02 01 00 Centro de Saúde 01 04 1 3 02 Outros 07 1 2 02 '02 : I Total 22 21 1 7 04 I Ressalta-se que quatro pacientes necessitaram de até quatro atendimentos para a confirmação do diagnóstico e a aplicação do tratamento específico, o que aponta a necessidade de uma reciclagem dos profissionais em relação à essa doença, como forma de contribuir para o diagnóstico mais precoce da mesma. No decorrer da entrevista, foi perguntado aos pacientes se apresentaram alguma reação quando tomaram a medicação prescrita no tratamento (Glucantime) e todos foram unânimes em responder que não, apenas queixaram-se de dor nos locais de aplicação e da quantidade necessária ao tratamento. Como o tratamento específico é feito exclusivamente no Centro de Saúde, a Secretaria de Estado da Saúde repassa para o Escritório Regional de Saúde da Região o medicamento N-Metil-Glucamina (Glucantime) e que o redistribui para os Centros de Saúde e estes para os pacientes, conforme prescrição médica. Os tratamentos, de acordo com os dados obtidos nos prontuários, não. foram iniciados com posologia gradativa conforme o preconizado, e sim com 02 ampolas diárias desde o início, sendo que a maioria dos pacientes recebeu duas séries de 20 ampolas, com intervalos de duas semanas entre os mesmos. Não foram observados efeitos colaterais ou reacionais nos pacientes e todas as lesões regrediram após o tratamento, não sendo necessária a hospitalização para a administração da Anfotericina B. R. Bras. Enferm. Brasília, v. 49, n. 3, p. 445-458, jul./set. 1996 455 CONSIDERAÇÕES FINAIS na· unidade de saúde, pois as mesmas encontram-se incompJetas, dificultando uma investigação epidemiológica; A Secretaria de Estado da Saúde ficaria com a ineumbência de melhorar as informações contidas na Ficha de Investigação Epidemiológica obrigjitória e que está em vigor, como também melhorar o fluxo de encaminhamento dessas fichas, para que os órgãos governamentais envolvidos tomem ciência do problema o mais rápido possível e assim possam desenvolver ações junto ao município, visando o controle da doߨnça. R. Bras. Enferm. Brasília, v. 49,.n.,3,.p. 445-458, jul./set. 1996 456 ABSTRACT: Since 1 98 1 , the incidence of Leishmaniasis is incrŋasing in the district of Porto Ferreira, due to favorable environmental oonditions for phlebotomini breeding. The data were collected from the Epi<demiological Investigation Card and also through interviews with local inhabitants cases. U ntil the first semester of 1 993, forty cases had been recorded and they basically occurred in an area we called criticaI. The epidemiological features of the disease in the counfry are different trom thQse listed in literature by the observation that women working at home, were more affected, which characterizes an autoctone, domiciliar and urban transmission. The sequence of assistance of patients h as, also been evaluated, identifying some quesfions concerning the time eklpsed from the first symptoms to the specific treatment. Actions of epidemic-sanitary confrol were proposed in order to conter the incidence the disease in the district. ABSTRACT: Since 1 98 1 , the incidence of Leishmaniasis is incrŋasing in the district of Porto Ferreira, due to favorable environmental oonditions for phlebotomini breeding. The data were collected from the Epi<demiological Investigation Card and also through interviews with local inhabitants cases. U ntil the first semester of 1 993, forty cases had been recorded and they basically occurred in an area we called criticaI. The epidemiological features of the disease in the counfry are different trom thQse listed in literature by the observation that women working at home, were more affected, which characterizes an autoctone, domiciliar and urban transmission. The sequence of assistance of patients h as, also been evaluated, identifying some quesfions concerning the time eklpsed from the first symptoms to the specific treatment. Actions of epidemic-sanitary confrol were proposed in order to conter the incidence the disease in the district. KEYWORDS: Leishmaniasis - Epidemiology KEYWORDS: Leishmaniasis - Epidemiology R. Bras. EČferm. Brasília. v. 49, o. 3, p. 445-458, jul./set. 1 996 1 0. WANDERLEY, D.M. et aI. Relatório do Grupo de Estudos do Programa de Leishmaniose Tegumentar Americana, criado através da Portaria da Superintendência nO 1 38/88, publicada no Diário Oficial do Estado de São Paulo de 1 5 de junho de 1 989. São Paulo: Secretaria de Estado · da Saúde, Superintendência de Controle de Endemias, 1 990, 49p. REFERÊNCIAS BIBLIOGRÁFICAS 1 . ACHA, P. N . et aI. Zoonosis y enfermedades transmissibiles com unes aI hombre y a los animales. Washington, D.C. 1 977, p. 26. 2. BERGO, E. S. et aI. Foco de Leishmanionese Tegumentar Amaricana no Município de Porto Ferreira, SP. Ribeirão Preto. 1 992 (Relatório Preliminar, SUCEN). 3. DINIZ, A. F. et ai Aspectos da Municipalização da Saúde no Município de Porto Ferreira. Relatório de Trabalho de Campo Multiprofissional. São Paulo: USP, Faculdade de Saúde Pública, 1 991, 1 33 p. 4. PESSOA, S.B. et aI. Parasit% gia Médica. 8a ed. Rio de Janeiro. Guanabara Koogan, 1 972. 5. SABROSA, P. C. O domicílio como fator de risco da Leishmaniose Tegumentar Americana. Rio de Janeiro. 1 981 . Dissertação de Mestrado, Escola Nacional de Saúde Pública. 6. SECRETARIA DE ESTADO DA SAÚDE. Centro de Informações em Saúde. Manual de Vigilância Epidemiológica: Normas e Instruções. São Paulo. 1 978, 31 3p. R. Bras. Enferm. Brasília, v . 49. n . 3, p. 445-458, jul.lset. 1996 457 7. . Relatório do grupo de trabalho de Leishmaniose Tegumentar Americana, S.P. São Paulo. 1 986. 7. 8. . Superintendência de Controle de Endemias. Situação atual de Leishmaniose Cutâneo Mucosa na SR-6. Ribeirão Preto. 1 989. 8. 9. VERONESI, R. Doenças Infecciosas e Parasitárias. Rio de Janeiro: Gua­ nabara, 1 972, 694p. 1 0. WANDERLEY, D.M. et aI. Relatório do Grupo de Estudos do Programa de Leishmaniose Tegumentar Americana, criado através da Portaria da Superintendência nO 1 38/88, publicada no Diário Oficial do Estado de São Paulo de 1 5 de junho de 1 989. São Paulo: Secretaria de Estado · da Saúde, Superintendência de Controle de Endemias, 1 990, 49p. 458
https://openalex.org/W2112362183
https://indieskriflig.org.za/index.php/skriflig/article/download/1796/2469
English
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A missional reformed refocus
In die skriflig/In die Skriflig
2,014
cc-by
1,472
A missional reformed refocus The door of a very dark world, which is relatively close to all of us but of which most of us are blissfully unaware, is unlocked: do we really understand the extent of and the way in which sexual abuse of children should be handled? The magnitude of this problem is much bigger than we think. In the second part of the book, the intentional focus is to explore a practical missional ministry within the harsh reality of our broken world. It is clear that each of the authors struggled with several practical issues and they offer solutions to consider. They generously share their insight and practical knowledge with the reader. There is a section that focuses on how the issue of poverty and social injustice should be handled. The reality and challenges that the modern city presents to the church of Jesus Christ is also discussed, and a practical way for a missional focus on health issues in the modern city is explained. There is also an excellent article about a missional response to the global crisis of violence against women. The door of a very dark world, which is relatively close to all of us but of which most of us are blissfully unaware, is unlocked: do we really understand the extent of and the way in which sexual abuse of children should be handled? The magnitude of this problem is much bigger than we think. There is also the reality of so-called ‘diaspora churches’ in our midst. The context of the New Testament church is once again a reality in our own time. What is the church called to do when it faces the diversity of the secularised world around us? How should the church of Jesus Christ approach the challenges of ministry to Muslims? An article in the book offers valuable insights. Do we know of the ‘hidden Christians’ in our world? How should we care for them? How should churches minister to the homosexual community in our midst? A wealth of practical ministry experience is shared with us. We are indeed privileged to sit at the feet of these servants of God. Copyright: © 2014. The Authors. Licensee: AOSIS OpenJournals. This work is licensed under the Creative Commons Attribution License. The implications of a conscious decision to focus missionally on the world around us, is huge. Book Review Page 1 of 2 Book Review Page 1 of 2 Page 1 of 2 Book Review A missional reformed refocus Logan, Jr (ed.) ISSN: 978-1-938267-75-8 Publisher: Greensboro: New Growth Press, 2013, R243.52* *Book price at time of review Review Title: A missional reformed refocus Reviewer: Ignatius W. Ferreira1 Affiliation: 1Faculty of Theology, North-West University, Potchefstroom Campus, South Africa Email: 10322086@nwu.ac.za Postal Address: PO Box 674, Wingatepark 0153, South Africa How to cite this book review: Ferreira, I.W., 2014, ‘A missional reformed refocus’, In die Skriflig 48(1), Art. #1796, 2 pages. http:// dx.doi.org/10.4102/ids. v48i1.1796 Copyright: © 2014. The Authors. Licensee: AOSIS OpenJournals. This work is licensed under the Creative Commons Attribution License. Scan this QR code with your smart phone or mobile device to read online. Read online: This is the reason for the origin of the book titled Reformed means missional. It is the result of an initiative of the World Reformed Fellowship (WRF). This is not a book in which Reformed thinkers are considering vague possibilities of careful action. This is a book in which Reformed servants have stepped over the threshold of the well known, and entered into the unknown turbulent world of our time. The subtitle of the book confirms their intentional commitment: ‘Following Jesus into the world’. That is exactly the practical contribution that each of the authors is doing in their own ministry. It starts with an introductory orientation about the Biblical meaning of being a missional church. This is followed by a thorough discussion of the Reformed theologian Jonathan Edwards’ missionary approach, wherein he emphasises the right relationship between Christians’ Orthodox faith and their moral conduct. Another article in the first part of the book explains the missionary mandate of the book of Romans. In the second part of the book, the intentional focus is to explore a practical missional ministry within the harsh reality of our broken world. It is clear that each of the authors struggled with several practical issues and they offer solutions to consider. They generously share their insight and practical knowledge with the reader. There is a section that focuses on how the issue of poverty and social injustice should be handled. The reality and challenges that the modern city presents to the church of Jesus Christ is also discussed, and a practical way for a missional focus on health issues in the modern city is explained. There is also an excellent article about a missional response to the global crisis of violence against women. 1.Christendom: The known world was culturally Western and religiously Christian. A missional reformed refocus This is the reason why there is a specific section that exclusively focuses on the critical challenge of an evangelical, Reformed and missional theology that must be (re)designed for the 21st century. We will not know what is lacking practically and theologically unless we intentionally and incarnationally start to cross our own comfort zones into the unknown and harsh reality of the world we live in. It will not be easy, but is imperative that it must be done. This is also the reason for a clear and timely call to the church and the academy to ‘bridge the gap’ and start working together in future. Scan this QR code with your smart phone or mobile device to read online. Read online: A missional reformed refocus The treasure of our Reformed confessions was formulated in a time that is today commonly referred to as the time of Christendom.1 We search these writings in vain for an intentional missional theological expression that focuses on the non-believing world outside of Christendom. The reason may be the fact that these confessions were essentially intended as an urgent call to a deformed Christian church living within the cultural boundaries of Christendom. Today, however, the time of Christendom has passed. The content of our Reformed tradition is still foundational for the church of Jesus Christ, but it is now confronted with the near overwhelming reality of a non-Christian world − which is not somewhere in the fading distance of our planet, but has become a confronting reality in the context of our own everyday existence. The time has come for our Reformed confessions to be missionally refocused. Book Title: Reformed means missional. Following Jesus into the world Book Cover: Author: S.T. Logan, Jr (ed.) ISSN: 978-1-938267-75-8 Publisher: Greensboro: New Growth Press, 2013, R243.52* *Book price at time of review Review Title: A missional reformed refocus Reviewer: Ignatius W. Ferreira1 Affiliation: 1Faculty of Theology, North-West University, Potchefstroom Campus, South Africa Email: 10322086@nwu.ac.za Postal Address: PO Box 674, Wingatepark 0153, South Africa How to cite this book review: Ferreira, I.W., 2014, ‘A missional reformed refocus’, In die Skriflig 48(1), Art. #1796, 2 pages. http:// dx.doi.org/10.4102/ids. v48i1.1796 Copyright: © 2014. The Authors. Licensee: AOSIS OpenJournals. This work is licensed under the Creative Commons Attribution License Book Title: Reformed means missional. Following Jesus into the world Book Cover: Author: S.T. Logan, Jr (ed.) ISSN: 978-1-938267-75-8 Publisher: Greensboro: New Growth Press, 2013, R243.52* *Book price at time of review Review Title: A missional reformed refocus Reviewer: Ignatius W. Ferreira1 Affiliation: 1Faculty of Theology, North-West University, Potchefstroom Campus, South Africa Email: 10322086@nwu.ac.za Postal Address: PO Box 674, Wingatepark 0153, South Africa How to cite this book review: Ferreira, I.W., 2014, ‘A missional reformed refocus’, In die Skriflig 48(1), Art. #1796, 2 pages. http:// dx.doi.org/10.4102/ids. v48i1.1796 Copyright: © 2014. The Authors. Licensee: AOSIS OpenJournals. This work is licensed under the Creative Commons Attribution License. Scan this QR code with your smart phone or mobile device to read online. Read online: world Book Cover: Author: S.T. Scan this QR code with your smart phone or mobile device to read online. Read online: Read online: 1.Christendom: The known world was culturally Western and religiously Christian. tendom: The known world was culturally Western and religiously Christian doi:10.4102/ids.v48i1.1796 http://www.indieskriflig.org.za Book Review This book confronts my own beliefs and dramatically enhances my own ministry. To be Reformed is foundational to being the church of Jesus Christ − but something is still missing. An intentional, outward, missional focus is needed. We must follow Jesus Christ into the world (Heb 13:13–14). Everyone who is serious about being Reformed should read and reread this book. However, not all is said and done – yet. This book only provides the introductory paragraphs of a Reformed theology that urgently needs to fill and supplement what has been lacking so far: a missional Reformed refocus. http://www.indieskriflig.org.za doi:10.4102/ids.v48i1.1796 doi:10.4102/ids.v48i1.1796
https://openalex.org/W115187930
https://eprints.qut.edu.au/81862/27/CRF-2015-1-75.pdf
English
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Development of an evidence-based symptom checklist for symptoms of recurrence in women with endometrial cancer.
Cancer research frontiers
2,015
cc-by
8,147
Development of an evidence-based symptom checklist for symptoms of recurrence in women with endometrial cancer Audra de Witt 1,2, Monika Janda2*, Andreas Obermair3 chool of Health Research, Epidemiology and Health Systems, Spring Hill, Brisbane, Queensland 000 1 Menzies School of Health Research, Epidemiology and Health Systems, Spring Hill, Brisbane, Queensland, Australia, 4000. 2 Queensland University of Technology, School of Public Health, Institute of Health and Biomedical Innovation, Brisbane, Queensland, Australia, 4059. 2 Queensland University of Technology, School of Public Health, Institute of Health and Biome Innovation, Brisbane, Queensland, Australia, 4059. 2 Queensland University of Technology, School of Public Health, Institute of Health and Biomedical Innovation, Brisbane, Queensland, Australia, 4059. 3 Queensland Centre for Gynaecological Cancer, Royal Brisbane and Women's Hospital, Herston, Brisbane, Queensland, Australia, 4029. Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Research Article Research Article *Corresponding author: Monika Janda, Queensland University of Technology, School of Public Health and Social Work, Institute of Health and Biomedical Innovation, Victoria Park Road, Kelvin Grove, Queensland, Australia, 4059. E-mail: m.janda@qut.edu.au Citation: de Witt A, et al. Development of an evidence-based symptom checklist for symptoms of recurrence in women with endometrial cancer. Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Copyright: @ 2015 de Witt A, 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. Competing Interests: The authors declare that they have no competing interests. Received Nov 10, 2014; Revised Feb 8, 2015; Accepted Feb 16, 2015; Published Feb 28, 2015. Competing Interests: The authors declare that they have no competing interests. R i d N 10 2014 R i d F b 8 2015 A t d F b 16 2015 P bli h d F b 28 2015 75 *Corresponding author: Monika Janda, Queensland University of Technology, School of Public Health and Social Work, Institute of Health and Biomedical Innovation, Victoria Park Road, Kelvin Grove, Queensland, Australia, 4059. E-mail: m.janda@qut.edu.au Citation: de Witt A, et al. Development of an evidence-based symptom checklist for symptoms of recurrence in women with endometrial cancer. Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Copyright: @ 2015 de Witt A, 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. Competing Interests: The authors declare that they have no competing interests. Received Nov 10, 2014; Revised Feb 8, 2015; Accepted Feb 16, 2015; Published Feb 28, 2015. endometrial cancer. Cancer Research Frontiers. 2015 Feb; 1(1): 75 88. doi: 10.17980/2015.75 Copyright: @ 2015 de Witt A, 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. Introduction subsequent three years (13, 14, 26). Recently published guidelines suggest that patient education on symptoms, rather than investigations such as medical imaging and tumour markers should be the cornerstone for patients’ follow-up, as prompt and thorough investigation of symptoms is more likely to increase the chances of early detection of disease recurrence (27, 28). Endometrial cancer is the sixth most common cancer in women worldwide and is the fourteenth most common cancer overall with 320,000 new cases diagnosed in 2012 (1). A higher incidence of this cancer occurs in more developed countries with Northern America and Europe experiencing the highest incidence and Africa and Asia experiencing the lowest incidence (2) however in 2008, more than half of deaths (64%) were in developing countries (3). Survival varies widely depending on patients’ age, stage at diagnosis, grade and cell type (4) and appears to be poorer in developing countries (3). To date, three symptom checklists have been developed for endometrial cancer, two for the purpose of educating patients about symptoms of recurrence, and the other for surveillance (28-30). However, the symptoms recorded in these checklists differ, and it is unknown how comprehensive these lists are. There are various types of endometrial cancer, the most common being adenocarcinomas (cancers that begin in glandular cells) (5). Other types of endometrial cancers include adenosquamous carcinoma, serous carcinoma and clear cell carcinoma, which are typically more aggressive forms of cancer (5). Tests to detect endometrial cancers typically include physical examination, transvaginal ultrasound, hysteroscopy and biopsy, computerized tomography (CT), and magnetic resonance imaging (MRI) scans (6). The overall aim of this paper was to conduct, a systematic review of relevant contemporary literature to comprehensively generate an updated list of symptoms potentially associated with a recurrence of endometrial cancer. Literature searches undertaken suggests that controversies currently exist in regard to what constitutes effective surveillance management for endometrial cancer patients (22). This review explores alternative evidence based options to detect recurrences in women with endometrial cancer that will ultimately improve survival. Based on articles included in this review, this paper also explores the definition of endometrial cancer ‘recurrence’, determines recurrence rates in women with endometrial cancer, provides an overview of the leading symptoms of recurrence and identifies the post treatment surveillance schedules in these studies. Primary treatment of endometrial cancer is surgical, and includes hysterectomy and bilateral salpingo-oophorectomy, with or without surgical staging. Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Research Article Introduction A small number of patients are considered at high risk of local, regional or distant relapse and will be recommended postoperative radiation, chemotherapy or a combination of both. Overall 13% of patients will develop recurrence (7) with the highest risk within the first three years after primary treatment (7-9). However cancer survival is lower in less developed countries due to a range of factors including lack of access to treatment (10), lack of resources (11) and economic disadvantage (12). Abstract Objective: Women treated for endometrial cancer commonly attend clinic-based follow-up for up to five years even though there is evidence of discrepancies on effectiveness of this approach to improve survival. Furthermore, recent guidelines recommend patient education be the cornerstone for follow-up practices rather than clinical investigations such as medical imaging and tumor markers as prompt and thorough investigations of symptoms are more likely to improve survival. This current practice is based on little evidence and thus alternative models need to be investigated. The overall aim of the study is to identify currently available symptom checklists, determine the comprehensiveness of identified checklists, and generate an updated list of symptoms potentially associated with a recurrence for future testing that will lead to early recurrence detection ultimately improving survival. This paper also explores the definition of recurrence, determines recurrence rates, and identifies post treatment surveillance schedules in reviewed studies. Methods/materials: We conducted a systematic review of the literature extracting; routine follow-up schedules; proportion of patients with symptomatic or asymptomatic recurrence; symptoms of recurrence; prevalence of these symptoms at recurrence. Results: Overall, three previous checklists, and 12 retrospective studies were identified meeting the selection criteria. The average rate of recurrence across the studies was 13% (range 3%-19%). The proportion of patients identified with a symptomatic recurrence varied widely (overall average 67%; range 41% to 91%). The most commonly reported symptoms were vaginal bleeding (25%), pain [not further described] (16%) and abdominal pain and/or discomfort and swelling (15%) which combined, represented 56% of the total reported symptoms. The three previous checklists listed 14 and this review identified an additional 24 symptoms (e.g. vaginal discharge, extremity/bone pain and constipation) not previously identified. Conclusion: The newly developed symptom checklist expands previous ones and will be used in a prospective study to assess sensitivity and specificity to identify recurrence compared to current standard follow-up examinations. Upon completion of successful testing in a prospective study, it could potentially provide an alternative form of follow-up to the traditional surveillance method to detect recurrences in endometrial cancer survivors. Keywords: Endometrial cancer; symptom checklist; recurrence; follow-up care; institutional follow-up protocol. - 75 - Literature search strategy EBSCO host (Medline complete, CIHAHL full text, PsychINFO, Academic search elite), PubMed, Web of science and Google Scholar databases were searched. Based on information obtained from article abstracts, studies conducted on the adult population and published between 1982 and 2012 were retrieved. Key search terms used included “endometr*” “cancer or neoplasm,” or “carcinoma” “recurrence”, “relapse”, “signs,” “symptoms”, “surveillance” and use of MeSH terms “Endometrial neoplasms/diagnosis, rehabilitation, surgery, mortality, therapy, epidemiology, complications, nursing, prevention and control”. Reference lists of articles were searched and relevant articles retrieved. Case reports, letters, editorials and papers published in a language other than English were not considered for inclusion in this review. EBSCO host (Medline complete, CIHAHL full text, PsychINFO, Academic search elite), PubMed, Web of science and Google Scholar databases were searched. Based on information obtained from article abstracts, studies conducted on the adult population and published between 1982 and 2012 were retrieved. Key search terms used included “endometr*” “cancer or neoplasm,” or “carcinoma” “recurrence”, “relapse”, “signs,” “symptoms”, “surveillance” and use of MeSH terms “Endometrial neoplasms/diagnosis, rehabilitation, surgery, The majority of patients who develop recurrences present with symptoms (51%-100%) (9, 13-19) and only a minority are diagnosed during routine follow- up visits (17-21). Furthermore, there are reports of routine follow-up leading to a delay in diagnosing recurrence as some patients wait until their next clinic visit to report symptoms (14, 18, 19, 22). Current follow-up regimes are largely based on individual hospitals’ protocols. According to some authors, this has not shown to improve survival and may be ineffective (13, 14, 16, 23-25). Despite this lack of evidence, typically women are offered follow-up commonly consisting of physical examinations every three to four months for about two years, then extending to six monthly for the - 71 - -76- Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. Study selection criteria and data extracted recurrence, recurrence symptoms and prevalence, and routine follow-up schedules of institutions reported in studies. All articles selected for inclusion in this review (Table 1) reported on original data, were from patients who received successful curative treatment for endometrial cancer, and were disease-free prior to commencing follow-up. Studies needed to provide data on; duration, year of follow-up, stage using the International Federation of Gynecology and Obstetrics (FIGO) staging systems, routine investigations undertaken, recurrence rates including the proportion of symptomatic recurrences, leading symptoms of recurrence, and time frames of scheduled follow-up protocols. We extracted data on patients’ initial diagnosis (stage), the proportion of patients with symptomatic or asymptomatic Literature search strategy doi: 10.17980/2015.75 Research Article Figure 1: Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) flow diagram of article selection process Records identified through database searching (n = 14017) Additional records identified through article references sources (n = 16) Screened records excluded as studies not meeting inclusion criteria removed and duplicates removed (n = 10931) Abstracts of records screened (3102) Records excluded due to not meeting study criteria & duplicates removed (n =3022) Full-text articles assessed for eligibility (n = 80) Full-text articles excluded as did not report on symptoms of endometrial cancer and/or did not report on follow up protocol (n = 68) Studies included in quantitative analysis (n =12) and articles where symptoms check list identified (n=3) Records identified through database searching (n = 14017) Screened records excluded as studies not meeting inclusion criteria removed and duplicates removed (n = 10931) Abstracts of records screened (3102) Records excluded due to not meeting study criteria & duplicates removed (n =3022) Full-text articles excluded as did not report on symptoms of endometrial cancer and/or did not report on follow up protocol (n = 68) Full-text articles assessed for eligibility (n = 80) Additional records identified through article references sources (n = 16) Additional records identified through article references sources (n = 16) Studies included in quantitative analysis (n =12) and articles where symptoms check list identified (n=3) Figure 1: Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) flow diagram of article selection process Statistical analysis Frequencies and proportions of recurrence presenting with or without symptoms were extracted. To estimate the proportion of patients experiencing a presenting symptom we used the formula x/y x 100 where x = the number of patients reporting the symptom and y = the total population sample that experienced recurrence symptoms. When calculating decimal points for this study, figures were rounded up if ≥ 0.5, or rounded down if ≤ 0.4 to the nearest whole number. Results The initial search resulted in 14,017 potentially eligible abstracts. In the first screen, abstracts of articles were reviewed against study eligibility criteria. Articles not meeting the study criteria were excluded, as were duplicate studies. The second screen involved review of full text articles by two independent assessors of which again, duplicate articles and those not meeting study criteria were removed. As a result, 12 retrospective studies were identified (Figure 1). Summary of study characteristics are presented in Table 1. Extraction of data Data was extracted from each study. One study (26) focused on vaginal recurrences only, therefore was separated from other studies in Table 2, or excluded (Table 3) as it was not comparable to other studies which reported recurrences of all body sites. Another study was not included in Table 3 as it did not report details on symptoms of recurrences in the study population (28). In Table 3, several symptom categories from individual studies were combined into larger categories. For example, abdominal discomfort (one case), abdominal swelling (11 cases) -77- Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Research Article and abdominal pain (84 cases) were combined into a larger category of abdominal pain/discomfort/swelling (see Table 3 footnote for other combined categories). recurrence definition (8, 13, 17, 21) but provided local (disease limited to the pelvic cavity or vagina), and distant recurrence definitions (any disease occurring outside of the pelvis (8). In contrast, Aalders and colleagues define local recurrence as ‘tumor regrowth anywhere in the pelvic structures or in the lymph nodes located below the pelvic brim’ (31). In this review, where no definition was provided for disease recurrence, we assumed the definition described by Podczaski applied (22), and where no definition for local or distant recurrence was provided, we assumed the definition described by Ueda applied (8), both as described above. In the event where cases may belong to more than one category, cases were counted in all relevant categories. For example, the chest/chest wall pain category (14 cases) was derived from a combination of categories reported in individual studies as follows; ‘pain in chest wall’ (7 cases) (19), ‘cough/chest wall pain’ (1 case) (14), and ‘dyspnoea, dry cough, chest pain’ (6 cases) (18). In the cough category, cases described above that were included in a combined category of cough and chest pain were counted in both categories under chest/chest wall pain and cough category (see Table 3 footnote). The definition of symptomatic and asymptomatic disease was consistent across articles (‘symptoms prior to clinical exams’ and ‘no complaints or symptoms but disease was detected by routine examinations’ respectively) (13, 17, 21, 22, 26, 28, 31, 32). Four articles did not provide definitions, (8, 14, 18, 19) for these we assumed the definition above applied. Recurrence rates Of the 12 studies reviewed, three (21, 28, 32) included patients with stage 1 and stage 1-2 disease only, four studies included patients with stages 1-3, (13, 17, 19, 22) while five studies included all cancer stages (8, 14, 18, 26, 31) (Table 2). Overall, the average rate of recurrence was 13% (range 3%-19%). The recurrence rate was lower in studies that included stage 1 (7%) and stage 1-2 (8%) cases only, compared to studies that included participants with stage 3 (16%) or stage 4 disease (14%). The proportion of patients identified with symptomatic recurrence varied widely across the studies from 41% (28) to 91% (17). On average, symptomatic recurrence rates were lower in studies that included stage 1 participants only (58%) compared to studies that included stage 1-2 (81%), stage 1-3 (68%) or all stages (67%). Definitions Overall, 10 of the 12 studies described in detail symptoms that led to identification of recurrence (Table 3). The most commonly reported symptoms were vaginal bleeding (25%), pain [not further described] (16%), and abdominal pain and/or discomfort and swelling (15%). Combined, these three symptoms represented 56% of the total reported symptoms in this study. In contrast, a large number of symptoms such as, fever, cardiac arrhythmia, and hemianopsia were reported in less than 1% of recurrences. Symptoms grouped into ‘other’ category are included in Table 3 with the contents of the Five of the 12 studies used a consistent definition of recurrence (‘confirmed and documented disease following a minimum three month disease free interval from the time of primary treatment’) (14, 18, 19, 22, 31, 32). Two studies required one month of being clinically disease free before being considered a recurrence (28), and one study defined recurrence as ‘regrowth or reappearance of tumor in cases where no visible tumor was left at the completion of the operative procedure’ with no time interval specified (22). Some studies did not provide an overall - 78 - Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Research Article Table 1. Summary characteristics of the 12 studies reviewed Author Year of treatment Duration of follow- up of participants Routine investigations indicated by protocols Retrospective cohort studies: FIGO Stage I & II Morice et al. (2001) (32) 1986-1995 Range: 12 to 137 months (Median: 42 months) Gynaecological examination and pap smear every 3 months for the first year, 4 months during second year, 6 months during the third year and yearly thereafter. Chest X-ray and abdomino-pelviccultrasonography annually. Salani et al. (2011) (21) 1997-2007 Range: 24 to 77 months (Mean 46 months) Not indicated. *Reddoch et al. (1995) (28) 1985-1992 (Median: 64 months) Physical examination and pap smear every 3 months for the first 2 years, every 4 months for the third year, and every 6 months for the fourth and fifth years. A chest radiograph was ordered annually. Selected patients were also followed with tests of serial CA-125 levels. Computed tomographic (CT) scans were ordered based on the patients’ complaints and physical findings. Many physicians also requested blood work, such as complete blood count and serum chemistries at follow-up visits. Retrospective cohort: All cancer stages (FIGO) Bristow et al. Definitions (2006) (26) 1997-2005 Range: 3.4 to 143.9 months (Median: 30 months) Pelvic examination and pap test every 3 months during the first year, 3 to 4 months during the second year and 6 monthly thereafter for total of 5 years. Chest radiograph and CT at the discretion of the treating physician, clinical risk factors and patient symptomatology. Ueda et al. (2010) (8) 2000-2006 Range: 2 to 108 months (Median: 43 months) Routine physical examinations, including a pelvic-rectal examination, vaginal vault cytology, and transvaginal ultrasonography (TV-USG), were performed at every visit. CT scan and chest X-ray performed biannually in the first year and annually thereafter. Tumor markers, including CA-125, one to four times annually in a subset of the cases. Ng et al. (1997) (14) 1987-1994 Range: 3 to 90 months (Median: 43 months) History. General examination. Cytological smear of the vault. Bimanual and recto-vaginal examination. Chest X-ray (not as routine). At every 1 to 2 months for first 2 years, every 3 months for the third year and every 6 months thereafter for 5 years. Agboola et al. (1997) (13) 1982-1991 Range: 3 to 138 months (Median: 55 months) Pelvic examination at each visit. Pap smear from the vaginal vault at each visit. Chest radiograph annually. Abdominal ultra sonograms, CT scans of the pelvis and abdomen and biopsies when clinically indicated. Shumsky et l (1994) 1981-1986 Not stated History at each visit. Table 1. Summary characteristics of the 12 studies reviewed Author Year of treatment Duration of follow- up of participants Routine investigations indicated by protocols Retrospective cohort studies: FIGO Stage I & II Morice et al. (2001) (32) 1986-1995 Range: 12 to 137 months (Median: 42 months) Gynaecological examination and pap smear every 3 months for the first year, 4 months during second year, 6 months during the third year and yearly thereafter. Chest X-ray and abdomino-pelviccultrasonography annually. Salani et al. (2011) (21) 1997-2007 Range: 24 to 77 months (Mean 46 months) Not indicated. *Reddoch et al. (1995) (28) 1985-1992 (Median: 64 months) Physical examination and pap smear every 3 months for the first 2 years, every 4 months for the third year, and every 6 months for the fourth and fifth years. A h t di h d d ll Table 1. Summary characteristics of the 12 studies reviewed Computed tomographic (CT) scans were ordered based on the patients’ complaints and physical findings. Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Research Article Podczaski et al. (1992) (22) 1977-1987 (Median: 56 months) Other examinations (not specified) every 3 months for 2 years, every 6 months for 3 years and yearly thereafter. Pap smear every 6 months. X-ray for first 5 years. Patients undergoing postoperative pelvic radiotherapy also underwent yearly intravenous pyelogram for 5 years after completion of treatment. Salvesen et al. (1997) (17) 1981-1990 Range: 48 to 192 months (Median: 108 months) Gynaecological examination and Cytological smear every 3 months for the first year, every 6 months in the second year then yearly for 8 years. Chest X-ray yearly with individually increasing intervals for 10 years with routine attendance. Ultrasound investigation were perform when clinically indicated. Aalders et al. (1984) (31) 1960-1976 Range: 36 to 228 months Follow-up exams every 3 months during first year. Every 6 months during second year and annually thereafter. Case series Smith et al. (2007) (19) 1990-2006 (Median: 109 months) Clinical examinations every 3 months for 2 years. Vaginal vault smears performed twice a year for first 2 years. Clinical review is then extended to once every 6 months until 5 years with vaginal vault cytology being performed once a year during this period. Additional investigations used intermittently within the follow- up period included CT scans and serum CA-125 as clinically indicated. Table 1. (Continued) Clinical review is then extended to once every 6 months until 5 years with vaginal vault cytology being performed once a year during this period. Additional investigations used intermittently within the follow- up period included CT scans and serum CA-125 as clinically indicated. category varying between authors as noted in the footnote. 33% four monthly follow-ups (range one to six months) whereas in the third year, 50% of studies offered a six monthly follow-up schedule (three to 12 months). Symptoms checklist Based on the reported symptoms of recurrences from these 12 studies listed in Table 3, a checklist was derived. This was compared to the three currently published symptom checklists (27, 28, 30). Table 4 demonstrates this review found 14 symptoms that are common to all three checklists and additional 24 symptoms of recurrence that were not previously identified. Examples of these additional symptoms include; vaginal discharge, extremity/bone pain and constipation. Discussion Compared to previously published symptom checklists, this review identified 24 additional symptoms of recurrence that were not previously included, and confirmed the 14 symptoms that were common to all three checklists published earlier. Only five of these symptoms were identified in one or more of these symptom checklists. Some of these additional symptoms found in this paper occurred in small numbers when studies reviewed in this paper were combined; these symptoms include fever, cardiac arrhythmia and urethral lesion. Symptoms common to all checklists include; vaginal bleeding, abdominal pain/discomfort/swelling and cough, which were identified within the top four most commonly reported symptoms in this review. Pain [not further described] was the second most common symptom in this review. Although the three previous symptom checklists included back, hip and abdominal pain, it did not include neck, leg, bone, shoulder and chest Definitions Many physicians also requested blood work, such as complete blood count and serum chemistries at follow-up visits. p Retrospective cohort: All cancer stages (FIGO) Bristow et al. (2006) (26) 1997-2005 Range: 3.4 to 143.9 months (Median: 30 months) Pelvic examination and pap test every 3 months during the first year, 3 to 4 months during the second year and 6 monthly thereafter for total of 5 years. Chest radiograph and CT at the discretion of the treating physician, clinical risk factors and patient symptomatology. Ueda et al. (2010) (8) 2000-2006 Range: 2 to 108 months (Median: 43 months) Routine physical examinations, including a pelvic-rectal examination, vaginal vault cytology, and transvaginal ultrasonography (TV-USG), were performed at every visit. CT scan and chest X-ray performed biannually in the first year and annually thereafter. Tumor markers, including CA-125, one to four times annually in a subset of the cases. Ng et al. (1997) (14) 1987-1994 Range: 3 to 90 months (Median: 43 months) History. General examination. Cytological smear of the vault. Bimanual and recto-vaginal examination. Chest X-ray (not as routine). At every 1 to 2 months for first 2 years, every 3 months for the third year and every 6 months thereafter for 5 years. Agboola et al. (1997) (13) 1982-1991 Range: 3 to 138 months (Median: 55 months) Pelvic examination at each visit. Pap smear from the vaginal vault at each visit. Chest radiograph annually. Abdominal ultra sonograms, CT scans of the pelvis and abdomen and biopsies when clinically indicated. Shumsky et al. (1994) (18) 1981-1986 Not stated History at each visit. Cytology smears of vaginal vault at each visit. Bimanual and recto-vaginal examination at each visit. Chest X-ray biannually. Retrospective cohort: All cancer stages (FIGO) - 79 - Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Study follow up schedules reported in articles Additionally, and in recognition of the ongoing debate on effectiveness of follow-up schedules, we also reviewed the reported post-treatment surveillance schedules of studies in this review which varied in the provision of details (Table 5). In the first year, 75% of treatment centers provided one to three monthly surveillance. In the second year, 33% offered three monthly follow-ups and another - 80 - Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Research Article In this study, on average, symptomatic recurrence rates were lower in studies that included stage 1 participants only (58%) compared to studies that included stage 1-2 (81%), stage 1-3 (68%) or all stages (67%). Further studies need to be conducted to determine whether women with lower grades of endometrial cancer have less symptomatic recurrences compared to those with more advanced cancers. It may also suggest that women with lower grade cancers experience recurrence symptoms but these may not have significant impact on their life/lifestyle. For example, little/intermittent pain experienced and/or minor/ intermittent vaginal bleeding compared to symptomatic recurrences that occur in women with more advanced cancers, who may experience more severe pain and/or heavy vaginal bleeding. This may result in women with lower grade cancer under-reporting recurrence symptoms. However, as stated above, further investigation is required prior to reaching clear conclusions. pain, which are examples of additional symptoms identified in this review. Some symptoms were reported in one of the three checklists only; such as neuropathy, lethargy, (30) dizziness and skin lesions (28). These variations in reported symptoms may be due to reasons, such as patients’ recall ability, health professionals’ acuity (prompting for symptoms), health service (time constraints at appointment), and/or clinical notes recording systems (automated drop down menus restricting options). These findings are significant as previous studies have consistently demonstrated that the majority of endometrial cancer recurrences are symptomatic (7, 9, 13-17, 19, 21, 25, 26, 28, 31, 32). This review applied stringent inclusion criteria and carefully dissected symptom reports across studies including women with differing stage of disease, and found an overall symptomatic recurrence rate of 67% which is within the 50% to 70% range reported in other studies (7, 27). Table 2. Endometrial cancer recurrence rates in the identified studies Sample/FIGO Total recurrences/sample (%) Recurrence identified by symptoms (%) Stage 1 only Salani et al. (21) 4/154 (3) 3/4 (75) Reddoch et al. Study follow up schedules reported in articles (28) 39/398 (10) 14/39 (41) Average (7) (58) Stage 1 and 2 only Morice et al. (32) 27/351 (8) 22/27 (81) Stage 1-3 only Agboola et al. (13) 50/432 (12) 30/50 (60) ^Smith et al. (19) 438/2637 (17) 199/280^ (71) Salvesen et al. (17) 47/249 (19) 43/47 (91) Podczaski et al. (22) 47/300 (16) 23/47 (49) Average (16) (68) Stage 1-4 (all) Aalders et al. (31) 379/3393 (11) 258/379 (68) Ueda et al. (8) 29/271 (11) 13/29 (45) Ng et al. (14) 14/86 (16) 11/14 (79) Shumsky et al. (18) 53/317 (16) 40/53 (75) Average (14) (67) °Overall rate: Stage 1-4 (13) (67) * Bristow et al. (26) 61/377 (16) 9/11* (82) ^Detailed recurrence data were only available to analyse 280 patients ° Not including study by Bristow *Study reported on vaginal recurrences only Table 2. Endometrial cancer recurrence rates in the identified studies - 81 - Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Research Article Table 3. Symptoms of recurrence Symptoms tally of 10 studies reviewed1* Reported symptoms No of occurrences (combined) Total occurrences/total symptomatic reoccurrences of studies combined (%) (n=642) Vaginal bleeding˜ 158 25 Pain 105 16 Abdominal pain/discomfort/swelling˘ 96 15 Cough˙ 46 7 Hemoptysis (cough blood) 35 6 Dyspnoea 32 5 Self detected/palpable mass¨ 30 5 Lethargy/weight loss° 24 4 Lumbar/back pains 21 3 Urinary frequency 18 3 Pelvic pain 16 3 Pedal edema 16 3 Constipation 15 2 Chest/chest wall pain 14 2 Nausea 12 2 Gastrointestinal pain, ascites 12 2 Vaginal lesion 11 2 Vomiting 11 2 Pneumonia 11 1 Leg swelling 10 1 Diarrhea 9 1 Vaginal discharge 9 <1 DVT 8 <1 Extremity/bone painˠ 5 <1 Bowel/intestinal obstruction 4 <1 Headaches 4 <1 RUQ pain 3 <1 Pain, malignant pericardial effusion omentum 2 <1 Fever 1 <1 Cardiac arrhythmia 1 <1 Hemianopsia 1 <1 Urethral lesion 1 <1 Enlarged supraclavicular lymph node 1 <1 Crural pains 1 <1 Other# 76 12 Other^ 54 8 1 Some patients reported more than one symptom therefore total% exceeds 100% *Reddoch study (28) did not provide symptoms detail thus was excluded from this table * Bristow study (26) only reported on vaginal recurrences thus was excluded from this table Table 3. Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Research Article no more anxious as a result of foregoing clinic examinations and face-to-face consultations and reported higher levels of satisfaction than those attending traditional hospital clinics (36). The results of a systematic review of nurse-led versus conventional physician-led follow-up for cancer patients reported that patients appeared satisfied with nurse-led follow-up. Practical alternatives to conventional care included patient-initiated or telephone follow-up. While there were no statistically significant differences in survival, recurrence or psychological morbidity between the groups, further research is needed to determine suitability for gynecological cancer patients (37). In another study (n=36) which consisted of a clinical nurse specialist intervention among gynecological oncology patients, it was found that sexual functioning and quality of life were improved in the trial arm (38). Given the high symptomatic recurrence rate, educating survivors on commonly occurring symptoms that may indicate recurrence such as vaginal bleeding, pain, abdominal discomfort/swelling is crucial and could complement surveillance care. It is recommended that health professionals use their discretion in determining appropriate symptom recurrence information to provide to individual cancer survivors, and remind patients to promptly seek professional advice if any symptoms are experienced or concerns arise. A number of earlier reviews have suggested limited or no increased survival benefit of intensive hospital- based follow-up surveillance for endometrial cancer survivors after receipt of primary treatment (7, 33). Furthermore, at least half of gynecologic oncology experts in a recent e-survey considered less intensive follow-up adequate for low-risk patients (6). Other surveillance suggestions include; tailoring follow-up care according to low and high risk groups (7, 18, 19, 25, 32, 34), limiting the follow-up surveillance time period (13, 26) given that 80% of recurrences occur within the first three years of follow-up (25), or, abandoning routine follow-up schedules altogether (25). These results may suggest a readiness to explore alternate forms of surveillance for recurrence detection. In response to this, we have compiled an updated and expanded symptom checklist tool to aid clinicians and patients in the detection of symptoms of recurrence. This tool could be utilized in a variety of ways. Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 For example, it could be used by specialist consultants, general practitioners and registered nurses as a prompt at appointments to encourage discussion of symptom experiences, used as an educational tool checklist to increase patient awareness of common recurrence symptoms (based on discretion of health professional), and may also be used in less traditional methods to follow-up, such as over the telephone to discuss symptoms and/or to provide patient education. This symptom checklist is currently being tested for sensitivity and is used to trial a novel and less-intensive approach of surveillance that could potentially be used as an alternative to traditional surveillance method to detect recurrences as early as possible in endometrial cancer survivors. Current follow-up schedules (Table 5) commonly require survivors to attend about 14 hospital clinic visits within the first five years (39). In countries such as Australia, survivors often undertake extensive travel to the closest hospital for treatment. Survivors’ expectations of follow-up include the prevention of recurrence or diagnoses of recurrence as early as possible (40), whereas physicians’ mainly aim to see patients for on-going quality assurance and to diagnose complications as early as possible. Study follow up schedules reported in articles Symptoms of recurrence Symptoms tally of 10 studies reviewed1* *Reddoch study (28) did not provide symptoms detail thus was excluded from this table # Aalders study (31): edema, intestinal obstruction, anorexia, bone fracture, icterus and neurological symptoms ^ Smith study (19): fall, haematuria, hemiparesis, pain neck, discharge umbilicus, pain hip, bloating, anaemia, pleural effusion, respiratory infection, bone pain, malena, mobility problems and shoulder pain # Aalders study (31): edema, intestinal obstruction, anorexia, bone fracture, icterus and neurological symptoms - 82 - Limitations This review relied on studies that had undertaken retrospective data collection or chart reviews, and the accuracy of data extracted from clinical records may vary (36). Other issues commonly relating to retrospective studies, such as missing data may have an unknown impact on the review. Additionally, reporting was often done in overlapping categories therefore a prospective study is needed to confirm that symptoms extracted here accurately reflect relevant symptoms. Variations of definitions of disease recurrence across the reviewed studies were also evident. Available data for detailed analysis were limited in some studies due to; a lack of details, no evidence of a disease-free period (19), reporting on vaginal recurrences only (26) and minimal information on follow-up schedules (30). In breast cancer survivors, alternative follow-up regimes have already been tested systematically and in clinical trials. For example, the provision of follow- up care by general practitioners rather than in-hospital specialists has been shown to be effective and acceptable to breast cancer patients (35). In a trial using telephone follow-up by specialist nurses after treatment for breast cancer, the telephone group were It is recommended that prospective cohort studies be conducted to determine alternative approaches of follow-up care for endometrial cancer patients. Randomized trials may be conducted to determine whether less intensive out of hospital follow up, or telephone care is equally effective compared to current standard care. The role of nurses in the provision of cancer follow-up care has been shown - 83 - Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Research Article Table 4: Comparison of previous symptom checklists to symptoms identified in this review Comprehensive symptom checklist proposed Previous symptom checklists *SGO (30) *NCCN (27) Reddoch et al. The symbols “” indicates symptom listed in the checklist and “-”denotes absence. *SGO –Society of Gynaecologic Oncologist; *NCCN – National Comprehensive Cancer Network *NCCN – National Comprehensive Cancer Network *SGO –Society of Gynaecologic Oncologist; p The symbols “” indicates symptom listed in the checklist and “-”denotes absence. *SGO –Society of Gynaecologic Oncologist; Limitations (28) Local Vaginal bleeding    Vaginal discharge - - - Vaginal lesion - - - Urethral lesion - - - Urethral bleeding    Frequent urination - - - Rectal bleeding/malena    Rectal lesion - - - Pelvic pain    Self detected/palpable pelvic mass - - - Hip pain    Distant (extremities) Leg pain - - - Bone/extremity pain - - - Shoulder pain - - - Swollen leg(s)    Fracture - - - Distant (abdomen & back) Abdominal pain/discomfort    Abdominal swelling/ascites    Abdominal mass - - - Loss/decreased appetite -   Nausea    Vomiting    Constipation - - - Diarrhoea - - - Back pain/lumber pain    Distant (thoracic & upper body) Chest/right upper quadrant pain - - - Neck pain - - - Headache - - - Enlarged clavicular lymph node - - - Cough    Cardiac arrhythmia (irregular heartbeat) - - - Shortness of breath/dyspnoea    Distant (systemic) Fever - - - Weight loss    Lethargy/fatigue  - - Anaemia/looking pale - - - Dizziness/blackout - -  Hemiparesis/muscle weakness - - - Neuropathy/numbers  - - Skin lesions - -  Hemianopsia/blindness - - - Self detected mass - - - *SGO –Society of Gynaecologic Oncologist; *NCCN – National Comprehensive Cancer Network The symbols “” indicates symptom listed in the checklist and “-”denotes absence. Table 4: Comparison of previous symptom checklists to symptoms identified in this review - 84 - Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. Limitations doi: 10.17980/2015.75 Research Article Table 5: Follow-up surveillance protocol of the 12 studies included in this review Post primary treatment follow up surveillance in the 12 identified studies Follow-up time Salani* (21) Reddoch (28) Morice (32) Agboola (13) Smith (19) Salvesen (41) Podczaski (22) Aalders (31) Ueda (8) Bristow (26) Ng (14) Shumsky (18) Year 1  Monthly  1-2 monthly  3 monthly          3-4 monthly Year 2  1-2 monthly  3 monthly    3-4 monthly 3-6 monthly  4 monthly    6 monthly Year 3 3 monthly  3-6 monthly  4 monthly  6 monthly      6 monthly thereafter  Annually  Annually thereafter  Year 4 6 monthly         Annually   Year 5 6 monthly         Annually  Annually thereafter  Year 6  6 monthly    Annually   Annually thereafter   Year 7 6 monthly  Annually     Year 8 6 monthly  Annually  Annually thereafter  Year 9 Annually  Year 10 Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Research Article Table 5: Follow-up surveillance protocol of the 12 studies included in this review - 85 - Cancer Research Frontiers. 2015 Feb; 1(1): 75-88. doi: 10.17980/2015.75 Research Article 4. Creutzberg CL, van Putten WL, Koper PC, Lybeert ML, Jobsen JJ, Warlam-Rodenhuis CC, et al. Survival after relapse in patients with endometrial cancer: results from a randomized trial. Gynecol Oncol. 2003 May;89(2):201-9. to be valuable, cost-effective and acceptable in other cancer populations (42) thus, further research to determine suitability for endometrial cancer patients is recommended. Exploring patients’ and health professionals’ perspectives of follow-up care in the primary health care setting upon hospital discharge may also be of significant value to determine suitability and acceptability of this method for cancer patients. 5. Cancer Australia. Endometrial Cancer: Types of endometrial cancer Australia: Cancer Australia and Australian Government [updated 14 April 2014; cited 2015 1 February ]. Available from: http://canceraustralia.gov.au/affected-cancer/cancer- types/gynaecological-cancers/endometrial-cancer. Acknowledgements 10. W W. Research collaboration boosts women's health in Ethiopia. Lancet. 2008;372(1534). Monika Janda is funded by a National Health and Medical Research Council Career Development Award (#1045247). 11. Reeler A, Qiao Y, Dare L, Li J, Zhang AL, Saba J. Women's cancers in developing countries: from research to an integrated health systems approach. Asian Pac J Cancer Prev. 2009 Jul-Sep;10(3):519-26. 12. Pinotti JA, Faundes A. Obstetric and gynecological care for Third World women. Int J Gynaecol Obstet. 1984 Dec;22(6):449-55. Conclusions The management of endometrial cancer follow-up remains controversial (22, 43, 44). There is an urgent need for a more efficient, effective and streamlined approach for endometrial cancer surveillance. 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Pitting Influence on Electrical Capacitance in EHL Rolling Contacts
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Research Article Keywords: EHL (Elasto-Hydrodynamic lubrication), FSI (Fluid-Solid-Interaction), Tribology, Rolling bearing Posted Date: March 21st, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2695178/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Abstract This work presents an investigation on the influence of pitting in EHL rolling contact. The pit- ting geometry comes as an extension on the architecture for multi-physical numerical calculations of rolling element bearing contacts based on OpenFOAM. The model utilized is built according to the state-of-the-art for fluid-solid interaction and electro-quasi-static fields. In this framework, the con- tact is reduced to be two dimensional in order to reduce the computing costs needed. The changes in the electric properties, that are caused by pittings, are measured using the electric capacitance of the contact and put in perspective to the EHL contacts geometry. The investigation delivers an evaluation on how surface degradation, in both the pitting width w and pitting depth d of the contact, affects the electric properties. It can be seen that the electrical capacitance change of different initial geometries has different gradients; both in terms of absolute pitting geometries like pitting width w and pitting depth d and relative compared to the reduced radius w/Rr. Keywords: EHL (Elasto-Hydrodynamic lubrication), FSI (Fluid-Solid-Interaction), Tribology, Rolling bearing Pitting Influence on Electrical Capacitance in EHL Rolling Contacts Anatoly Zaiat1*†, Karim Ibrahim1† and Eckhard Kirchner1 1Product Development and Machine Elements, Technical University Darmstadt, Otto-Berndt-Straße 2, Darmstadt, 64287, Germany. Anatoly Zaiat1*†, Karim Ibrahim1† and Eckhard Kirchner1 1Product Development and Machine Elements, Technical University Darmstadt, Otto-Berndt-Straße 2, Darmstadt, 64287, Germany. Anatoly Zaiat1*†, Karim Ibrahim1† and Eckhard Kirchner1 1Product Development and Machine Elements, Technical University Darmstadt, Otto-Berndt-Straße 2, Darmstadt, 64287, Germany. *Corresponding author(s). E-mail(s): zaiat@pmd.tu-darmstadt.de; Contributing authors: karim.k.ibrahim@web.de; kirchner@pmd.tu-darmstadt.de; †These authors contributed equally to this work. DOI: https://doi.org/10.21203/rs.3.rs-2695178/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Springer Nature 2021 LATEX template Springer Nature 2021 LATEX template Keywords: EHL (Elasto-Hydrodynamic lubrication), FSI (Fluid-Solid-Interaction), Tribology, Rolling bearing 1.2 Scope and assumptions This paper builds upon the work done by Neu et al. [19, 22] in the field of EHL and aims to fur- ther bridge the gap between the numerical data, that has been known for several decades, and the recent experimental data. The CFD model devel- oped by Neu et al., in the simulation environment OpenFOAM, simplifies the rolling bearing as an analogous model of a parallel plate capacitor, as was already experimentally done [8, 12, 23]. In this model the inner raceway and the rolling element represent the parallel plates of the capacitor, while the lubricant is considered to be the dielectric material, as shown in figure 1. Springer Nature 2021 LATEX template damaging mechanisms like discharges through the tribological contacts in the load zone of adjacent bearings [5, 6]. The degree of harmfulness of bear- ing currents can be determined using the apparent bearing current density, which is the electric cur- rent passing through the bearing divided by the Hertzian contact area. Such a phenomenon is investigated in the work done by Muetze [7], where the author defines threshold values for the current density below which no electric damage occurs (J < 0.1A/mm2 ). Therein, the apparent current density J is defined by the ratio of applied electric current and the Hertzian contact area Ahz. There- fore, such harmless electric signals can be applied and are considered suitable for sensory utilization [8, 9] in both new developed products as well as retrofitted machines [10]. Ergo an approach can be derived to be able to use the change in the electric signal for the purposes of using a rolling bearing as an in situ sensor and thus achieve condition moni- toring. The advantage of this method is the direct monitoring of the machine element or the damage location without further structural components in the chain of action that can influence the measure- ment, compared to currently used methods such as vibration measurements [11]. semi-analytical models, the advantage of higher accuracy and more versatile boundary conditions becomes increasingly usable with increasing com- puting power. A major advantage of numerical models is the independence of the surface under consideration. While analytical models can hardly or only con- sider certain regular surface changes, the geometry of the bodies is much more variable in numer- ical models. Already in ideal equations of state like the numerically solvable Reynolds equation, originally derived for hydrodynamic journal bear- ings, the description of the fluid film is possible also including irregular surfaces, see [21]. However, this can only be used to represent changes in the contact partners along the gap height. Numerical computational fluid dynamics (CFD) models, on the other hand, overcome this disadvantage, see [20]. Thus, numerical models can be used to inves- tigate, for example, the influence of pittings and their initiation cracks. 1 Introduction rolling bearings are often replaced prematurely. Thus causing an increase in downtime and the overall maintenance costs. The common method used in the industry to esti- mate the rating life of a rolling bearing follows the prescribed model provided by the interna- tional standard ISO 16281. This method follows a statistical model that is primarily based on the work done by Lundberg and Palmgren [1, 2]. The model only considers the rolling contact fatigue as a cause of damage. Other damage mechanisms such as wear and corrosion are neglected, so are multiple factors such as electric current, water in lubricating oil and hydrogen embrittlement [3]. Hence, due to the statistical nature of the model, The phenomenon of electric current induced damage affects all power-dense systems. The rise of electric mobility means that this type of dam- age in the powertrain of electric vehicles has a higher significance for the industry. The electric damage in such systems is caused by the volt- ages induced from the inverters which are needed to convert direct current into alternating cur- rent [4]. Additionally, a voltage difference between the driveshaft and the motor housing induces 1 Springer Nature 2021 LATEX template Springer Nature 2021 LATEX template Springer Nature 2021 LATEX template Springer Nature 2021 LATEX template 3 3 Fig. 2 Schematic overview of the domains involved [19]. A long term rolling bearing damage mecha- nism is the occurrence of pittings. To counteract these, rolling element bearings are either oversized or replaced at an early stage. Hence, it is crucial for the bearing life and for condition monitoring to be able to detect such damage as reliable as possible, improving resource efficiency. Therefore, in framework of this paper, the model developed by Neu et al. is utilized and extended to accom- modate for surface irregularities in the form of pittings. The effect of pittings on the EHL contact is investigated, with a particular focus being laid on the changes observed to the electric properties of the contact. Fig. 2 Schematic overview of the domains involved [19] 2 Fundamentals The resulting reduced central film thickness h leads to a higher electrical capacitance C of the modeled capacitor, resulting in a change in the impedance signal. The calculation of EHL systems such as rolling bearing contacts is generally regarded as a multi-physical phenomenon and classified as fluid-structure interaction (FSI) problem. Sev- eral source codes have emerged for the numer- ical calculation of EHL contacts over the past two decades. Starting with the calculations by Almqvist et al. [18] over the numerical exten- sions made by Hartinger et al. [25] and later by Hajishafiee et al. [26], phenomena such as cav- itation and temperature distributions inside the surfaces and rolling elements have been added. Although these approaches exist, the numerical solution of EHL problems is still challenging in terms of calculation time and convergence, espe- cially if additional domains are added. Approach- ing the described problem, the main FSI coupling is extended with a thermodynamic and electrical domain as shown in figure 2. F The describing differential equations and ana- lytical terms for the material properties are given in the following. 1.1 Analytical and numerical models Approaches to model the electrical behaviour of rolling bearings have existed for several years. One of the first analytical models were described by Prashad in 1988 [12], for example, and later devel- oped further semi-analytically by Schirra [13, 14], and Puchtler [15]. These description models have analytical or empirical conditions in common, such as an ideal surface of the contact partners or a purely radial load on the contact. Major con- tributions in this field can be attributed to the work done by Dowson and Higginson [16] or later Hamrock and Dowson [17]. Still, deviations from these requirements, for example irregular or dam- aged surfaces or combined load conditions, cannot be represented with such models. Numerical mod- els of the elastohydrodynamic lubrication (EHL) contact, however, are not bound to such sim- plifications. Work by Almqvist [18], Neu [19] or Singh [20] has already been able to overcome this limit of ideal contact surfaces. Although the dura- tion of a numerical calculation is several orders of magnitude longer than that of analytical and Fig. 1 Capacitor model of the EHL contact from Martin [9]. based on the work done by Gemeinder [24] Fig. 1 Capacitor model of the EHL contact from Martin [9]. based on the work done by Gemeinder [24] 2.1 Fluid domain The basic differential equations for a compressible calculation of the fluid domain are the mass trans- port in equation 1 and the momentum transport in equation 2. ∂ρ ∂t + ∇· (ρu) = 0 (1) (1) ∂ ∂t(ρu) = −(∇· ρuu) −∇ρ −∇· τ (2) (2) The compressible calculation of the fluid domain requires an equation of state (EOS) to set pressure temperature and fluid density into con- text. In this case the Tait EOS in equation 3 is used. The coupling between the fluid domain ΩF and the solid domain ΩS is realized through the pres- sure and drag forces that the fluid imposes on the solid, which in return is deformed, thus changing the geometry of the fluid domain. While high shear rates are present at the inlet of the contact zone, viscous heating contributes to the thermodynam- ics ΩT of the system. In turn, the properties of the fluid, namely the dynamic viscosity µ and the density ρ change with temperature T and pressure p. The impact extends towards the permittivity e of the fluid. V V0 = ρ0 ρ = 1 − 1 1 + K′ 0 ln(1 + p K0 (1 + K′ 0)) (3) Equation 3 is accurate across all pressure regions and is extended to the temperature through the bulk modulus according to Fakhred- dine et al. [27]. The Tait EOS is seen to be the most accurate representation for density unless no empirical equation is used, according to Bair [28]. In this context, surface irregularities of the solid domain produce an effect on the load-bearing capacity of the lubricating film of the fluid domain. Springer Nature 2021 LATEX template Springer Nature 2021 LATEX template Building on this, the Doolittle equation [29], [30] is used to describe the viscosity over the pressure and temperature range according to equation 4. Building on this, the Doolittle equation [29], [30] is used to describe the viscosity over the pressure and temperature range according to equation 4. elements E2 as well as their Poisson ratios ν1 and ν2, respectively. and temperature range according to equation 4. E′ = 1 2 1 −ν2 1 E1 + 1 −ν2 2 E2 −1 (9) (9) µ = µRexp " BR0 V∞ V∞R V VR −R0 V∞ V∞R ! 2.1 Fluid domain − 1 1 −R0 # (4) Since the problem to be investigated is a contact mechanical problem, the inertia forces neglected, which reduces the basic differential equation 5. Inserting equations 6, 7, 8 and 9 into equation 5 delivers the final form of the differential equation 10 to solve for the elastic deformations, # (4) ( ) Apparently, the equation does not depend on pressure or temperature explicitly rather indi- rectly via the specific volume V and how it changes with temperature and pressure. There- fore, the Doolittle equation is only applicable in conjunction with an EOS and has been designed to be used with the mentioned Tait EOS from equation 3. ∂2 ∂t2 ρD = 0 = ∇· (µs∇D + µs(∇D)T + λsI(∇D)) + ρsfb (10) (10) This legitimate simplification is advantageous for the stability of the coupling problem as oscil- lations between the fluid domain and the solid domain are reduced significantly but can still occur and impact the calculation. 2.2 Solid domain For the calculation of the solid domain, the elas- tic deformation of the contact area, commonly referred to as the Hertzian contact area [31], is described via equation 5. 2.3 Electrostatic domain ∂2 ∂t2 ρsD = ∇· σ + ρsfb (5) (5) (5) τE = l c = hc c ≪TAC 4 (11) ∂2 ∂x2 (ϵ · Φ) = −ρE (12) τE = l c = hc c ≪TAC 4 (11) (11) ∆h0 = (Dmax −Dmin) · Faim −Fp Faim · ∆t tcd rd (13) ∂2 ∂x2 (ϵ · Φ) = −ρE (12) (12) (13) Equation 12 describes the potential of the elec- tric field E = ∇Φ, which is impacted by the charge density ρE inside of the domain. In this case, all boundaries of the solid domain are moved along the vertical and the fluid domain is adapted accordingly. Afterwards, the strong coupled iteration between the fluid and solid domain is calculated iteratively until changes in the handover parameters (force Fp, deformation D) drop below the convergence criterion. 3 Implementation The calculation procedure evolves around the FSI and is shown in detail in figure 3. Contrary to regular FSI calculations where macroscopic elastic deformations of the solid are induced, the present calculations originate from a contact mechanic problem. The aim is to achieve an equilibrium of forces between the fluid film load capability Fp and an externally imposed target force Faim. The first one is determined by the pressure and elastic deformation of the contacting area, which in turn depends on the initial lubrication gap height h0. ) g The change in the initial gap height ∆h0 is determined via the deformation difference Dmax − Dmin the load fraction (Faim −Fp)/Faim the time fraction ∆t/tcd, where tcd is a time constant derived from the speed of sound of the solid domain. The equation is relaxed with the same factor rd used for the FSI coupling. Even though this equation accounts for the main aspects of time-stepping, deformation, and load, it still needs limitation towards a maximum deformation per time increment to stabilize the iterative proce- dure. Up until now, the respective value ∆h0 ≤ ∆hmax has been found iteratively and reliable values are provided for the respective calculations. Fig. 3 Calculation procedure [19] (5) The descriptive equations for the fluid and solid regions are state of the art for numerical calcu- lations in tribology. A description of the electro- dynamic part takes place according to the work of Neu et al. [19]. In general, several equations are needed to fully describe the interacting elec- tric and magnetic field of a system. Thus, the fully electrodynamic description of the electric field would entail an increased computational effort or consideration of electrodynamic effects within the micro/nanofluidics of the lubrication gap. In contrast to the fluid domain, which is gov- erned by a Eularian description, the solid domain is described as Lagrangian, leading to the known arbitrary Lagrangian Eularian (ALE) formulation for the coupling problem in FSI. Therefore, the displacement of the volume elements is calculated via the Cauchy Stress Tensor and probable exter- nal volume forces. The Cauchy tensor is defined according to Hooke’s law as σ = 2µsϵ + λstr(ϵ)I (6) L ´ t t d λ (6) / g p To determine whether an electrodynamic or quasi-electrostatic formulation is required, the characteristic time tE of the electrodynamic domain is calculated. τE is the time taken by an electromagnetic wave of velocity c = 1/√µeϵ (permeability µe and permittivity ϵ) over the char- acteristic length of the region under consideration l (lubricant film thickness h0). The time constant, compared to the time it takes for the exter- nally imposed electric or magnetic field to change, determines whether a quasi-static approximation is applicable. For AC fields, this is one quarter of the duration of the period TAC. This quasi- static assumption in equation 11 applies to the with the Lam´e constants µs and λs with the Lam´e constants µs and λs µs = E 2(1 −ν) (7) (7) λs =  νE (1+ν)(1−ν) for plane stress νE (1+ν)(1−2ν) for plane stress and 3D cases (8) ( ) To reduce the complexity of the solid calcula- tion approach, the deformation of the solid region is mapped onto the rolling element, utilizing the reduced Youngs modulus E′ while including the elasticity modulus of the rings E1 and the rolling Springer Nature 2021 LATEX template 5 electric field in tribological contacts and is stored in equation 12. [25] has been adapted. During the calculation the initial gap height is adjusted by the height ∆h0 according to equation 15, while the calculation advances in time ∆t. 3.2 Reference and modified case lubricant, which is forced into the lubrication gap by the surface velocity of the two bodies. The process of transferring the roller bearing geometry into the simulation environment of OpenFOAM must be executed in a manner that is optimum for the prevailing flow conditions. How- ever, certain simplifications have to be made in order to ease the complexity and the computa- tional effort. In the framework of academic EHL simulations, such as the work done by Almqvist et al. [32], Neu et al. [22], and Hajishafiee et al. [25], a geometrically unified case setup is used. In the model the geometry of the contact is reduced to a two dimensional numerical grid. Due to the solver being based on the finite volume method, the numerical grid generated is a pseudo-2D grid with one element in the depth direction. Further- more, a simplification is made to the raceway as it is considered to be flat, and only a partial rolling element with a reduced contact radius Rr is utilized, as depicted in figure 4 and equation 14. Fig. 4 2D simplification of EHL contact [22] Fig. 4 2D simplification of EHL contact [22] To further reduce complexity, the rolling ele- ment is modeled to be elastic, while the raceway is considered to be rigid. Again, this is done in accordance to the Hertzian stress theory. Addi- tionally, the inlet and outlet were shifted closer to the centre of the EHL zone, resulting in the overall contact length being 35% of the reduced radius Rr. In this condition, the computational costs remain low, while a numerical starvation effect, as first described by Hamrock and Dowson [33] is ruled out. To rule out phenomena based on the mesh settings, the cell density is based on previous work done by Neu et al. [19]. Rr =  1 RCylinder + 1 RRaceway −1 (14) Rr =  1 RCylinder + 1 RRaceway −1 (14) (14) According to Hertzian theory, the contact between two curved bodies is modelled as an equivalent contact consisting of a body with the reduced radius Rr and a flat counter body. The reduced radius of 10 mm established in the lit- erature [18, 20, 25, 26] would correspond, for example, to the contact of a cylinder of RCylinder = 6 mm and an inner ring raceway of RRaceway = 15 mm radius. Springer Nature 2021 LATEX template Springer Nature 2021 LATEX template 3.1 Numerical Setup The calculation of multi physical FSI problems is resource demanding even for two-dimensions, such that the three-dimensional representations are typically out of scope, as in this case. Moreover, a three-dimensional calculation would not offer more insights for the topic discussed in this contri- bution. Therefore, the total calculation domain Ω is shown in figure 4 right side. It is divided into the solid and fluid subdomain. The fluid region also contains the thermodynamic domain as no heat transfer into the solid is calculated as well as the electrostatic domain ΩE. The coupling of fluid and solid takes place at the interface Γ, which repre- sents the EHL contact and its transition towards the fluid far field. Fig. 3 Calculation procedure [19] For the implementation of a load dependent displacement, the approach by Hartinger et al. 3.2 Reference and modified case Transferred to real cylindrical bear- ings, it is noticeable that this ratio is not common. This is particularly evident compared to a real NU1020 cylindrical roller bearing with a 6 mm cylinder roller radius, but a real inner race radius of 56.6 mm. Fig. 5 Fluid and solid domain of the reference case Fig. 5 Fluid and solid domain of the reference case In the context of this paper, the modelled reduced radius of 10 mm is therefore considered as the upper limit at which the model is ver- ified. Furthermore, the numerical investigations are transferred to smaller reduced radii of 7.67 mm and 5.35 mm. This requires higher comput- ing costs or longer computing times, but achieves a EHL contact closer to that of more common rolling bearings. In this framework, the model shown in figure 5 is used as an undamaged Reference case. This model is then extended to accommodate a pitting geometry, which its effect on the electric proper- ties of the EHL contact is investigated. In this work, the geometry was initially defined using a semi-ellipse that is embedded in the fluid domain based on the work done by Pausch [34], where the long semi-axis represents the pitting width while the short semi-axis represents the pitting depth. However, by removing the arcs and making the geometry block-shaped, it is observed that the Next, the rolling element is assumed to be rotating in space, while the raceway moves transi- tionally. The fluid domain is completely filled with Springer Nature 2021 LATEX template 7 7 Table 1 Investigated parameters Rr [mm] d [µm] w [mm] 5.35 d1 = 1 w1 = 0.0004 · Rr 7.67 d5 = 5 w2 = 0.0008 · Rr 10 d9 = 9 w3 = 0.001 · Rr w4 = 0.0015 · Rr w5 = 0.002 · Rr w6 = 0.003 · Rr w7 = 0.004 · Rr Table 1 Investigated parameters difference in results is minimal, as shown by the pressure distribution shown in figure 6, while the overall stability of the mesh is increased. Fig. 6 Pressure distribution of ellipse-shaped versus brick-shaped pitting geometry at ω=250 rad/s; T=353 K; d=9 µm; w=15 µm; Rr=10 mm of 8 cells in the vertical, contact height, in order to get valid and stable results. Due to the nature of the investigated parameters, the geometry of the EHL contact is continuously altered between the simulated cases. 3.2 Reference and modified case Hence, in order to maintain a constant aspect ratio between the number of cells in the contact length and the contact height across all the simulations, the cell density in the horizontal contact length of the individual blocks is defined as a linear function of said parameters. In the pitting case, shown in figure 7, the fluid- block 1, and consequently fluid-block 3, an aspect ratio of 10:1 is implemented in order to main- tain the skewness and the stability of the mesh, while having reasonable computing time. Fluid- block 2 is considered to be more crucial for the accuracy of the results, hence a higher cell den- sity is applied, as an aspect ratio of 1:1 is used. The cell density of the outer fluid-blocks 0 and 5 remains unchanged from the model developed Neu [22], where it is defined as 15·Rr. Furthermore, in order to be able to vary the pitting geometry the vertices must have the ability to be shifted along the EHL contact. Fig. 6 Pressure distribution of ellipse-shaped versus brick-shaped pitting geometry at ω=250 rad/s; T=353 K; d=9 µm; w=15 µm; Rr=10 mm Hence, the pitting geometry used throughout the investigation carried out in this paper along with the pitting case are shown in figure 7. The ratio of the pitting size to the reduced radius Rr shown in figure 7 is not representative of the actual pittings investigated in this work, as the figure only serves as a method to illustrate the geome- try. The investigated parameters are given in table 1, where Rr is the reduced radius, di is the pit- ting depth and wi is the pitting width. The ratios of pitting width to the reduced radius wi/Rr are chosen to represent the spectrum of different pit- ting geometries. The spectrum ranges from the phenomenon of micropittings, where the intervals are kept at a minimum, to the limit at which the simulations can run stably. Fig. 7 Fluid and solid domain of the pitting case Table 2 Boundary conditions Table 2 Boundary conditions Furthermore, it is observed that initially for the small to medium sized pitting widths, w1 to w5, the difference between the pitting depths d1 and d5 is minimal. The calculated difference in film thickness ranges between 2.7 % to 4.8 %. However, the differences between the pitting depths d5 and d9 can be considered negligible for these cases. Fig. 8 Film thickness at different pitting width to reduced radius ratios, at a fixed reduced radius Rr = 7.67 mm Table 3 FVA 3A dynamic viscosity and SAE 20 modelling parameters taken from Bair [28] Table 3 FVA 3A dynamic viscosity and SAE 20 modelling parameters taken from Bair [28] Parameter Variable Value Doolittle parameter B 3.520 V∞R/VR R0 0.6980 Thermal expansion of occupied volume εt −1.034× 10−3 K−1 Thermal expansion av 8 × 10−4 K−1 Dynamic viscosity at reference state T = 293K µr 0.1089 kg1m−1s−1 FVA 3A dynamic viscosity at reference state T = 313K µr 0.0807 kg1m−1s−1 Fig. 8 Film thickness at different pitting width to reduced radius ratios, at a fixed reduced radius Rr = 7.67 mm Upon approaching the upper limit of the pit- ting geometries that can be stably simulated, w6 and w7, it is observed that the differences between the pitting depths d5 and d9 become more pronounced. 3.3 Boundary conditions The boundary conditions implemented in the sim- ulations are based on the experimental work done by Martin et al. [9] and the numerical work done by Neu et al. [19]. The boundary conditions for density are zero gradient at all boundaries. The pressure gradient is zero at the walls and pres- sure is fixed to ambient pressure at inlet and outlet. There, the velocity gradient is zero. Since the reduced radius Rr is not fixed throughout the simulations, the angular velocity is varied in accor- dance to the rolling condition, in order to ensure that the same surface velocity is maintained in all the simulated cases. Fig. 7 Fluid and solid domain of the pitting case In the work done by Neu [22], it is shown that the mesh of the fluid domain requires a minimum Springer Nature 2021 LATEX template Springer Nature 2021 LATEX template Furthermore, the parameters of the SAE 20 lubricant [28] are used to model it for the simula- tions. The boundary conditions used and the SAE 20 modelling parameters are given in tables 2 and 3 respectively. The effect of the different pitting geometries investigated is shown in figure 7, where the mea- sured film thicknesses are compared using a fixed reduced radius of Rr = 7.67 mm. The results of the performed FSI simulations show that increasing the pitting width causes a decrease in the mea- sured film thickness. By comparing the reference case to the largest simulated pitting case, at a pitting depth of d9, it is calculated that the film thickness drops by about 42 %. Table 2 Boundary conditions Parameter Variable Value Lubricant temperature T 333 K1 Electric potential φ 2.5 kg1m2A1s−3 Surface velocity uR 2.5 m1s−1 Youngs modulus E1,2 2.1 × 1011 kg1m−1s−2 Relative permittivity εR 2.10 Oil Density (At 288 K) ρoil 878 kg1m−3 Table 2 Boundary conditions 4 Results 9 Comparing different reduced radii; top: at pit- ting width to reduced radius ratio w/Rr = 0.0004; bottom: w/Rr = 0.004 4 Results Since the three reduced radii have different central lubricant film thicknesses due to their geometry, the change in electrical capacitance relative to the reference case of ideal surface is used for this purpose. First, the influence of absolute pitting widths, at constant pitting depth, on the electrical prop- erties of the simulated EHD contacts is consid- ered. Here the pitting width, which can be stably modeled, increases with the reduced radius, see figure 11. Beyond the respective pitting width, the numerical mesh is distorted too much or the respectively. However, the drop in the film thick- ness for the reduced radius Rr = 5.35 mm is only 4 %. respectively. However, the drop in the film thick- ness for the reduced radius Rr = 5.35 mm is only 4 %. ness for the reduced radius Rr = 5.35 mm is only 4 %. Fig. 9 Comparing different reduced radii; top: at pit- ting width to reduced radius ratio w/Rr = 0.0004; bottom: w/Rr = 0.004 Fig. 10 Relative capacitance at fixed pitting depth. d = 5 µm. All capacitance are relative to the reference case with Rr = 10 mm. geometry and in the ratio of the pitting width to the reduced radius. Since the three reduced radii have different central lubricant film thicknesses due to their geometry, the change in electrical capacitance relative to the reference case of ideal surface is used for this purpose. First, the influence of absolute pitting widths, at constant pitting depth, on the electrical prop- erties of the simulated EHD contacts is consid- ered. Here the pitting width, which can be stably modeled, increases with the reduced radius, see figure 11. Beyond the respective pitting width, the numerical mesh is distorted too much or the contact of both bodies occurs. This can also be observed in the increasing gradient of capacity for wider pitting, indicating a greater reduction in load bearing pressure. Furthermore, it can be seen that for the same pitting width, the change in capacitance is larger for smaller radii than for larger ones. This can also be expected due to the geometric conditions. However, it can be seen that the difference in capacitance change increases from an initial few percent to more than 30 % at pitting widths of 30 µm. Fig. 4 Results The magnitude of the effect that the pitting geometry has on the electric properties of the EHL contact is determined using the capacitance of the contact. Based on the simplified analogous elec- tric model of the parallel plate capacitor [24], the capacitance can be calculated using equation 15. Since the dielectric ε and the Hertzian area Ahz are assumed to be constant throughout the simu- lations [13, 14], the capacitance C is considered to be solely dependable on the central film thickness hc. Therefore, the capacitance is considered to be anti-proportional to the central film thickness. This phenomenon is further investigated by comparing the three reduced radii at the highest and lowest ratios w1 and w7 as shown in figure 8 top and bottom respectively. For the smallest pit- ting width w1, the trend observed prevails in all the reduced radii. The differences between the pit- ting depths d5 and d9 are negligible. In the biggest pitting ratio w7, the film thickness drops notice- ably between the pitting depths d5 and d9 across all reduced radii. The drop in the film thickness for the reduced radius Rr = 7.67 mm and Rr = 10 mm lie within similar ranges of 8.2 % and 7.5 % C = εAhz hc (15) (15) Springer Nature 2021 LATEX template 9 respectively. However, the drop in the film thick- ness for the reduced radius Rr = 5.35 mm is only 4 %. Fig. 10 Relative capacitance at fixed pitting depth. d = 5 µm. All capacitance are relative to the reference case with Rr = 10 mm. Fig. 10 Relative capacitance at fixed pitting depth. d = 5 µm. All capacitance are relative to the reference case with Rr = 10 mm. respectively. However, the drop in the film thick- ness for the reduced radius Rr = 5.35 mm is only 4 %. Fig. 9 Comparing different reduced radii; top: at pit- ting width to reduced radius ratio w/Rr = 0.0004; bottom: w/Rr = 0.004 Fig. 10 Relative capacitance at fixed pitting depth. d = 5 µm. All capacitance are relative to the reference case with Rr = 10 mm. geometry and in the ratio of the pitting width to the reduced radius. 5 Discussion All capacitances changes are relative to the reference case of the individual reduced radius. radius Rr = 7.67 mm for the same pitting width at all pitting depths, and smallest at the largest reduced radius Rr = 10 mm. In general, once a pitting is present, the change in electrical capac- itance is found to be more dependent on pitting width wi than pitting depth di. The maximum increase in electrical capacitance in the case of the medium and large reduced radius is 20 % at the respective maximum pitting widths of 30 µm and 40 µm. For pitting widths below 20 µm, which can be referred to as micropittings, the difference is less than 5 % for all reduced radii considered. It can thus be concluded that pittings with a width of up to 20 µm cause a change in electrical capac- itance of about 20 - 30 %, regardless of the size of the rolling element. The pitting depth di also plays a rather minor role in this range. radius Rr = 7.67 mm for the same pitting width at all pitting depths, and smallest at the largest reduced radius Rr = 10 mm. In general, once a pitting is present, the change in electrical capac- itance is found to be more dependent on pitting width wi than pitting depth di. The maximum increase in electrical capacitance in the case of the medium and large reduced radius is 20 % at the respective maximum pitting widths of 30 µm and 40 µm. For pitting widths below 20 µm, which can be referred to as micropittings, the difference is less than 5 % for all reduced radii considered. It can thus be concluded that pittings with a width of up to 20 µm cause a change in electrical capac- itance of about 20 - 30 %, regardless of the size of the rolling element. The pitting depth di also plays a rather minor role in this range. Next, the influence of pitting widths relative to the reduced radius is considered. For this, the same data is used as already in figure 11. Here, a much more uniform progression of the three reduced radii considered can be seen. 5 Discussion First, the influence of pittings on the capacitance signal is considered. Figure 10 shows the variation of the capacitance with different, reduced radii Rr and pitting widths wi. The pitting depth d5 is constant with d = 5 µm. All capacities refer to the lowest capacitance, which occurs at the largest reduced radius Rr = 10 mm without a pitting. An expectable progression can be seen here. The smaller the reduced radius, the larger the capaci- tance. This behavior does not change even if the pittings become wider. Also, a stagnant plateau is evident for all reduced radii, with little change in capacitance for smaller pitting width changes. This is partic- ularly evident in the top and middle graphs in figure 11, for pitting widths between 5 µm and 15 µm. It can be assumed that the flow of oil tends to be turbulent under these boundary conditions and partly compensates for the reduced carrying capacity. The regular machining of the raceway surface to take advantage of this effect had, for example, already Pausch [34]. Nevertheless, it can already be seen that the gradients show different behaviour, which is why all capacitance changes are related to the refer- ence value of the respective reduced radius in the following. The results show that the electrical properties of the three selected reduced radii respond dif- ferently to the simulated surface damage. This is shown both in the absolute view of the pitting It can also be seen that the effects on electrical capacitance are greatest at the medium reduced Springer Nature 2021 LATEX template Fig. 11 Relative capacitance at fixed pitting depth. Top: d = 1 µm; middle: d = 5 µm; bottom: d = 9 µm. All capacitance changes are relative to the reference case of the individual reduced radius. Fig. 12 Relative capacitance at fixed pitting depth. Top: d = 1 µm; middle: d = 5 µm; bottom: d = 9 µm. All capacitances changes are relative to the reference case of the individual reduced radius. Fig. 11 Relative capacitance at fixed pitting depth. Top: d = 1 µm; middle: d = 5 µm; bottom: d = 9 µm. All capacitance changes are relative to the reference case of the individual reduced radius. Fig. 12 Relative capacitance at fixed pitting depth. Top: d = 1 µm; middle: d = 5 µm; bottom: d = 9 µm. 5 Discussion Despite the compression of the abscissa, it can be seen here that, with the same ratio of pitting width to reduced radius wi/Rr, the capacity change of the medium reduced radius is still just about the largest, see figure 12. In contrast to the absolute plot in figure 11, the course of the large and mean reduced radius lags are mostly similar and differ by a maximum of 10 % for d = 5 µm. In contrast to the previous plot, the smallest reduced radius shows the largest deviation from the other two curves, regardless of the pitting depth. Springer Nature 2021 LATEX template Springer Nature 2021 LATEX template 11 6 Conclusion • It can be stated that a regularity can be seen in the behaviour of the electric capacitor model, once certain pitting geometries are investigated. As a next step, the model at hand needs to be enhanced into a three-dimensional model and the pitting geometries need to be investigated experimentally. In conclusion, this paper extends the state of research on the influence of surfaces irregularities on the electrical capacitor model of rolling bear- ings. The numerical model used herein, bench- marked and explained in more detail in previous works [19, 22], is extended to implement different variations of reduced radii and pitting geometries. In a parameter study, the behaviour of the electri- cal capacitor model is investigated in relationship to the modelled reduced radius, the pitting depth and the pitting width. The operating conditions remain constant. In conclusion, this paper extends the state of research on the influence of surfaces irregularities on the electrical capacitor model of rolling bear- ings. The numerical model used herein, bench- marked and explained in more detail in previous works [19, 22], is extended to implement different variations of reduced radii and pitting geometries. I d h b h i f h l i One of the aims of describing the electrical properties of rolling element bearings is to iden- tify damage mechanisms. Work done by Almqvist et al. [32] and Singh et al. [20] already presented the influence of surface damages on the EHL state. Pausch [34], on the other side, was able to show that different changes in topography can influ- ence the EHL state positively. To conclude, this paper connects the disciplines of EHL research and extends them through the electrical capacitance model. The changes in electrical capacitance are com- pared absolutely and relatively to the ratio of pitting width to reduced ratio wi/Rr. The conclu- sions are as follows: • The appearance of a pitting-like structure dis- turbs the lubricant flow, changing the pressure profile over the EHL contact length. Once a pit- ting is existing, its pitting width w has a more pronounced effect than the pitting depth d, as can be seen in figure 8 and 9. Declarations All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Anatoly Zaiat and Karim Ibrahim. The first draft of the manuscript was written by Anatoly Zaiat and Karim Ibrahim and all authors commented on previous versions of the manuscript. Review and editing was done by Prof. Eckhard Kirchner. All authors read and approved the final manuscript. • As known from empirical studies [16, 17], the geometry of the contact partners also affects the lubricant film height. A smaller, reduced radius has a shorter contact length, a higher maximum pressure and a lower lubricant film height. However, if the effects of pitting are com- pared in terms of the electrical capacitance of the individual EHL contacts, the behavior is approximately the same, see figure 11. From the knowledge of the geometry of the rolling bear- ing used, the severity of the surface damage can thus be approximately concluded. References [1] Lundberg, G., Palmgren, A.: Dynamic capac- ity of rolling bearings. IVA Handlingar Vol- ume 196 (1947). https://doi.org/10.1115/1. 4009930 • As can be seen in figure 10, the change in electric capacitance lies in a similar range for all three reduced radii. This indicates a regularity that needs to be clarified in further studies. [2] Lundberg, G., Palmgren, A.: Dynamic capac- ity of roller bearings, acta polytechnica. Mechnical Engineering Series (1952) • Still, as shown in figures 11 and 12, the relative change in electrical capacitance of the medium reduced radius is similar or up to about 10 % greater than that of the largest reduced radius. This behaviour is attributed to the interaction between the rolling elements radius and the fluid flow, as investigated in a different manner by Pausch [34], but a distinctive relationship is still to be developed. [3] Halme, J., Andersson, P.: Rolling contact fatigue and wear fundamentals for rolling bearing diagnostics - state of the art. Pro- ceedings of the Institution of Mechanical Engineers, Part J: Journal of Engineering Tribology Volume 224(4), 377–393 (2010). https://doi.org/10.1243/13506501JET656 Springer Nature 2021 LATEX template Journal of Product Lifecycle Management Volume 13(1), 48–65 (2021). https://doi. org/10.1504/IJPLM.2021.115700 [4] Willwerth, A., Roman, M.: Electrical bearing damage — a lurking problem in inverter- driven traction motors. In: 2013 IEEE Trans- portation Electrification Conference and Expo (ITEC), pp. 1–4 (2013). https://doi. org/10.1109/ITEC.2013.6573484 [12] Prashad, H.: Theoretical evaluation of Impedance, Capacitance and Charge Accu- mulation on Roller Bearings Operated under Electrical Fields. Wear (125), 223–239 (1988) [5] Lin, F., Chau, K.T., Chan, C.C., Liu, C.: Fault diagnosis of power components in elec- tric vehicles. 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Jour- nal of Lubrication Technology Volume 99, 264–279 (1977). https://doi.org/10.1115/1. 3453074 [10] Vorwerk-Handing, G., Martin, G., Kirchner, E.: Integration of measurement functions in existing systems – retrofitting as basis for dig- italization. DS 91: Proceedings of NordDesign 2018, Link¨oping (Sweden) (2018) [18] Almqvist, T., Larsson, R.: The navier- stokes approach for thermal ehl line contact solutions. Tribology International Volume 35(4), 163–170 (2002). https://doi.org/10. 1016/S0301-679X(01)00112-8 [11] Hausmann, M., Koch, Y., Kirchner, E.: Man- aging the uncertainty in data-acquisition by in situ measurements: a review and evalua- tion of sensing machine element-approaches in the context of digital twins. International [19] Neu, M., Zaiat, A., Harder, A., Kirchner, E., Hussong, J.: Multi-physics modeling of Springer Nature 2021 LATEX template Springer Nature 2021 LATEX template 13 13 //doi.org/10.1016/j.triboint.2016.12.046 417 D¨usseldorf 2013 (2013) [26] Hajishafiee, A., Kadiric, A., Ioannides, S., Dini, D.: A coupled finite-volume cfd solver for two-dimensional elasto- hydrodynamic lubrication problems with particular application to rolling ele- ment bearings. Tribology International Volume 109, 258–273 (2017). https:
https://openalex.org/W4387612930
https://link.springer.com/content/pdf/10.1007/s10988-023-09396-z.pdf
English
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Keeping context in mind: a non-semantic explanation of apparent context-sensitivity
Linguistics and philosophy
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cc-by
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Linguistics and Philosophy (2024) 47:191–209 https://doi.org/10.1007/s10988-023-09396-z Linguistics and Philosophy (2024) 47:191–209 https://doi.org/10.1007/s10988-023-09396-z ORIGINAL RESEARCH Keeping context in mind: a non-semantic explanation of apparent context-sensitivity Mark Bowker1 Accepted: 17 August 2023 / Published online: 13 October 2023 © The Author(s) 2023 Accepted: 17 August 2023 / Published online: 13 October 2023 © The Author(s) 2023 Abstract Arguments for context-sensitivity are often based on judgments about the truth val- ues of sentences: a sentence seems true in one context and false in another, so it is argued that the truth conditions of the sentence shift between these contexts. Such arguments rely on the assumption that our judgments reflect the actual truth values of sentences in context. Here, I present a non-semantic explanation of these judgments. In short, our judgments about the truth values of sentences are driven by heuristics that are only fallible reflections of actual truth values. These heuristics can lead to different truth-value judgments in different contexts, even when the sentence at issue is not semantically context-sensitive. As a case study, I consider Sterken’s (Philos. Imprint, 15, 2015a) argument for the context-sensitivity of generic generalisations. I provide a non-semantic explanation of Sterken’s truth-value judgments, which builds on Leslie’s (Philos Perspect 21(1):375–403, 2007; Philos Rev 117(1):1–47, 2008) theory of default generalisation. Keywords Generic generalisations · Primitive generalisation · Default generalisation · Psychological heuristics · Application conditions B Mark Bowker markalanbowker@gmail.com 1 Lund University, Lund, Sweden B Mark Bowker markalanbowker@gmail.com 1 Lund University, Lund, Sweden 1 Introduction Arguments for context-sensitivity are often based on judgments about the truth values of sentences in context. The author of the argument invites us to agree with their judgment that some sentence has different truth values in different contexts, despite there being no significant change in the state of the world between those contexts. The author concludes that the truth conditions of the sentence vary with context. This line of argument assumes that our judgments accurately reflect actual truth values. There is reason to think, however, that our judgments are often based on 123 192 M. Bowker heuristics that only fallibly reflect actual truth values. This opens up an alternative explanation of the data that allegedly establishes context-sensitivity: our heuristics might lead to different truth-value judgments in different contexts, even when the sentence at issue is not semantically context-sensitive. This possibility will be illus- trated by considering Rachel Sterken’s (2015a) argument for the context-sensitivity of generic generalisations. Sterken’s argument is a particularly good case study for two reasons. First, Sterken’s argument is more nuanced than other arguments for context- sensitivity, relying not only on judgments about the truth values of generics in context but also on judgments about the truth values of other expressions. Second, heuris- tics for the assessment of truth values are generally an understudied phenomenon but Sarah-Jane Leslie (2007, 2008) has proposed a very clear series of heuristics for the assessment of generic generalisations. This paper is not intended to refute Sterken’s argument for a context-sensitive account of generics but rather to show that it is incomplete, as there is an alternative explanation that she has not considered. A sentence may appear to change its truth value between contexts, not because its truth conditions change between contexts, but because our heuristics for assessing truth and falsity lead us to different answers in different contexts. More broadly, this paper presents a challenge for accounts of context-sensitivity in general. Authors of context-sensitivity arguments should either provide reasons to think that the relevant truth-value judgments do not stem from fallible heuristics, or they should give reasons to think that those heuristics are accurate in the cases at issue. The following section will describe the distinction between truth conditions and heuristics in more detail, drawing on work by Johnston and Leslie (2012, 2019). 1 Introduction It will be argued that truth-value judgments are fallible evidence for context-sensitivity because context might affect our truth-value judgments without affecting truth con- ditions. Section 3 will introduce Sterken’s argument for the context-sensitivity of generics. Section 4 will then describe the heuristics that Leslie takes to determine our truth-value judgments about generic generalisations and Sect. 5 will explain how these truth-value judgments might be influenced by context. 2.1 Heuristics vs truth conditions Some questions are difficult, or even impossible, to answer with complete reliability. To answer a question like that, we need to use a heuristic, i.e., “a simple procedure that helps find adequate, though often imperfect, answers to difficult questions” (Kah- neman, 2012, p. 98). How often do house fires break out when their residents go on holiday? I don’t know the answer to this question and don’t have nearly enough infor- mation to answer this question through, say, a completely reliable algorithm. Instead, I have to rely on some heuristic to estimate the answer. Tversky and Kahneman (1974) identified several heuristics that are used to answer questions about probability and frequency. Using the availability heuristic, for example, your judgment will be based on the ease with which you can recall instances of similar fires; the easier it is, the 193 Keeping context in mind… more likely you will judge it to be. This heuristic is not completely unrelated to the question. If fires are rare, you’ll likely have heard of very few and find them difficult to recall; if fires are common, you will probably find it easier. But the heuristic is fallible. Perhaps fires are generally rare but people close to you have been particularly unlucky. more likely you will judge it to be. This heuristic is not completely unrelated to the question. If fires are rare, you’ll likely have heard of very few and find them difficult to recall; if fires are common, you will probably find it easier. But the heuristic is fallible. Perhaps fires are generally rare but people close to you have been particularly unlucky. Heuristics influence our thinking in vast numbers of judgments. Some heuristics are applied consciously, as when i estimate 589 + 10, 059 by adding 600 to 10, 000. Others are used unconsciously. When estimating probabilities, for example, you might not notice that your judgment is based on ease of recall. In this paper, I’ll be focusing on the heuristics that we use to decide whether a sentence is true. In discussing the heuristics that guide our truth-value judgements, I will extend a distinction made by Johnston and Leslie (2012, 2019) between heuristics and applica- tion conditions. Application conditions are the conditions under which a term applies. A theory of application conditions might tell us, for example, that the word ‘gold’ applies only to the element with atomic number 79. 1 In the psychology literature, which is usually concerned with heuristics rather than application conditions, the combination of these heuristic features can be referred to as my concept or prototype of gold. See Knobe (2003, pp. 314–5) for two different notions of a concept. For the classic pioneering work on prototypes, see Rosch (1973, 1978) and Rosch & Mervis (1975). 2 See Knobe (2003, pp.314–5) for this way of distinguishing application conditions from heuristics, though Knobe doesn’t use these terms. 3 There is debate about what kind of error this is. See Wikforss (2001) and Hattiangadi (2006) for arguments that application conditions are not prescriptive and Fennell (2013) for a response. 1 In the psychology literature, which is usually concerned with heuristics rather than application conditions, the combination of these heuristic features can be referred to as my concept or prototype of gold. See Knobe (2003, pp. 314–5) for two different notions of a concept. For the classic pioneering work on prototypes, see Rosch (1973, 1978) and Rosch & Mervis (1975). 2 See Knobe (2003, pp.314–5) for this way of distinguishing application conditions from heuristics, though 3 There is debate about what kind of error this is. See Wikforss (2001) and Hattiangadi (2006) for arguments that application conditions are not prescriptive and Fennell (2013) for a response. 2.1 Heuristics vs truth conditions Bowker not, there is no possibility of impugning their usage; I am simply wrong about the operative heuristics. I extend Johnston and Leslie’s distinction from words to sentences. The application conditions of a sentence are its truth conditions. Just as the application conditions of a word like ‘gold’ tell us the conditions under which the word applies, the truth conditions of a sentence tell us the conditions under which the sentence applies. Just as the heuristics associated with a term are our ways of deciding whether its application conditions are satisfied, the heuristics associated with a sentence are our ways of deciding whether its truth conditions are satisfied. Suppose, for example, that my heuristic for identifying gold is sensitive only to its hallmarks. Then I will judge ‘This is gold’ to be true only when the referent of ‘this’ is stamped with the correct hallmarks. Clearly, however, being stamped with the correct hallmarks does not turn a copper- zinc alloy into gold. Though my heuristics for assessing the truth of the sentence are satisfied, the truth conditions of the sentence are not. The heuristics we use to assess the truth of the sentence are therefore distinct from its truth conditions. 2.1 Heuristics vs truth conditions Such theories are often phrased in terms of the ‘reference’, ‘semantic content’, or ‘extension’ of the term. Speakers often want to avoid using words whose application conditions are not satisfied (excepting, e.g., cases of lies, jokes, metaphor etc.) but often have no way to tell whether these conditions are satisfied without relying on heuristics. Suppose I am describing a novel piece of jewelry and have to decide whether it counts as ‘gold.’ I don’t have direct access to its chemical composition, or the expertise the assess it with certainty, so I have to rely on some fallible procedure for deciding whether the application conditions of ‘gold’ are satisfied. These heuristics might include colour, texture, shape, and hallmarks.1 While these heuristics are good enough in normal circumstances, they are clearly fallible; a substance might satisfy all these superficial criteria and yet fail to be gold. Theories of application conditions and theories of heuristics also differ in their relationship to linguistic behaviour.2 Here’s a very modest theory of the application conditions of ‘bear’: the term does not apply to dogs. Suppose someone says, in earnest, ‘Watch out! There’s a bear!’ upon seeing a particularly large dog. They have used the term ‘bear’ with reference to a dog but this fact doesn’t refute the modest theory above. It isn’t the theory of application conditions that is wrong in this case, it is the speaker.3 Of course, we shouldn’t always conclude that a speaker is in error when their usage fails to match a theory of application conditions. Theories can be wrong too and language use might be a good source of evidence against a theory. The point is only that speaker error is a possible explanation of a mismatch between a theory of application conditions and linguistic behaviour. A theory of heuristics is more directly falsifiable by appeals to linguistic behaviour, however. If my theory suggests that people will apply ‘gold’ under certain conditions, but they in fact do 1 In the psychology literature, which is usually concerned with heuristics rather than application conditions, the combination of these heuristic features can be referred to as my concept or prototype of gold. See Knobe (2003, pp. 314–5) for two different notions of a concept. For the classic pioneering work on prototypes, see Rosch (1973, 1978) and Rosch & Mervis (1975). 12 123 194 M. 2.2 Heuristics in action Thedistinctionbetweenapplicationconditionsandheuristicshashistoricalantecedents. Putnam (1975), for example, argued that meanings (application conditions) are not in the head. There could be two individuals with identical psychologies who refer to dif- ferent things by ‘gold’ due to their environments. In one’s head are not meanings but, for Putnam, “stereotypes”, which are “features which in normal situations constitute ways of recognizing if a thing belongs to the kind” (p. 147). Application conditions are the conditions under which a thing belongs to a kind and Putnam’s stereotypes are the heuristics that we use to decide whether application conditions are satisfied. Likewise, Kripke (1980) argued (although not in these terms) that the application conditions of proper names are not determined by the heuristics that we use to identify their bearers (Johnston and Leslie, 2019, p. 197). I might identify Gödel as the first person to prove the incompleteness of arithmetic, yet for all I know it is possible that Gödel in fact stole the proof, which shows that this heuristic is a fallible means of assessing whether the application conditions of ‘Gödel’ are satisfied. The distinction between heuristics and application conditions opens up the study of language in a similar way to Grice’s (1989) notion of implicature. Grice taught us that semantic theories are not totally at the mercy of truth-value intuitions because those intuitions might result from implicatures. Likewise, even when no implicature is present, truth-value intuitions might result from heuristics, rather than knowledge of application conditions. Some sentences might seem true or seem false, not because they are, but because of the defeasbile heuristics that we use to assess truth and falsity. Williamson (2020), for example, suggests that the truth conditions of the indicative conditional are captured by the material conditional but that various features of the indicative are explained by the fallible heuristics that we use to decide whether a conditional is true. Just as Grice was able to explain various intuitions about truth and falsity through general features of rationality, we can explain some of these intuitions through general features of cognition, namely the use of heuristics. 123 195 Keeping context in mind… 2.4 Summary This section has introduced the distinction between application conditions and heuris- tics. The application conditions of a linguistic term tell us what the term applies to and the heuristics associated with a term are our fallible ways of deciding whether those application conditions are satisfied. The application conditions of a sentence are its truth conditions and the heuristics associated with the sentence are our fallible ways of deciding whether the sentence is true. These heuristics can lead to different judgments in different contexts, which allows us to explain contextual variation in truth-value judgments without positing semantic context-sensitivity. The following section describes Sterken’s indexical analysis of generics and the data she presents in favour of it. Sections 4 and 5 will then offer a non-semantic explanation of this data in terms of the heuristics by which we judge the truth values of generics. 2.3 Heuristics and context-sensitivity Arguments for context-sensitivity often rely on the judgment that the truth value of some sentence differs between contexts. As our truth-value judgments are the result of heuristics, however, they might be explained by the effect of context on those heuristics, rather than semantic context-sensitivity. Context can clearly affect the outcome of a heuristic-driven process. We have already noted the availability heuristic, through which probability and frequency are estimated by ease of recall. Features of context that do not affect probability or preva- lence can influence ease of recall. Gabrielcik and Fazio (1984), for example, presented participants with a questionnaire asking them to compare the frequency of two letters, e.g., “Do more words contain T or S?” Participants responded on a nine-point scale anchored to “Many more contain S” and “Many more contain T”. They found that participants primed with words containing the letter T gave higher estimates for the relative frequency of that letter. Gabrielcik and Fazio suggest that priming increases the availability of words begin- ning with T, which leads to higher estimates due to the availability heuristic. For our purposes, it doesn’t matter which heuristic is being used. Whatever the heuristic at play, its result is affected by features of context. There is an implied linguistic result here: subjects primed with the letter T are more likely to judge that ‘Slightly more words contain T’ is true, but the priming is semantically irrelevant to the truth of the sentence; exposure to certain words does not affect the frequency with which letters appear in English. Priming effects have been demonstrated for a variety of tasks and a great many of those will extend into linguistic results, showing how semantically irrelevant features of context can influence truth-value judgments. Indians: Indians eat beef.6 The truth value of this generic seems to vary between contexts, even when Indian beef consumption is held constant. Consider, for example: The truth value of this generic seems to vary between contexts, even when Indian beef consumption is held constant. Consider, for example: Context 1: Beef is popular all over the world. Europeans eat beef. They eat beef in South America. Indians eat beef. Context 1: Beef is popular all over the world. Europeans eat beef. They eat beef in South America. Indians eat beef. Context 2: Despite the cultural taboo prevalent in India, Indians eat beef, but it is less common than in Europe. Context 2: Despite the cultural taboo prevalent in India, Indians eat beef, but it is less common than in Europe. Indians seems false in Context 1 but same generic seems true in Context 2. Indians seems false in Context 1 but same generic seems true in Context 2 5 See Leslie (2008, 2007) and Sterken (2015b) for problems with some of the most influential accounts to date. 6 The example is inspired by one in Cohen (2004). 7 This analysis of adverbially-quantified generalisations was developed by Heim (1982), expanding on Lewis (1975). Kamp (1981/1984) independently developed a theory very similar to Heim’s. 3.1 Generics and context-sensitivity If I tell you that some contemporary philosophers are boring, you know how to tell whether I speak the truth: go look for a boring philosopher. If you find one, then I 123 196 M. Bowker have spoken the truth. If I tell you that all contemporary philosophers are boring, you can follow the same procedure: go check the philosophers. If each of them is boring then I have spoken truly. Other generalisations are more complex. If I tell you that ‘many’ contemporary philosophers are boring, for example, then you have to decide what counts as ‘many’. Once this issue is settled, however, you can follow the same procedure: go and check the philosophers. These generalisations all tell us something about the world, so we can go and check the world to figure out whether they have told us something true. What if I say simply that contemporary philosophers are boring? How would you verify that claim? The problem is vexed because this generalisation includes no quantifier (like ‘some’, ‘all’, or ‘many’) that tells you how far the generalisation extends. This is the mark of what are called generic generalisations or simply generics.4 It is usually assumed that a generic, like a quantified generalisation, tells us something that is true under certain conditions. Research has then focused on trying to identify these truth conditions. But, despite the prevalence of generics in natural language, there remains no satisfactory account of the conditions under which they are true or false.5 Sterken (2015a) points to a phenomenon that makes the truth conditional analysis of generics still more difficult: a single generic can seem to have different truth values in different contexts. Consider, for example: Indians: Indians eat beef.6 4 Here, I restrict discussion to bare plural generics. Interesting complications are raised by, say, definite and indefinite generics. See Greenberg (2007) for discussion of indefinite singulars. 6 The example is inspired by one in Cohen (2004). 7 Gen [Contemporary philosopher x] [Boring x]. Here, the variable-binding role is played by an unpronounced operator Gen. The question then is how Gen determines the truth conditions of the generic as a function of the explicitly pronounced words.8 Sterken argues that the context-sensitivity of generics is best explained by analysing Gen as an indexical quantifier. The paradigm indexicals are words like ‘I’, ‘here’, and ‘now’, which vary in reference depending on the context in which they are used. ‘I am hungry’, for example, is true when uttered by a hungry person but false when uttered by a non-hungry person because ‘I’ refers to different people in different contexts. Sterken suggests that the indexical quantifier Gen is context-sensitive in two ways. The context must set the quantificational force of the operator as well as what Sterken callsthe‘lexicalrestrictor’.‘Sometimes’forexamplehasexistentialforce(like‘some’) and is restricted to actual situations. In contrast, ‘Normally’ plausibly has universal force (like ‘all’) but is restricted to normal situations that may not be actual. Sterken’s account allows for the lexical restrictor and quantificational force of Gen to vary independently, creating complex interpretations that might not be easy to pin down using natural language quantifiers. g g g q As an example of variation in the lexical restrictor, consider: Post: Workers in Sorting Room 6 handle the mail from Antarctica.9 Suppose that there has never been any mail from Antarctica but that the mail system is set up in such a way that Sorting Room 6 would deal with mail from Antarctica, were it to arrive. Now consider the following two linguistic contexts: Context 1: What do they do in Sorting Room 6 do all day? Workers in Sorting Room 6 handle the mail from Antarctica. Context 1: What do they do in Sorting Room 6 do all day? Workers in Sorting Room 6 handle the mail from Antarctica. Context 2: The mail system is prepared for every eventuality. We have assigned people to handle mail from the unlikeliest of place. Workers in Sorting Room 6 handle the mail from Antarctica and workers in Sorting Room 7 handle the mail from Mars! In Context 1, the generic seems false. No mail has ever arrived from Antarctica, so that certainly isn’t keeping Sorting Room 6 busy. For the generic to be true in that context, there must be actual situations in which Sorting Room 6 handles mail from Antarctica. In Context 2, however, the generic seems true. 3.2 Sterken’s indexical account The standard syntactic analysis takes generics to be structurally identical to adverbially-quantified generalisations like ‘Contemporary philosophers are mostly boring,’ which are analysed as exhibiting the form: Mostly [Contemporary philosopher x] [Boring x] The ‘Mostly’ operator binds the variables, resulting in a sentence that is true just in case most variable assignments that satisfy ‘Contemporary philosopher’ also satisfy ‘Boring’.7 The generic ‘Contemporary philosophers are boring’ is then analysed as: 4 Here, I restrict discussion to bare plural generics. Interesting complications are raised by, say, definite and indefinite generics. See Greenberg (2007) for discussion of indefinite singulars. 5 See Leslie (2008, 2007) and Sterken (2015b) for problems with some of the most influential accounts to date. 12 123 197 Keeping context in mind… Gen [Contemporary philosopher x] [Boring x]. 8 The idea that generics involve an unpronounced binding operator is suggested by Heim (1982, pp. 127– 128). 9 This is a variation of an example from Krifka et al. (1995, p. 72). 8 The idea that generics involve an unpronounced binding operator is suggested by Heim (1982, pp. 127– 128). 9 This is a variation of an example from Krifka et al. (1995, p. 72). Dobermans: Dobermans have floppy ears. Dobermans: Dobermans have floppy ears. The ears of the Doberman are naturally floppy but are cut by breeders to give the pointy shape they are often associated with. Sterken notes that Dobermans is intuitively true when uttered in the context of evolutionary biology: Context 1: Some breeds of dogs have evolved to focus on their hearing. These breeds have pointy ears. Dobermans, however, mostly rely on their sense of smell, which is why Dobermans have floppy ears. This same generic is intuitively false when uttered in the context of a dog show in which all the Dobermans present have pointy ears, rendering the negation true: This same generic is intuitively false when uttered in the context of a dog show in which all the Dobermans present have pointy ears, rendering the negation true: Context 2: Welcome to this year’s meeting of the Westminster Kennel Club. Once again, we’ve got a great range of dog appearances. While Labradors and golden retrievers have floppy ears, Dobermans do not. Dobermans have pointy ears.10 Context 2: Welcome to this year’s meeting of the Westminster Kennel Club. Once again, we’ve got a great range of dog appearances. While Labradors and golden retrievers have floppy ears, Dobermans do not. Dobermans have pointy ears.10 Sterken argues for the context-sensitivity of generics through a process of elim- ination. So what are the alternatives to be eliminated? How else might we explain the difference between Contexts 1 and 2? We might think that one of the pronounced words is responsible. Perhaps, in Context 1, ‘Dobermans’ is interpreted to mean some- thing like ‘Dobermans that haven’t been tampered with by humans’, while in Context 2, ‘Dobermans’ is interpreted to mean something like ‘Dobermans at this dog show’. Or perhaps, in Context 1, ‘have floppy ears’ is interpreted to mean ‘have floppy ears at birth’, while in Context 2 it is interpreted to mean ‘have floppy ears at this dog show’. Alternatively, we might think that our interpretation of the pronounced words is stable but that differences in truth value can be explained through standard features of quantifiers such as domain restriction. Gen might only quantify over Dobermans at the dog show in Context 2, for example, but be unrestricted in Context 1. Gen [Contemporary philosopher x] [Boring x]. Although no mail has ever arrived from Antarctica, Sorting Room 6 would handle that mail, were it ever to arrive. In this case, the generalisation is not restricted to actual situations but quantifies over possible situations as well. As an example of variation in quantificational force, recall Indians. In Context 1, the level of beef-eating in India does not seem sufficient to make the generalisation true. In Context 2, however, the same level of beef consumption does seem sufficient, suggesting that the quantificational force has varied between these contexts. 123 3 12 198 M. Bowker 10 This phrasing is taken from Nickel (2016). 11 11 The wording of the test is a synthesis of Sterken’s three discussions of the test; two in Sterken (2015a) and one in Sterken (2015b). 3.3 Sterken’s argument for the indexical account Sterken’s primary example of generic context-sensitivity, drawn from Nickel (2008), is: Dobermans: Dobermans have floppy ears. Dobermans: Dobermans have floppy ears. Finally, we might think that the sentences itself is interpreted identically in both contexts but that some pragmatic phenomenon, like implicature or presupposition, leads to an apparent shift in truth value. Sterken intends to discredit these suggestions through her A-Quantifier Test. A-Quantifier Test: Check whether explicitly adverbially-quantified sentences vary their truth value across the same contexts as generics. If there is no difference in truth value, this is evidence that generics shift their truth values as a result of distinctive context-sensitivity in the semantics of the implicit quantifier Gen.11 Suppose that our interpretation of the subject or predicate were shifting between con- texts. In that case, Sterken suggests, we would expect the same shift to occur for adverbially-quantified variations of the generic (Sterken, 2015b, p. 2505), e.g.,: 123 12 199 Keeping context in mind… A-Dobermans: Typically/Generally/Normally Dobermans have floppy ears. It is true, for example, that typically Dobermans that have not been tampered with by humans have floppy ears but it is false (let us suppose) that typically Dobermans prepared for a dog show have floppy ears. According to Sterken, however, the quan- tified generalisations in A-Dobermans sound false in both contexts, showing that the interpretation of the subject term is not shifting in this way. Sterken uses the same form of argument to suggest that standard features of quan- tification like domain restriction are not responsible for the shift in apparent truth value. If they were, we would expect to see it mirrored in adverbially-quantified gen- eralisations (Sterken, 2015a, pp. 14–15). Likewise for pragmatic mechanisms like implicature or question-sensitivity.12 Sterken assumes that generics of the form ‘Ks are F’ are “close in meaning” to adverbially-quantified generalisations like ‘Typi- cally/Generally/Normally Ks are F’ (Sterken, 2015a, p. 10). If the generics shifted their truth values because of non-semantic factors like implicature, Sterken argues that those same factors would lead adverbially-quantified generalisations to shift their truth values relative to the same contexts. Through this process of elimination, Sterken concludes that the context-sensitivity of generics is due to the semantics of the implicit Gen operator and that this operator displaysadistinctiveformofcontext-sensitivitythatisn’tsharedbyexplicitquantifiers, opting ultimately for an analysis on which the operator is an indexical quantifier. I find Sterken’s judgment about A-Dobermans hard to accept. To my ear, the adverbially-quantified generalisation is false in the context of the dog show but true in the context of evolutionary biology. 12 Post, for example, is uttered in contexts with very different questions at issue. In Context 1, ‘what is it that people in Mail Room 6 do all day?’ and, in Context 2, something like ‘Who would sort mail from Antarctica?’ Dobermans: Dobermans have floppy ears. In the latter context, it seems natural to focus on what is evolutionarily normal, typical, or generally true, which excludes Dobermans that have suffered accidents, or have been tampered with by humans. Looking at the Dobermans that remain, they typically/generally/normally have floppy ears. I therefore find it hard to agree that the A-Quantifier Test, as applied to this case, provides evidence that Gen displays distinctive context-sensitivity. While I don’t share Sterken’s intuitions regarding A-Dobermans, that example is not necessary for her argument. Sterken’s claim is not that there are no contexts relative to which generics and their adverbially-quantified generalisation both shift their truth values. Adverbially-quantified generalisations can be context-sensitive for a variety of reasons (e.g., implicature, domain-restriction, and context-sensitivity of the subject or predicate). Her claim is that generics exhibit an additional context-sensitivity that is not shared by their adverbially-quantified counterparts. For this, it is sufficient that there are some contexts relative to which generics shift their truth values, while their adverbially-quantified counterparts do not. Indeed, Sterken presents other examples that I find more plausible. Take for example: Frenchmen: Frenchmen eat horse meat. 13 See Graham et al. (2004) for more on prelinguistic generalisation. 14 Hollander et al. (2002) suggest that this learning process takes place around four years of age. In their studies, three-year-olds responded to generics, existentials and generics in way that four-year-olds and adults respond only to generics. 15 This view of generics as defaults is supported by Gelman and Brandone (2010), Gelman (2010), and Hollander et al. (2009). Frenchmen: Frenchmen eat horse meat. This is plausibly true in a context that requires relatively few Frenchmen to eat horse meat, as when a contrast is set up with another nationality: 12 3 200 M. Bowker Context 1: Traditional French food differs from the traditional food of even their closest neighbours. Frenchmen eat horse meat, for example, whereas Englishmen find the idea incredible (my example). Sterken cites another context in which the generic is intuitively false, rendering the negation true: Context 2: A group of nutritionists is querying the unhealthy eating patterns of the French population …“Frenchmen eat croissants and baguettes. They don’t eat traditional food, like horse meat and grains” (Sterken, 2015a, pp. 314–5) My ear agrees with Sterken here. The adverbial variants sound false in both contexts: My ear agrees with Sterken here. The adverbial variants sound false in both contexts: A-Frenchmen: Typically/Generally/Normally Frenchmen eat horse meat. Frenchmen do not generally/typically/normally eat horse meat, even if it is a traditional food. While adverbially-quantified generalisations may exhibit some sensitivity to context, Sterken concludes that generics display an additional, distinctive context- sensitivity. Sterken suggests that this distinctive generic context-sensitivity is best explained by analysing Gen as an indexical quantifier. The following section will present an alternative source of the intuitive difference in truth value between generics and their adverbially-quantified counterparts. It is worth bearing in mind that this alternative source of context-sensitivity is not intended to completely undermine the A-Quantifier Test. Indeed, I will make use of the test in Sect. 5 to argue that the default generalisa- tion account should explain our truth-value judgments through heuristics, rather than linguistic context-sensitivity. The test also provides some evidence for the indexical analysis, in that it successfully eliminates some competing accounts. It does not, how- ever, eliminate all competitors and future applications of the test should bear in mind the full range of theories that can accommodate the data. 4.1 The default mechanism of generalisation Leslie (2007, 2008) argues that humans have a prelinguistic mechanism of general- isation that associates kinds with properties.13 With the acquisition of language, we learn alternative ways to generalise, such as those associated with the quantifiers ‘all’, ‘most’ and ‘some’.14 Leslie hypothesises that the prelinguistic mechanism remains our default mode of generalisation, however. Because generics include no explicit quantifier to override the default, they are interpreted through the prelinguistic mecha- nism.15 Leslie therefore takes the default mechanism of generalisation to explain both our dispositions to utter generics and our dispositions to assess them as true: 123 12 201 Keeping context in mind… If a speaker’s knowledge and experiences with members of a kind K leads her default mechanism to generalize the property of being F to that kind, then she will express this with the generic ‘Ks are F’. Similarly, her hearer would judge the utterance to be true if, given his knowledge and experience, his default mechanism would generalize the property of being F to the Ks. Leslie (2008, p. 22) Leslie (2008, p. 22) If the default mechanism of generalisation is responsible for the shift in generics’ apparent truth values, the stability of adverbially-quantified generalisations can be explained as a result of shifting away from the default mechanism. If the default mechanism of generalisation is responsible for the shift in generics’ apparent truth values, the stability of adverbially-quantified generalisations can be explained as a result of shifting away from the default mechanism. Leslie argues that the default mechanism is sensitive to several contingent psy- chological factors. Leslie suggests, for example, that we are disposed to agree with generics only when the counterinstances are negative, and that we agree with gener- ics more easily when they attribute striking or characteristic properties. These terms require some explanation. First, counterinstances to ‘Ks are F’ are negative when Ks that are not F do not display any psychologically salient alternative property. So, for example, counterin- stances to ‘Lions have manes’ are negative. The lions that lack manes don’t have any particularly interesting alternative property: they simply lack manes. Counterexam- ples to ‘Humans have dark hair’, however, are positive. The people without dark hair exhibit salient alternatives properties, such as having red hair. According to Leslie, our default mechanism of generalisation only associates a property F with a kind K when the counterinstances to ‘Ks are F’ are negative. 16 Our response to this case may in fact be overdetermined, given that male ducks constitute a positive counterinstance to ‘Ducks are female’ but a negative counterinstance to ‘Ducks lay eggs’. 17 Leslie doesn’t detail the required disposition but it faces significant problems. Note, for example, that we don’t agree with ‘Humans have HIV’. Perhaps some humans are immune to HIV (Ni et al., 2018) but the required mutation is not known to most people. Indeed, the susceptibility of mosquitoes to viruses, and the degree to which they are disposed to transmit viruses, vary both between and within mosquito species (Hardy, 1988). See Sterken (2015b, pp. 2500–3) for reasons to doubt that Leslie’s disposition requirement can be finessed to include only the intuitively true generics. 4.2 Heuristics for generics So, according to Leslie, we are disposed to agree with generics when our default mech- anism associates the right property with the relevant kind and our default mechanism does so only under the following conditions: Generic Truth: The counterinstances are negative,18 and: If F lies along a characteristic dimension for the Ks, then some Ks are F, unless K is an artifact or social kind, in which case F is the function or purpose of the kind K; If F is striking, then some Ks are F and the others are disposed to be F; Otherwise, almost all Ks are F. (Leslie, 2008, p. 43) Generic Truth: The counterinstances are negative,18 and: If F lies along a characteristic dimension for the Ks, then some Ks are F, unless K is an artifact or social kind, in which case F is the function or purpose of the kind K; K; If F is striking, then some Ks are F and the others are disposed to be F; Otherwise, almost all Ks are F. (Leslie, 2008, p. 43) If F is striking, then some Ks are F and the others are disposed to be F; Otherwise, almost all Ks are F. If F is striking, then some Ks are F and the others are disposed to be F; Otherwise, almost all Ks are F. (Leslie, 2008, p. 43) Otherwise, almost all Ks are F. (Leslie, 2008, p. 43) Leslie’s view of generics so far involves three key claims: 1. There is a default mechanism of generalisation that associates kinds with proper- ties. 2. The default mechanism associates kinds and properties under the conditions iden- tified in Generic Truth. 3. When the default mechanism associates property F with kind K, we are disposed to treat the generic ‘Ks are F’ as true. 3. When the default mechanism associates property F with kind K, we are disposed to treat the generic ‘Ks are F’ as true. These claims characterise the heuristics associated with generics. The first posits a cognitive mechanism, the second describes the working of this mechanism, and the third identifies this mechanism as the way that we decide whether generics are true or false. 4.1 The default mechanism of generalisation This explains why we are not disposed to agree with ‘Humans have dark hair’, despite the proportion of people with dark hair being far higher than the proportion of lions with manes. Second, characteristic properties. The default mechanism is disposed to generalise about kinds more easily along certain dimensions. We expect, for example, that mem- bers of the same animal species share a characteristic diet, mode of reproduction, and so on. Where a generic concerns one of these characteristic properties, we generalise based on very little evidence. We might agree with ‘Ducks lay eggs’, for example, based on experience of a single egg-laying duck. We don’t associate animal species with particular sexes, however, explaining why we are not disposed to agree with ‘Ducks are female’, despite there being more female ducks than egg-laying ducks.16 Characteristic properties vary depending on the kind of kind in question. For artifacts, their characteristic property is their function. Leslie suggests that the default mecha- nism always associates a kind of artifact with its function, regardless of whether there are any instances that fulfil this function, explaining why we might agree with ‘Cold fusion reactors produce power’ even if one has never been constructed. Finally, some properties are particularly striking. Leslie’s usual examples are prop- erties that pose a danger to humans. Where a property is particularly striking, Leslie suggests that our default mechanism associates it with a kind so long as some members of the kind have the property and all members of the kind are disposed to have it. This is intended to explain why we agree with ‘Mosquitoes carry West Nile virus’, despite the fact that very few mosquitoes are in fact carriers, but don’t agree with ‘Animals 123 202 M. Bowker carry West Nile virus’. Leslie (2008, p. 41) assumes that all mosquitoes are disposed to carry West Nile virus but, of course, not all animals are.17 carry West Nile virus’. Leslie (2008, p. 41) assumes that all mosquitoes are disposed to carry West Nile virus but, of course, not all animals are.17 18 Note that this can be read as a generic, albeit in definite rather than bare plural form. It isn’t entirely clear, therefore, exactly what proportion of counterinstances must be negative, according to this condition. If some small number of lions had spikes in place of manes, would we judge ‘Lions have manes’ to be true? 4.2 Heuristics for generics If these heuristics are responsible for the context-sensitivity of our judgments about the truth of generics like Frenchmen, but adverbially-quantified sentences like those in A-Frenchmen are interpreted through different heuristics, then we have an explanation of our diverging truth-value judgments that doesn’t posit semantic context- sensitivity. y To these three claims, Leslie adds a claim about the truth conditions of generics: 4. The truth conditions of generics are given by the conditions under which the default mechanism associates kinds and properties. 4. The truth conditions of generics are given by the conditions under which the default mechanism associates kinds and properties. As Leslie (2008, p. 43) puts it, “Since this mechanism is responsible for our under- standing of generics, providing an account of this mechanism has also allowed us to 12 123 203 Keeping context in mind… understand the circumstances in which generics are true or false.” Sterken (2015b, p. 2494) describes Leslie’s account as assuming that the “primitive cognitive mech- anism of generalisation has certain accuracy conditions” and records in a footnote that a reviewer suggested “a better interpretation is that certain conditions make the primitive mechanism activate.” Here, Sterken draws our attention to the application conditions of the theory, while the reviewer draws our attention to the heuristics. In what follows, however, I will focus on Leslie’s theory of heuristics, rather than her theory of truth conditions. Johnston and Leslie caution against drawing conclusions about application conditions from speakers’ intuitions about correct usage. As they put it, “the method of appealing to our judgments as to whether we should apply or withhold a term in a variety of imaginary cases is obviously a way of manifesting our criteria or ways of telling whether the term applies. It is not obviously a way of manifesting our ‘implicit grasp’ of the application conditions of terms” (Johnston and Leslie, 2012, pp. 135–6).19 That is, intuitions about cases might be a good way of revealing the heuristics by which we judge whether a term applies but we cannot infer the application conditions of a term from these heuristics. Leslie’s inference from truth-value intuitions to truth-conditions is not obviously more acceptable than the inference that Johnston and Leslie (2012, pp. 134–6) identify as “a bad verificationist error”: identifying the application conditions of ‘dog’ with the heuristics that we use to judge whether something is a dog. 4.2 Heuristics for generics Those heuristics might work well enough in ordinary circumstances but fail when we encounter “A coiffed squirrel …made to look like a chihuahua”. Likewise, the heuristics that we use to judge whether a generic is true or false might work well enough in ordinary circumstances, but may also be prone to error, leading us to mistakenly treat false generics as true, or vice versa. None of this is to argue that Leslie’s truth conditional theory is incorrect. For the purposes of this paper, however, it is important to separate Leslie’s theory of heuristics from her theory of truth conditions. The following section will set the truth conditional aspect of Leslie’s theory to one side and consider whether Sterken’s context-sensitivity data might be explained entirely by Leslie’s heuristics. 19 See also Williamson (2020, p. 26). 5 An alternative to context-sensitivity Consider the following generic, uttered by a worker at Store A, which sells expensive coffee, many cheap second-hand books, and a few expensive rare editions, in response to a customer who asks what they can buy for two dollars: Books: Books are cheap. Books: Books are cheap. Suppose the worker goes into another store, Store B, on their day off. Store B sells cheap coffee and rare, highly-priced books. They ask what they can buy with limited funds and are told Books. ‘That’s not true!’, our protagonist replies, and they leave the store in a state of incredulous shock. How can Leslie’s view explain the behaviour of our protagonist? The first utterance supposedly shows that our protagonist’s default mechanism of generalisation asso- 12 123 3 204 M. Bowker ciates the kind books with the property being cheap. If they associate this kind and property, however, they should be willing to accept the utterance of the assistant in Store B. It seems, therefore, that Leslie is committed to the view that our protagonist both does and does not associate books both with the property of being cheap. How can we make sense of this? Suppose that ‘Books’ amounts to something like ‘books in this store.’ Books would then express different default generalisations in each context. In Store A, it would express an association between the kind books in Store A and the property being cheap. As our protagonist’s default mechanism of generalisation associates this kind with the property, they would judge it true. In Store B, the generic would express an association between the kind books in Store B and the property being cheap. As our protagonist’s default mechanism of generalisation does not associate this different kind with the property, they would judge it false. Alternatively, the predicate could be context-sensitive, amounting to something like ‘cheap in this store’. Again, Books would then express different default generalisations in different contexts. In Store A, it would express an association between the kind books and the property being cheap in Store A. As our protagonist’s default mechanism of generalisation associates the kind with this property, they would judge it true. In Store B, the generic expresses an association between the kind books and the property being cheap in Store B. As our protagonist’s default mechanism of generalisation does not associate the kind with this different property, they would judge it false. Either way, we would have an explanation of why our protagonist is willing to utter Books to their customers in Store A, but not willing to accept its utterance by the assistant in Store B. Books: Books are cheap. According to Sterken’s A-Quantifier Test, if the subject or the predicate is respon- sible for Books changing its apparent truth value between contexts, then we should expect the same difference to hold for its adverbially-quantified variants: A-Books: Typically/Generally/Normally books are cheap. A-Books: Typically/Generally/Normally books are cheap. It seems to me that these adverbially-quantified generalisations do shift their truth value between contexts. In Store A, books are typically cheap and A-Books is true. In Store B, books are not typically cheap and A-Books is false. So we can explain why Books and A-Books change their apparent truth values across contexts by posit- ing context-sensitivity in either the subject or predicate. The same explanation cannot easily be extended to Frenchmen, however. If context-sensitivity of either the sub- ject or predicate were responsible for Frenchmen changing its apparent truth value between contexts, we would also expect A-Frenchmen to change its apparent truth value across contexts but it does not. An alternative explanation is available, however: the kinds and properties associated by the default mechanism of generalisation change between contexts. As applied to Books, when in Store A, the worker associates the kind books with the property being cheap but this changes when they move to Store B. Changing their associations allows them to navigate two different environments. The worker follows exactly the same heuristic in both contexts, relying on their default mechanism of generalisation, but their truth-value judgments change as a function of changes in their associations. Likewise, in Context 1, the kind Frenchmen is associated with the property of eating horse meat. If the associations of the default mechanism vary between contexts, we 123 123 12 Keeping context in mind… 205 can explain different truth-value judgments in different contexts without positing any semantic context-sensitivity. In both contexts, the subject picks out the same kind and the predicate picks out the same property but that property is cognitively associated with that kind in some contexts and not in others. This leads to differing truth-value judgments because our heuristics for assessing truth and falsity operate based on these contextual associations. can explain different truth-value judgments in different contexts without positing any semantic context-sensitivity. In both contexts, the subject picks out the same kind and the predicate picks out the same property but that property is cognitively associated with that kind in some contexts and not in others. This leads to differing truth-value judgments because our heuristics for assessing truth and falsity operate based on these contextual associations. Leslie’s account already has some flexibility with respect to the conditions under which kinds and properties are associated.20 Generic Truth tells us, for example, that when a property F lies along a characteristic dimension for some kind K, the property is associated with the kind so long as some Ks are F. It isn’t clear, however, that these characteristic dimensions are always independent of context. Context 1 sets up a context in which we associate nationalities with traditional cuisine. In that context, traditional cuisine is thought of as a characteristic dimension and the kind Frenchmen is associated with the property eating horse meat. In the second context, however, traditional cuisine is explicitly rejected as a basis for generalisation and isn’t considered a characteristic dimension. Instead, associations are made based on the prevalent eating habits of the population. The view that generics are affected by context has the resources to account for the results of the A-Quantifier Test. To account for the results, we need two things. First, we need to explain how judgments about truth-values of generics could vary between contexts. Second, we need to explain how judgments about the truth-values of their adverbial variants could remain stable across the same contexts. The first is secured by the possibility of contextual variation in associations. The second is secured by the notion of a default mechanism. 21 That is not to say, however, that this function is always fulfilled. Leslie et al. (2011), for example, observed the “generic overgeneralization effect” in which English speakers agreed to seemingly false sentences like ‘All ducks lay eggs’. They take this as evidence in favour of the default generalisation hypothesis. If quantifiers aim to override the generic default, then we should expect failures in which speakers nevertheless interpret quantified statements as generics. 22 I actually take the generalisation to be slightly more complicated. What is it, after all, for an individual to eat horse meat and so count as conforming to the generalisation? Is it for them to have eaten horse meat once in their lives? Regularly? How regularly? Is a bite on special occasions, barring exceptional circumstances, sufficient? The inclusion of ‘generally’ does not eliminate all elements of genericity from the sentence. My suspicion is that I judge the sentence false because I assume there are sufficiently many Frenchmen who I would not characterise as generic horse-meat-eaters. 20 Thanks to an anonymous reviewer for this suggestion. I actually take the generalisation to be slightly more complicated. What is it, after all, for an individual to eat horse meat and so count as conforming to the generalisation? Is it for them to have eaten horse meat once in their lives? Regularly? How regularly? Is a bite on special occasions, barring exceptional circumstances, sufficient? The inclusion of ‘generally’ does not eliminate all elements of genericity from the sentence. My suspicion is that I judge the sentence false because I assume there are sufficiently many Frenchmen who I would not characterise as generic horse-meat-eaters. 123 According to Leslie’s theory, the very purpose of quantifiers is to shift interpretation away from the default mechanism of generalisation.21 When assessing ‘Generally, Frenchmen eat horse meat’, for example, the term ‘generally’ functions to shift interpretation from the prelinguistic default mechanism to whatever mode of generalisation we have learned to associate with ‘generally’. Even if I currently associate the property eating horse meat with the kind Frenchmen, I can judge ‘Generally, Frenchmen eat horse meat’ to be false because I take ‘generally’ to require a majority of cases to conform to the generalisation and I do not believe that most Frenchmen eat horse meat.22 The hypothesis that associations vary between context is not unrealistic. Contex- tual effects have been found for many conceptual processing tasks, including word recall (Barclay et al., 1974; Anderson & Ortony, 1975; Greenspan, 1986; Zeelenberg, I actually take the generalisation to be slightly more complicated. What is it, after all, for an individual to eat horse meat and so count as conforming to the generalisation? Is it for them to have eaten horse meat once in their lives? Regularly? How regularly? Is a bite on special occasions, barring exceptional circumstances, sufficient? The inclusion of ‘generally’ does not eliminate all elements of genericity from the sentence. My suspicion is that I judge the sentence false because I assume there are sufficiently many Frenchmen who I would not characterise as generic horse-meat-eaters. 12 3 3 3 206 M. Bowker 2005), object identification (Biederman et al., 1982; Palmer, 1975; Boyce et al., 1989; Murphy & Wisniewski, 1989), property generation (Tabossi and Johnson-Laird, 1980; Barsalou, 1993, p.32), typicality judgments (Barsalou, 1993, p. 33; Roth and Shoben, 1983), lexical decision (Greenspan, 1986; Kellas et al., 1991), reading times (Tabossi & Johnson-Laird, 1980), association judgements (Greenspan, 1986), property veri- fication (Barsalou, 1982), generic agreement (Tabossi & Johnson-Laird, 1980) and category judgments (Tobia et al., 2020; Machery and Seppälä, 2011; Zarl and Fum, 2014).23 Machery and Seppälä (2011), for example, found that many subjects were willing to assent to seemingly incompatible category judgments e.g., ‘tomatoes are vegetables’ and ‘tomatoes are not vegetables’ when both were qualified with ‘In a sense’. This may be because they are aware that there are some contexts (e.g., biology) in which they would not associate the kind tomatoes with the property of being a vegetable but there are other contexts (e.g., a grocery store) in which they would. 23 See the literature review in Yeh and Barsalou (2006) for further examples of these effects. 24 We can get things wrong, even when they are encoded within our brains. We might fail to retrieve this information accurately, for example, due to inattentiveness or high cognitive load. Given that these disagreements can arise for competent speakers in ideal conditions, however, I claim that only the use of heuristics could explain them. 23 See the literature review in Yeh and Barsalou (2006) for further examples of these effects. 24 123 This interpreta- tion is strengthened by experiments conducted by Zarl and Fum (2014), who found that participants are more willing to accept both statements when they are presented individually, rather than in the same context, and that more explicit sentential context (e.g., ‘In a technical sense…’) reduced the number of incompatible judgments. The hypothesis that contextually-varying cognitive associations determine our contextually-varying truth-value judgments about generics is speculative, requiring empirical support. Regardless, our default position should be that contextual differ- ences in truth-value judgments about generics result from some effect of context on heuristics, not that generics are semantically context-sensitive. Competent speakers can disagree about the truth-values of generics. As noted in Sect. 3.3, for example, Sterken and I disagree about the truth-value of Dobermans in some contexts. Parties to such a disagreement cannot both be right. Given that competent speakers can so easily be wrong, they must be relying on fallible heuristics, rather than, say, seman- tic knowledge encoded within their brains.24 If that is the case, then differences in truth-value judgments must result from contextual effects on those heuristics and it is a further claim, requiring further argument, that these heuristics identify the actual truth-values of the generics and that generics are therefore context-sensitive. Indeed, any substantial context-sensitivity argument will be about expressions for which dis- agreements can arise, suggesting that our truth-value judgments must be the result of fallible heuristics. The burden of proof is therefore on the author of any such argu- ment to show that these heuristics deliver the correct result in the cases central to their argument. 12 3 207 Keeping context in mind… 6 Conclusion Arguments for context-sensitivity usually begin with data about our judgments of truth and falsity in different contexts. Often, this data is suppressed in that authors talk directly about the truth values, but these assertions must be rooted in their judgments about truth values. This paper has presented a way of explaining these judgments without positing context-sensitivity. Judgments about the truth and falsity of sentences depend directly on the heuristics that we use to judge truth and falsity. These heuristics may lead to different truth-value judgments in different contexts. y j g This explanation has been illustrated through Sterken’s argument for her indexical analysis of generics. Leslie has suggested that generics might be interpreted through heuristics that differ from those we use to interpret adverbially-quantified generalisa- tions. Contextual variations might therefore affect our the former but not the latter. While this is not intended to completely undermine Sterken’s A-Quantifier Test, it does limit the degree to which that test provides support for Sterken’s indexical analysis of generics. My hope is that future authors will pay more attention to the role of heuristics in future discussions of context-sensitivity. The key point is this: Intuitions about truth and falsity are evidence most directly for the heuristics that we use to judge truth and falsity. Where some contextual difference affects the output of these heuristics, our truth-value judgments can change without any corresponding change in truth conditions. In future, authors of context-sensitivity arguments should consider this possibility and either provide reason to think that the relevant truth-value judgments are not the result of fallible heuristics, or reason to think that these heuristics are accurate in the cases central to their argument. Acknowledgements My thanks to Jake Quilty-Dunn, Andrei Cimpian, and Lawrence Barsalou for helpful literature recommendations. Thanks also to Jiwon Kim, members of NYU’s Cognitive Development Lab, Mirela Fus, Ravi Thakral, Oliver Lemeire, anonymous reviewers, and the editor for helpful comments and suggestions. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 101063848. Funding Open access funding provided by Lund University. Funding was received from the European Research Council (Grant No. 101063848). Declarations Conflicting interests No conflicting interests to disclose. OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,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/. 123 M. 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Towards the identification of carriers of the unidentified infrared (UIR) bands in novae
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Towards the identification of carriers of the unidentified infrared (UIR) bands in novae Izumi Endo1 , Itsuki Sakon1, Takashi Onaka2,1, Yuki Kimura3, Seiji Kimura4, Setsuko Wada4, L. Andrew Helton5, Ryan M. Lau6, Yoko Kebukawa7, Yasuji Muramatsu8, Nanako O. Ogawa9, Naohiko Ohkouchi9, Masato Nakamura10 and Sun Kwok11 1University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan 2Meisei University, 2-1-1 Hodokubo, Hino, Tokyo 191-8506, Japan 3Institute of Low Temperature Science, Hokkaido University, Kita-19, Nishi-8, Kita-ku, Sapporo 060-0819, Japan Izumi Endo1 , Itsuki Sakon1, Takashi Onaka2,1, Yuki Kimura3, Seiji Kimura4, Setsuko Wada4, L. Andrew Helton5, Ryan M. Lau6, Yoko Kebukawa7, Yasuji Muramatsu8, Nanako O. Ogawa9, Naohiko Ohkouchi9, Masato Nakamura10 and Sun Kwok11 1University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan 2Meisei University, 2-1-1 Hodokubo, Hino, Tokyo 191-8506, Japan 3Institute of Low Temperature Science Hokkaido University Kita 19 Nishi 8 Kita ku Izumi Endo1 , Itsuki Sakon1, Takashi Onaka2,1, Yuki Kimura3, Seiji Kimura4, Setsuko Wada4, L. Andrew Helton5, Ryan M. Lau6, Yoko Kebukawa7, Yasuji Muramatsu8, Nanako O. Ogawa9, Naohiko Ohkouchi9, Masato Nakamura10 and Sun Kwok11 1University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan 2Meisei University, 2-1-1 Hodokubo, Hino, Tokyo 191-8506, Japan 3Institute of Low Temperature Science, Hokkaido University, Kita-19, Nishi-8, Kita-ku Sapporo 060-0819, Japan 4The University of Electro-Communications, 1-5-1, Chofugaoka, Chofu, Tokyo 182-8585, Japan 5SOFIA Science Center/NASA Ames Research Center, MS 211-1, P.O. Box 1, Moffett Field, CA 94035-0001, USA 6Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency, 3-1-1 Yoshinodai, Sagamihara, 229-8510, Japan 7Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Ja 8University of Hyogo, 2167 Shosha, Himeji-shi, Hyogo, 671-2280, Japan 9Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-Cho, Yokosuka, 237-0061, Japan 10Nihon University, Narashinodai, Funabashi 274-8501, Japan 11The University of British Columbia, 2329 West Mall Vancouver, V6T 1Z4, Canada Abstract. The unidentified infrared (UIR) bands, whose carriers are thought to be organics, have been widely observed in various astrophysical environments. However, our knowledge of the detailed chemical composition and formation process of the carriers is still limited. We have synthesized laboratory organics named Quenched Nitrogen-included Carbonaceous Composite (QNCC) by quenching plasma produced from nitrogen gas and hydrocarbon solids. Infrared and X-ray analyses of QNCC showed that infrared properties of QNCC well reproduce the UIR bands observed in novae and amine structures contained in QNCC play an important role in the origin of the broad 8 μm feature, which characterizes the UIR bands in novae. The Origin of Outflows in Evolved Stars Proceedings IAU Symposium No. 366, 2022 L. Decin, A. Zijlstra & C. Gielen, eds. doi:10.1017/S1743921322000382 Towards the identification of carriers of the unidentified infrared (UIR) bands in novae QNCC is at present the best laboratory analog of organic dust formed around dusty classical novae, which carries the UIR bands in novae via thermal emission process (Endo et al. 2021). Keywords. (stars:) novae, cataclysmic variables, ISM: lines and bands, infrared: ISM c ⃝The Author(s), 2022. Published by Cambridge University Press on behalf of International Astronomical Union. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. c ⃝The Author(s), 2022. Published by Cambridge University Press on behalf of International Astronomical Union. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.1017/S1743921322000382 Published online by Cambridge University Press https://doi.org/10.1017/S1743921322000382 Published online by Cambridge University Press 1. Introduction The breakthrough brought by the space-based infrared observations, unaffected by atmospheric absorption, made it clear that organics ubiquitously exist throughout the universe from the solar system to distant galaxies. The unidentified infrared (UIR) bands, which consist of a series of emission features arising from aromatic and/or aliphatic C-C and C-H bonds (Allmandola et al. 1989; Tielens 2008), have been widely observed in c ⃝The Author(s), 2022. Published by Cambridge University Press on behalf of International Astronomical Union. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. 328 I. Endo et al. various astrophysical environments (e.g., Tokunaga 1997). The organics which carry the UIR bands must, therefore, be a major constituent of the circumstellar and interstellar medium of the Galaxy. However, our knowledge of their exact chemical nature is still quite limited. Polycyclic aromatic hydrocarbons (PAHs; Allmandola et al. 1989) and PAH-like molec- ular species have been commonly used to interpret the properties of the UIR bands observed in the interstellar medium. However, no individual PAH have successfully repro- duced the observed properties of UIR bands. Other than PAHs, bulk carbonaceous grains have also been proposed as possible candidates of the carriers of the UIR bands observed in circumstellar environment. Quenched Carbonaceous Composite (Sakata et al. 1984, 1987) and Hydrogenated Amorphous Carbons (HAC; Jones et al. 1990) are laboratory analogues, while coal (Guillois et al. 1996) and kerogen (Papoular 2001) are examples of organic solids in the terrestrial environments. The mixed aromatic-aliphatic organics nanoparticles (MAON; Kwok & Zhang 2011), which contain heteroatoms including nitro- gen in addition to hydrocarbon models have recently been suggested as a more realistic interpretation and the challenges to understand the nature of the carries of the UIR bands are still ongoing. Classical novae, the final evolutional stage of binary systems harboring a white dwarf, eject heavy elements through outburst events and a part of them exhibit signs of dust formation. Dusty classical novae offer a valuable opportunity to investigate the formation process of dust and organics in space thanks to their higher occurrence. Past studies have shown that the UIR bands observed around novae are characterized by the presence of a broad 8 μm feature (Helton et al. 2011; Sakon et al. 2016). https://doi.org/10.1017/S1743921322000382 Published online by Cambridge University Press 2. Quenched Nitrogen-included Carbonaceous Composite (QNCC); a laboratory analogue of organics around novae We have synthesized a laboratory organic, Quenched Nitrogen-included Carbonaceous Composite (QNCC), which can well reproduce the characteristics of the UIR bands observed in dusty classical novae. Figure 1 shows comparison of the infrared absorp- tion spectrum of QNCC with the UIR bands observed in the nova V2361 Cyg on Day 116 after the outburst. The overall spectral properties of the spectrum of QNCC, espe- cially the profiles of the broad 8 μm feature, are in good agreement with the UIR bands observed in V2361 Cyg. QNCC is synthesized by quenching plasma generated from nitro- gen gas and hydrocarbon solids, including filmy QCC (Sakata et al. 1990) and PAHs, by a 2.45 GHz microwave discharge. Any QNCCs produced from different hydrocarbon solids (e.g., coronene, anthracene) consistently exhibit a characteristic broad feature at around 8 μm, as well as other major features at 3.3, 6.3, and 11.4 μm. The synthesis method of QNCC qualitatively mimic a possible formation process of organics around novae, where a nitrogen-rich nova wind (e.g., N/N⊙∼201 for V84s Cen; Gehrz et al. 1998, N/N⊙∼219 for V2361 Cyg; Munari et al. 2008) interacts with pre-existing carbonaceous dust in the circumstellar medium. We performed X-ray Absorption Near-edge Structure (XANES) analyses of QNCC with the measurement station for X-ray absorption spectroscopy installed in the beam- line BL10 at the NewSUBARU synchrotron radiation facility at the University of Hyogo (Kuki et al. 2015). The result of the XANES analyses show the presence of amine struc- tures, which we conclude to be responsible for the broad 8 μm UIR band observed in novae. The N/C ratio of QNCC is 3-5% based on the measurement using the modified elemental analyzer/isotope ratio mass spectrometer (EA/IRMS; Ogawa et al. 2010). We conclude that QNCC is at present the best laboratory analog of organic dust formed in circumstellar environment of dusty classical novae (Endo et al. 2021). https://doi.org/10.1017/S1743921322000382 Published online by Cambridge University Press Carriers of the UIR bands in novae 329 Carriers of the U R bands in novae 3 9 Figure 1. Comparison of the absorption spectrum of QNCC with the UIR bands observed in the classical nova V2361 Cyg on Day 116 after the outburst. Figure 1. Comparison of the absorption spectrum of QNCC with the UIR bands observed in the classical nova V2361 Cyg on Day 116 after the outburst. 3. Can bulk organic dust carry the UIR bands in novae? Two major emission mechanisms have been considered to explain the UIR bands and their carriers. Gas-phase PAH molecules emit the UIR bands through the process of UV- pumped IR fluorescence (Leger & Puget 1984; Allmandola et al. 1985). On the other hand, bulk organic dust in the vicinity of a heating source can reach the energy equilibrium and carry the UIR bands via a thermal emission process (Dwek et al. 1980; Duley & Williams 2011). In the circumstellar environment of novae, bulk organic dust is likely to carry the UIR bands via a thermal emission process because gas-phase PAH molecules are not supposed to survive such harsh conditions around the white dwarf in a nova remnant (Evans & Rawlings 1994). Additionally, Kwok & Zhang (2011) indicated that the carriers of the UIR bands observed in novae V2361 Cyg and V2362 Cyg are expected to be com- plex organic dust that contains a mixture of miscellaneous aliphatic branches attached to the newly formed ring clusters rather than gas-phase pure PAH molecules. In order to further clarify whether bulk organic dust can carry the UIR bands in novae, we examined the results of multi-epoch observations of V2361 Cyg with the Infrared Spectrograph (IRS) on Spitzer Space Telescope. The blackbody temperatures (Td) of the continuum emission of V2361 Cyg are estimated as 660 K on τ = 102 days, 610 K on τ = 116, and 380 K on τ = 251 after the outburst according to Helton et al. (2011). Based on the very simple optically-thin dust emission model, the temporal evolution of the dust temperatures Td(τ) is determined by the energy balance between the input energy that the dust particle receives and the output energy that the dust particle radiates: πa2 ¯Q(a, T∗)L/4πr2 = 4πa2 ¯Q(a, Td)σT 4 d , (3.4) (3.4) where a is a grain size of spherical dust, T∗is the effective temperature of the heating source, L is the effective luminosity of the heating sources, σ is the Plank constant, ¯Q(a, T∗) and ¯Q(a, Td) are the blackbody mean of absorption coefficient at T = T∗and T = Td, respectively, and r is the distance between the heating source and dust. Assuming the constant expansion velocity of dust as v, r can be given by r = vτ. 3. Can bulk organic dust carry the UIR bands in novae? In the case of a blackbody, the temporal evolution of the dust temperature as a function of the epoch from the nova outburst, τ, is given by Td ∝τ −0.5 (3.5) Td ∝τ −0.5 (3.5) https://doi.org/10.1017/S1743921322000382 Published online by Cambridge University Press I. Endo et al. I. Endo et al. 330 Table 1. The temporal evolution of the height of 8 μm feature in V2361 Cyg. Table 1. The temporal evolution of the height of 8 μm feature in V2361 Cyg. τ T d Height of the 8 μm feature Evolution of the 8 μm feature relative to Day 102 [days] [K] [Jy] Observed Thermal equilibrium model 102 660 1.8 100 % 100 % 116 610 1.6 88.9 % 78.9 % 251 380 0.19 10.6 % 12.6 % Figure 2. The evolution of the blackbody temperature of the continuum emission in V2361 Cyg as a function of the epoch τ after the outburst. T d Height of the 8 μm feature Evolution of the 8 μm feature relative to Day 102 ays] [K] [Jy] Observed Thermal equilibrium model Figure 2. The evolution of the blackbody temperature of the continuum emission in V2361 Cyg as a function of the epoch τ after the outburst. As shown in Figure 2, the behavior of the blackbody temperatures of continuum emission observed at τ = 102, 116 and 251 days in V2361Cyg (Helton et al. 2011) roughly agree with this very simple model. As shown in Figure 2, the behavior of the blackbody temperatures of continuum emission observed at τ = 102, 116 and 251 days in V2361Cyg (Helton et al. 2011) roughly agree with this very simple model. The height of the 8μm feature, which characterize the UIR bands in novae, measured at τ = 102, 116 and 251 days are quoted from Helton et al. (2011) and are summa- rized in Table 1. If the thermal equilibrium model can be applied to the UIR bands in V2361 Cyg, the evolution of the height of the 8 μm feature should be proportional to [exp{hc/λkTd} −1]−1 , where λ = 8 μm, and h, c, and k are the Plank constant, light speed, and the Boltzman constant, respectively. The observed values of the 8 μm feature height and those of the thermal equilibrium model relative to that at τ = 102 are sum- marized in Table 1. 3. Can bulk organic dust carry the UIR bands in novae? The observed height of the 8 μm feature at τ = 251 drops to 10.6% of that at τ = 102. The thermal equilibrium model indicates that the value of 8 μm feature height at τ = 251 drops to 12.6% of that at τ = 102. This is roughly consistent with the observed value (i.e., 10.6%). https://doi.org/10.1017/S1743921322000382 Published online by Cambridge University Press 4. Conclusion We have synthesized QNCC by the quenched condensation of plasma generated from nitrogen gas and hydrocarbon solids. We conclude that QNCC is at present the best laboratory analog of organic dust formed around dusty classical novae, which carries the UIR bands via a thermal emission process. In the present discussion, we neglect the changing in the luminosity of the heating source (L) as well as the possible destruction of the carriers of the emission. Although long-term monitoring observations are needed to clearly conclude which emission process is dominant, at least, our interpretation that the 8 μm feature is emitted via the thermal emission process consistently explain the observed temporal variations of the 8 μm feature strength well. https://doi.org/10.1017/S1743921322000382 Published online by Cambridge University Press 331 Carriers of the UIR bands in novae https://doi.org/10.1017/S1743921322000382 Published online by Cambridge University Press References Allmandola, L. J., Tielens, A. G. G. M., & Barker, J. R. 1985, ApJL, 290, 25 Allmandola, L. J., Tielens, A. G. G. M., & Barker, J. R. 1989, ApJS, 71, 733 Duley, W. W., & Williams, D. A. 2011, ApJL, 737, L44 Dwek, E., Sellgren, K., Soifer, B. T., & Werner, M. 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Circulating Levels of Interferon Regulatory Factor-5 Associates With Subgroups of Systemic Lupus Erythematosus Patients
Frontiers in immunology
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ORIGINAL RESEARCH published: 17 May 2019 doi: 10.3389/fimmu.2019.01029 Edited by: Reviewed by: Carla Marie Cuda, Northwestern University, United States Betsy J. Barnes, Feinstein Institute for Medical Research, United States Reviewed by: Carla Marie Cuda, Northwestern University, United States Betsy J. Barnes, Feinstein Institute for Medical Research, United States Reviewed by: Carla Marie Cuda, Northwestern University, United States Betsy J. Barnes, Feinstein Institute for Medical Research, United States Reviewed by: Carla Marie Cuda, Northwestern University, United States Betsy J. Barnes, Feinstein Institute for Medical Research, United States *Correspondence: Elisabet Svenungsson elisabet.svenungsson@ki.se †These authors share first authorship ‡These authors share senior authorship 1 Division of Rheumatology, Department of Medicine Solna, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden, 2 SciLifeLab, Division of Affinity Proteomics, Department of Protein Science, KTH Royal Institute of Technology, Stockholm, Sweden, 3 Department of Medical Sciences, Akademiska Hospital, Uppsala University, Uppsala, Sweden, 4 Division of Physiological Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden, 5 Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden, 6 Department of Medical Sciences, Rheumatology, Uppsala University, Uppsala, Sweden, 7 Department of Medical Sciences, Clinical Chemistry, Uppsala University, Uppsala, Sweden, 8 Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, Uppsala, Sweden, 9 Science for Life Laboratory, Drug Discovery and Development & School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden *Correspondence: Elisabet Svenungsson elisabet.svenungsson@ki.se Specialty section: This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann–Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. Edited by: David Stephen Pisetsky, Duke University, United States Edited by: David Stephen Pisetsky, Duke University, United States Edited by: David Stephen Pisetsky, Duke University, United States Edited by: We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (padjusted = 3 × 10−9, 3 × 10−6, and 5 × 10−6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann–Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (padjusted = 3 × 10−9, 3 × 10−6, and 5 × 10−6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be Received: 14 December 2018 Accepted: 23 April 2019 Published: 17 May 2019 Helena Idborg 1†, Arash Zandian 2†, Elena Ossipova 1, Edvard Wigren 1, Charlotta Preger 1, Fariborz Mobarrez 1,3, Antonio Checa 4, Azita Sohrabian 5, Pascal Pucholt 6, Johanna K. Sandling 6, Cátia Fernandes-Cerqueira 1, Johan Rönnelid 5, Vilija Oke 1, Giorgia Grosso 1, Marika Kvarnström 1, Anders Larsson 7, Craig E. Wheelock 4, Ann-Christine Syvänen 8, Lars Rönnblom 6, Kim Kultima 7, Helena Persson 9, Susanne Gräslund 1, Iva Gunnarsson 1, Peter Nilsson 2, Elisabet Svenungsson 1*‡ and Per-Johan Jakobsson 1‡ INTRODUCTION division of SLE patients. In addition, experimental validation of biomarker candidates discriminating between SLE and control was performed. Our main focus was to identify molecular subgroups in SLE since these, despite similar clinical phenotypes, may benefit from different treatment perspectives. Systemic Lupus Erythematosus (SLE) is a heterogeneous systemic autoimmune disorder with a plethora of clinical manifestations. Clinical and immunological criteria, defined by the American College of Rheumatology (ACR) (1), are used to classify the disease for research purposes, but reliable diagnostic biomarkers are lacking. The diversity of the disease is a great obstacle and might reflect differences in pathogenesis between different subgroups. Several recent reviews highlight the importance of defining subgroups of SLE to better treat patients with tailored medicine, and in order to increase efficacy in clinical trials (2–5). Accordingly, there is a great need for exploring subgrouping and novel diagnostic biomarkers in SLE. Citation: Citation: Idborg H, Zandian A, Ossipova E, Wigren E, Preger C, Mobarrez F, Checa A, Sohrabian A, Pucholt P, Sandling JK, Fernandes-Cerqueira C, Rönnelid J, Oke V, Grosso G, Kvarnström M, Larsson A, Wheelock CE, Syvänen A-C, Rönnblom L, Kultima K, Persson H, Gräslund S, Gunnarsson I, Nilsson P, Svenungsson E and Jakobsson P-J (2019) Circulating Levels of Interferon Regulatory Factor-5 Associates With Subgroups of Systemic Lupus Erythematosus Patients. Front. Immunol. 10:1029. doi: 10.3389/fimmu.2019.01029 May 2019 | Volume 10 | Article 1029 Frontiers in Immunology | www.frontiersin.org 1 Idborg et al. SLE Subgroups Differ in IRF5 down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials. Keywords: Interferon regulating factor 5 (IRF5), antibody suspension bead arrays, subgroups, biomarker discovery, plasma proteomics, unsupervised clustering, hierarchical clustering, SLE - Systemic Lupus Erythematous Keywords: Interferon regulating factor 5 (IRF5), antibody suspension bead arrays, subgroups, biomarker discovery, plasma proteomics, unsupervised clustering, hierarchical clustering, SLE - Systemic Lupus Erythematous MATERIALS AND METHODS Plasma protein profiles were obtained for SLE patients and controls utilizing antibody suspension bead arrays for protein profiling. An overview of the study design can be found in Figure 1. Frontiers in Immunology | www.frontiersin.org Patient Cohort and Controls Few biomarkers have been implemented in clinical routine reflecting the difficulties of biomarker research in lupus (6). Screening of a large number of proteins (>50) but in a limited number (<50) of SLE patients have been performed to identify biomarkers in SLE (7–10). In this study we analyzed 281 proteins using a suspension bead affinity proteomics approach (11), in plasma samples from a total of 695 individuals comprising SLE and matched controls. Selection of proteins is crucial to obtain representative protein profiles. However, the current knowledge of protein functions is far from complete and transcription factors and other nuclear molecules could have unknown functions in the circulation or may, regardless of function, constitute novel biomarkers. The intra- and extracellular functions of a protein might be different and unconventional secretion is also possible (12). Therefore, both nuclear and cytoplasmic molecules are relevant to study in the circulation with the aim to identify potential biomarkers and possible pathogenic pathways. Fasting plasma samples were obtained from patients in the Karolinska SLE cohort consisting of 379 SLE patients and 316 population-based controls with matching age, gender and residential area. All SLE patients included in this cross-sectional study, were adults and diagnosed according to the ACR SLE criteria (1). Both patients and controls underwent a structured interview and physical examination as previously described (14). Clinical and serological data for the SLE patients are summarized in Table 1 and in previous work (13). Medication is reported in Supplementary Table S-1, and demographic data for the controls are shown in Supplementary Table S-2. Protein Profiling by Antibody Suspension Bead Arrays Bead Arrays A number of 281 proteins were selected as previously described (13), i.e., based on published data on suggested biomarkers in inflammation/SLE/myositis, microarray data comparing SLE and controls and an untargeted mass spectrometry-based proteomic analysis suggesting additional biomarker candidates. A customized set of 367 antibodies (Supplementary Table S-3) was utilized to target unique epitopes of these proteins in a screening experiment (Figure 1B) (13). The antibodies were selected from the Human Protein Atlas (HPA, www.proteinatlas. org) project and are affinity-purified polyclonal antibodies that In a previous study we presented protein profiles for two predefined SLE subgroups, delineated based exclusively on the autoantibody profiles, but also corresponding to clinical observations and experience (13). In the present study we used a different approach and performed unsupervised clustering of the obtained protein profiles to investigate an unprejudiced May 2019 | Volume 10 | Article 1029 2 Idborg et al. SLE Subgroups Differ in IRF5 FIGURE 1 | Overview of the experimental workflow. Plasma samples (A) were randomized in set 1 and set 2 for screening phase (B) followed by validation phase (C). Data were analyzed to investigate SLE subgroups (D) as well as comparing SLE and control in a multivariate (E) and univariate (F) manner, respectively, and main results can be viewed in the referred figures. FIGURE 1 | Overview of the experimental workflow. Plasma samples (A) were randomized in set 1 and set 2 for screening phase (B) followed by validation phase (C). Data were analyzed to investigate SLE subgroups (D) as well as comparing SLE and control in a multivariate (E) and univariate (F) manner, respectively, and main results can be viewed in the referred figures. have been extensively validated (17). Protein profiles were generated using antibody suspension bead array (18). In brief, the 367 HPA antibodies were attached to color-coded magnetic beads, then incubated with 45 µl diluted and biotinylated EDTA- plasma, followed by an addition of streptavidin-conjugated R-phycoerythrin (Invitrogen), and finally analyzed using a FlexMap3D instrument (Luminex Corp.). Data was evaluated as described below and 50 proteins (53 antibodies) were selected for further validation experiments (Figure 1C). In the validation experiment, additional HPA antibodies (n = 80) targeting other antigenic regions of these proteins were coupled to beads resulting in a validation assay of 133 antibodies toward the selected 50 proteins (Supplementary Table S-4). excluded from further analysis. Protein Profiling by Antibody Suspension Bead Arrays Subsequently, probabilistic quotient normalization (PQN) was performed on the MFIs to compensate for dilution errors and/or total amount of plasma proteins of the samples (21), followed by LOESS normalization on MA coordinates, per antibody, based on the MFIs to minimize the batch effects (22). Data quality was assessed by comparing replicates per 96-well plate, in combined 384-well plates and inter 384-well plates. Thereafter the data was split into two separate but comparable datasets (Figure 1B) with similar age and gender distribution and equal number of SLE patients and controls (Supplementary Table S-2). Set 1 consisted of 190 SLE patients and 158 controls, and set 2 of 189 SLE patients and 158 controls. This data is referred to as the data from the screening phase. Proteins reaching significance (after Bonferroni correction) comparing SLE and control, with the same direction in fold change between SLE/control, in both sample set 1 and set 2 in screening phase, were selected for validation (Figure 1C, n = 50). The validated proteins that were significantly different comparing SLE and controls (n = 15), were used for further interpretation. A generalized linear model with lasso regularization (R package: glmnet) Data Analysis of Antibody Suspension Bead Array Data The measured signals, reported as median fluorescent intensities (MFI) from FlexMap3D were imported into R (19). As previously described (20), outliers were identified in the raw data by robust principal component analysis (R package: rrcov) and May 2019 | Volume 10 | Article 1029 Frontiers in Immunology | www.frontiersin.org 3 SLE Subgroups Differ in IRF5 Idborg et al. TABLE 1 | Clinical and serological data are reported for the three molecular subgroups as well as for the entire cohort of SLE patients. Entire SLE cohorta Molecular SLE subgroupsa Comparing SLE subgroupsb n = 357 RF- IgM/SSA/SSB subgroup N = 51 IRF5 low subgroup N = 129 IRF5 high subgroup N = 177 RF- IgM/SSA/SSB vs. IRF5 low RF- IgM/SSA/SSB vs. IRF5 high IRF5 low vs. Data Analysis of Antibody Suspension Bead Array Data IRF5 high Age (years) 47.2 (34.3–58.1) 45.4 (33.6–56.8) 41.4 (31.1–54.2) 51.0 (37.8–60.3) P = 0.50 P = 0.07 p = 0.0003 Gender %F 87% 90% 87% 86% 0.62 0.65 0.92 Disease duration (years) 11.5 (4.4–21.7) 6.9 (1.5–14.4) 11.6 (4.5–20.6) 12.9 (5.3–23.2) P = 0.06 P = 0.005 P = 0.19 SLE ACR criteria 6 (5–7) 6 (5–7) 6 (5–7) 6 (5–7) P = 0.81 P = 0.43 P = 0.18 SLAM 6 (4–10) 8 (5–12) 6 (3.5–9.5) 6 (3.5–9.5) P = 0.02 P = 0.02 P = 0.90 SLEDAI-2k 4 (0–7) 4 (1–7) 3 (0.5–7.5) 4 (0–7) P = 0.74 P = 0.70 P = 0.96 C3a Kruskal–Wallis test p < 0.0001 268.4 (192.7–537.1) 351.8 (243.2–991.4) 434.8 (181.7–3092) 250.9 (191.3–324.2) P = 0.83 P < 0.0001 P < 0.0001 RF IgA (IU/ml) Kruskal–Wallis test p < 0.0001 5.3 (3.4–12) 16.6 (5.6–66.9) 4.1 (2.8–7.3) 5.7 (3.7–11.2) P < 0.0001 P = 0.0001 P = 0.0005 RF IgG (µg/ml) Kruskal–Wallis test p = 0.0001 11 (6.9–23) 20 (8.9–54.6) 10 (6.5–17.5) 11 (6.8–19.2) P < 0.0001 P = 0.0004 P = 0.23 RF IgM (IU/ml) Kruskal–Wallis test p < 0.0001 1.3 (0.63–4.7) 28 (13.5–44.6) 1.1 (0.6–2.4) 1.1 (0.5–2.1) P < 0.0001 P < 0.0001 P = 0.67 IgA total (mg/ml) 2.8 (2–3.9) 3.1 (2.1–4.2) 2.7 (1.9–3.6) 2.9 (2–3.9) P = 0.08 0.38 P = 0.15 IgG total (mg/ml) 12.8 (10.4–16.6) 16.7 (12.7–20.6) 11.7 (9.5–14.7) 12.8 (10.3–16.1) P < 0.0001 P < 0.0001 P = 0.05 IgM total (mg/ml) 0.92 (0.58–1.4) 1.2 (0.92–2.1) 0.96 (0.62–1.40) 0.8 (0.49–1.3) P = 0.0017 P < 0.0001 P = 0.03 ESR (mm/hour) 19 (11–33) 30 (16.5–46) 14 (9–27) 21 (12–36) P < 0.0001 P = 0.04 P = 0.0004 hsCRP (mg/l) 1.7 (0.68–5.3) 1.4 (0.51–5.7) 1.1 (0.48–4.7) 2.2 (0.83–5.8) P = 0.28 P = 0.18 P = 0.0003 Fibrinogen (g/l) 4.1 (3.4–5.0) 3.9 (3.1–4.6) 3.8 (3.2–4.8) 4.4 (3.6–5.2) P = 0.95 P = 0.006 P = 0.0005 TNF-α (pg/ml) 4.5 (3.3–6.2) 4.8 (3.5–6.7) 4.0 (2.8–5.7) 5.1 (3.6–6.4) P = 0.015 P = 0.77 P = 0.0005 Fibronectin (mg/ml) Kruskal–Wallis test p = 0.0008 0.38 (0.25–0.46) 0.40 (0.29–0.50) N = 33 0.41 (0.32–0.48) N = 80 0.31 (0.19–0.44) N = 95 P = 0.78 P = 0.03 P = 0.0002 Leptin (mg/ml) Kruskal–Wallis test p = 0.0002 14294 (4776–27938) 13321 (5026–21162) 7878 (2240–20389) 19502 (8474–48617) P = 0.23 P = 0.05 P < 0.0001 SLE American College of Rheumatology (ACR) classification criteria; SLAM, SLE Activity Measure (15); SLEDAI-2K, SLE Disease Activity Index (16). Data Analysis of Antibody Suspension Bead Array Data C3, Complement factor 3, RF IgA/G/M, Rheumatoid factor immunoglobulin A/G/M; ERS, erythrocyte sedimentation rate; hsCRP, high sensitivity C-reactive protein, aMedian (25% quantile - 75% quantile), NR, not reported. Serology data obtained as described in previous work. bMann–Whitney U-test for pairwise comparison of subgroups was used to characterize subgroups. P-values <0.001 without adjustment for multiple testing are highlighted in bold. Kruskal–Wallist test, i.e., comparing more than two groups and compensating for multiple testing, highlighted only RF-IgM, RF-IgG, RF-IgA, Leptin, Fibronectin and C3a as significantly different (names highlighted in italic). LE 1 | Clinical and serological data are reported for the three molecular subgroups as well as for the entire cohort of SLE patients. Comparing SLE subgroupsb SLE American College of Rheumatology (ACR) classification criteria; SLAM, SLE Activity Measure (15); SLEDAI-2K, SLE Disease Activity Index (16). C3, Complement factor 3, RF IgA/G/M, Rheumatoid factor immunoglobulin A/G/M; ERS, erythrocyte sedimentation rate; hsCRP, high sensitivity C-reactive protein, aMedian (25% quantile - 75% quantile), NR, not reported. Serology data obtained as described in previous work. bMann–Whitney U-test for pairwise comparison of subgroups was used to characterize subgroups. P-values <0.001 without adjustment for multiple testing are highlighted in bold. Kruskal–Wallist test, i.e., comparing more than two groups and compensating for multiple testing, highlighted only RF-IgM, RF-IgG, RF-IgA, Leptin, Fibronectin and C3a as significantly different (names highlighted in italic). clustering, emphasizing on the variables with greatest variance and the Calinski-Harabasz criterion was used to find the number of clusters in the data. was used to find panels of proteins to predict SLE patients and controls where the sample set 1 and set 2 corresponded to test set and training set, respectively. This was followed by analysis and visualization by performing receiver operating characteristic (ROC) analysis (R package: pROC) and confidence intervals (CI) for the area under the curve (AUC) were calculated (23). Production of Recombinant IRF5 Protein Multiple constructs of IRF5 (Uniprot ID Q13568) were sub-cloned into the expression vectors pNIC28-Bsa4 and pNIC-Bio3 (Genbank acc. No EF198106, JN792439). After performing small-scale screening for soluble recombinant protein expression as previously described (24), clones corresponding to constructs covering the regions M1-V120 and E232-L434 were selected for generation of single-chain fragment variable (scFv) binders. Frontiers in Immunology | www.frontiersin.org Generation of Antibody Fragments Against IRF5 IRF5 Single-chain fragment variable (scFv) clone J-IRF5-5 was generated by phage display technology using a human synthetic library denoted SciLifeLib. The phage selection procedure was performed basically as described earlier (27), but the first round of selection, including the steps of antigen-phage incubation to trypsin elution, was carried out in 1.5 ml tubes on a rotator with no automation. The number of washing steps was modified and increased with succeeding selection rounds; five in round one and seven in round four. Also, the recovered phages were propagated in XL1-Blue E. coli between the selection rounds. Re-cloning of the selected material in pool followed by transformation into TOP10 E. coli, small-scale expression of 94 randomly picked scFv and subsequent enzyme-linked immunosorbent assay (ELISA) for detection of recombinant full-length IRF5, i.e., verifying binding to target, and sequencing experiments were performed equivalent to previously reported (27). Affinity measurements were performed using a Biacore T200 biosensor instrument (GE Healthcare) as described in Supplementary Figure S-1. The top candidate (J-IRF5-5), binding the construct region E232-L434 of IRF5, obtained a measured affinity of 5 nM. Validation by ELISA and Biacore was extended by using IP-MS performed on cell lysate from HEK293 cells (300 µl) as previously described (27). The lysate was spiked with a small amount of recombinant IRF5 full-length protein (0.7 µg), as IRF5 is normally expressed at very low levels in HEK293 cells, and MS data was acquired in data dependent mode using a top 10 method. IRF5 was identified as the highest ranked protein in the obtained list of proteins (Supplementary Table S-5). This verifies that the antibody can capture its target in a complex mixture. The top candidate, J-IRF5-5, was then used in IP-MS on plasma samples as described below. Peptides were separated using an Ultimate 3000 RSLCnano system. Samples were trapped on an Acclaim PepMap nanotrap column (C18, 3 µm, 100 Å, 75 µm × 20 mm), separated on an NanoEaseTM M/Z HSS column (C18, 1.8 µm, 100Å, 75 µm × 250 mm), (Thermo Scientific) and analyzed on a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Peptides were separated using a gradient of A (3% ACN, 0.1% FA) and B (95% ACN, 0.1% FA), ranging from 3 to 40% B in 50 min with a flow of 0.25 µl/min. Immunoprecipitation Followed by Mass Spectrometry (IP-MS) of IRF5 in Plasma Spec o e y ( S) o 5 as a Heparin-plasma from a myositis patient and two SLE patients recruited at Karolinska University Hospital were analyzed by IP-MS as previously described (28) with a few adjustments. In brief, to an aliquot of 100 µl plasma, 400 µl of lysis buffer (1 mM Tris-HCl, 42 mM NaCl, and 0.01% NP-40 in water, pH 7.9) containing protease inhibitor cocktail (Roche) was added. Recombinant IRF5 protein (0.7 µg) was added to plasma and used as a positive control. An aliquot of 4 µg J-IRF5-5 was added to plasma samples and incubated overnight at 4◦C. As negative controls a scFv antibody, generated in the same way as J-IRF5-5 but targeting an unrelated antigen, was added to plasma and to another vial, J-IRF5-5 was added to a sample without plasma (lysis buffer only). Forty microliters of anti-FLAG M2 magnetic beads was added and incubated 2–5 h at 4◦C. The beads were washed three times (5–10 min in 4◦C) with low salt buffer (1 mM Tris-HCl, 10 mM NaCl, and 0.01% NP-40 in water, pH Data Analysis of Antibody Suspension Bead Array Data Expression and purification of selected clones and full-length IRF5 was performed essentially as previously described (25, 26), and a detailed protocol can be In order to identify molecular SLE subgroups, unsupervised clustering was applied on the screening data. To prepare the data for principal component analysis (PCA), the data for each dataset (190 SLE patients in set 1 and 189 SLE patients in set 2) was log2-transformed and centered on the mean of each antibody. In set 1 PC1 and PC2 explained 14 and 12% respectively of the variance, and in set 2 the explained variances by PC1 and PC2 were 18 and 16% respectively. Clustering of samples was done on the first two principal components by using K-means May 2019 | Volume 10 | Article 1029 4 SLE Subgroups Differ in IRF5 Idborg et al. found in Supplementary Methods and Results. Final protein batches were analyzed by SDS-PAGE and subsequently flash frozen in liquid nitrogen and stored at −80◦C until use. found in Supplementary Methods and Results. Final protein batches were analyzed by SDS-PAGE and subsequently flash frozen in liquid nitrogen and stored at −80◦C until use. 7.9) and two times with low salt buffer without NP-40. Elution was performed by 2 × 100 µl 0.5 M ammonium hydroxide and evaporated in Speedvac. Samples were reconstituted in 50 mM ammonium bicarbonate and subsequently reduced by 1 µl of 100 mM TCEP-HCl at 37◦C for 1 h, alkylated by 1 µl of 500 mM iodoacetamide in dark for 45 min and digested using 1 µg trypsin at 37◦C. Sample clean-up was performed in 50 mM ammonium bicarbonate using HiPPRTM Detergent Removal Spin Column Kit according to manufacturer’s instructions. Obtained peptide samples were desalted using ZipTip R⃝pipette tips or Pierce C18 Tips (Thermo Scientific) prior mass spectrometry analysis. A standard of IRF5 peptides were generated using full length IRF5 recombinant protein applying the same digestion protocol. Generation of Antibody Fragments Against IRF5 The Q Exactive was operated in a data dependent manner utilizing targeted SIM/ddMS2 method with an inclusion list containing masses corresponding to four unique IRF5 peptides. The survey scan was performed at 70.000 resolution from 400 to 1,200 m/z, with a max injection time of 100 ms and target of 1 × 106 ions. For generation of HCD fragmentation spectra, a max ion injection time of 250 ms and Automated Gain Control (AGC) of 3 × 106 were used before fragmentation at 30% normalized collision energy. Detection of IRF5 Positive Microparticles in Plasma In another set of SLE patients (n = 63), citrate plasma was analyzed for detection of microparticles (MPs) expressing IRF5. Characteristics of these SLE patients and details about the sample collection can be found in Supplementary Methods and Results. Healthy controls (n = 20) matched for age and gender to the SLE patients were also included in this study. Platelet-poor plasma were centrifuged (2,000 g for 20 min followed by 13,000 g for 2 min) and the supernatants were then incubated with polyclonal anti-IRF5-Fluorescein isothiocyanate (FITC) (Biorbyt, UK) as described in Supplementary Methods and Results. MPs were measured by flow cytometry on a Beckman Gallios instrument (Beckman Coulter, Bream CA, USA) and were defined as particles between ∼0.3 µm and 0.9 µm in size. Frontiers in Immunology | www.frontiersin.org General Biomarker Candidates of SLE General Biomarker Candidates of SLE Fifty-three antibodies, targeting 50 proteins, showed significant differences between SLE patients and controls in both sample set 1 and set 2, i.e., in two separate experiments performed in parallel containing samples from different patients/controls. In the following validation experiment, the plasma samples (n = 695) were analyzed using 133 antibodies targeting the 50 selected proteins. Protein profiles with low correlation (Spearman’s rho < 0.40) to the screening data were removed. The remaining 15 proteins, targeted by 16 antibodies (Table 2, Figure 1F), showed a median correlation to the screening data of rho = 0.78 with a minimum correlation of rho = 0.46. Antibody target sequence for all 15 proteins can be found in Supplementary Table S-6. Lipid Mediators and Cytokines Data Extracted From Related Projects In a previous study, sphingolipids were measured by LC-MS/MS in a selection of patients from our SLE cohort (29). Since one of the proteins characterizing the RF-IgM/SSA/SSB subgroup was ceramide synthase 5 (CERS5), which catalyzes the formation of C16 : 0-ceramide, we utilized data from this study where C16 : 0- ceramide was quantified. Among the analyzed patients (with data available from both C16 : 0-ceramide and antibody suspension bead array data), 16 patients were found to belong to the RF- IgM/SSA/SSB subgroup, 39 to IRF5 low subgroup and 44 patients belonged to the IRF5 high subgroup. p pp y The proteins yielding the largest fold change between SLE patients and controls (p < 0.05), were interferon regulatory factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2, organic cation transporter 2, OCT2) and S100 calcium binding protein A12 (S100A12, Calgranulin-C) (Figure 2). Of the 15 proteins in Table 2, three were found to be decreased, i.e., sterile alpha motif (SAM) pointed domain containing E26 transformation- specific (ETS) transcription factor (SPDEF), Apolipoprotein L6 (APOL6), and Cysteine-rich secretory protein 3 (CRISP3), and twelve were found to be increased in the SLE patients compared to controls. Seven of the proteins were classified as plasma proteins and two proteins, IRF5 and SPDEF, were transcription factors (Supplementary Table S-7). Levels of IRF5 were up- regulated and SPDEF were down-regulated in SLE compared to controls. Previously, 20 cytokines were analyzed in plasma from the entire Karolinska SLE cohort (14), and data from cytokines relevant in inflammation, i.e., TNF-α, IL-6, IL-8, Il-10, IL-16, and IP-10, were analyzed with respect to the identified molecular subgroups in this work. Interferon α (IFN-α) was measured by ELISA in the Karolinska SLE cohort in another study (30), and data was utilized in this work to study levels of IFN-α in IRF5 high and low subgroups. Data on IFN-α was obtained for 66% of the patients in the IRF5 low subgroup and in 70% of the patients in IRF5 high subgroup. Values below limit of quantification (LOQ) was set to LOQ/2. Detection of IRF5 in Plasma by Sandwich ELISA Nunc immobilizer amino plates (Thermo scientific) were coated with commercial mouse anti-human IRF5, antibody targeting May 2019 | Volume 10 | Article 1029 Frontiers in Immunology | www.frontiersin.org 5 SLE Subgroups Differ in IRF5 Idborg et al. aa 176–240 (Antibodies-online.com, ABIN121152). A standard curve was obtained using recombinant IRF5 protein at 0.156, 0.312, 0.625, 1.25, 2.5, 5.0, and 10 ng/ml (50 µl/well). Plasma samples from 25 SLE patients and 14 controls were diluted 1:2 in 0.1% BSA/PBS before adding 50 µl per well. As a secondary antibody, rabbit anti-human IRF5 (HPA046700, i.e., the antibody used in the antibody suspension bead array) was used. Donkey anti-rabbit IgG HRP-conjugated antibody was added for detection using TMB substrate and optical density was read at 450 nm. of antibody suspension bead array data, linear modeling and K-means clustering can be found in section Data Analysis of Antibody Suspension Bead Array Data. Calculations were performed using R (19), GraphPad Prism 7 and Excel 2016. IRF5 protein QTL analysis was performed on log10 transformed IRF5 protein levels using linear regression in R assuming an additive genetic model. Genetic Data on IRF5 The Karolinska SLE cohort had previously been genotyped using the Immunochip Illumina Infinium Assay (31, 32). Two previously reported independent IRF5 SLE risk variants rs4728142 and rs10488631 (a proxy to rs35000415) were selected from this data for association with IRF5 protein levels in quantitative trait locus (QTL) analyses. Genotype data was available for 253 SLE patients and 280 controls. Proteins that showed significant differences between SLE patients and controls (Table 2), were used to create a linear model (Figure 1E). Obtained model suggested a biomarker panel of 9 antibodies, targeting 8 proteins, i.e., GTP-binding protein Rhes (RASD2), S100A12, SLC22A2, Matrix metalloproteinase-1 (MMP1), CRISP3, complement component C6 (C6), Phospholipid phosphatase 1 (PPAP2A), SPDEF, achieving a ROC AUC of 0.78 (95% CI: 0.73–0.83) for prediction of SLE patients and controls (Figure 2). In comparison, the highest achieved AUC from a single protein was 0.73 (95% CI: 0.67–0.78) for MMP1 and 0.72 (95% CI: 0.66–0.77) for S100A12, and a panel of three proteins (S100A12, SLC22A2, and PPAP2A) yielding an AUC of 0.74 (95% CI: 0.69–0.80). This panel of 8 proteins is suggested as general biomarker candidates to differentiate between SLE and controls, independently of SLE subgroups. Frontiers in Immunology | www.frontiersin.org Statistics For comparison between SLE and controls Mann-Whitney U-test was used. Bonferroni-corrected p-value at a threshold of 0.05 was used as a measure of significance unless otherwise stated. When comparing three or more groups, i.e., when comparing the three molecular subgroups, Kruskal Wallis test or Fisher’s exact test (for categorical data) was used. In addition, in Table 1, Mann– Whitney U-test have been used for independent comparisons between molecular subgroups in favor for scientific reasoning of selected variables with cautious interpretation of p-values (33, 34). Spearman rank correlation was used to investigate correlations between variables. Additional details about analysis Applying strict statistical univariate analysis (Bonferroni correction) only two associations were identified between proteins and clinical data (i.e., serological data, clinical symptoms, disease activity scores). Lower levels of S100A12 were May 2019 | Volume 10 | Article 1029 Frontiers in Immunology | www.frontiersin.org 6 SLE Subgroups Differ in IRF5 Idborg et al. TABLE 2 | The 15 proteins (16 antibodies) differentially expressed comparing SLE and control. Protein name short Full protein name UniProta Correlation screening vs. validationb Fold change (SLE/Ctrl) p-value (SLE vs. Ctrl)c IRF5 Interferon regulatory factor 5 Q13568 0.96 0.48 4.5E-02 SLC22A2* Solute carrier family 22 (organic cation transporter), member 2 O15244 0.8 0.44 4.6E-06 S100A12* S100 calcium binding protein A12 P80511 0.77 0.28 3.3E-09 RASD2* GTP-binding protein Rhes Q96D21 0.86 0.26 1.7E-05 NOS3 Nitric oxide synthase 3 (endothelial) P29474 0.93 0.26 4.1E-02 MMP1* Matrix metallopeptidase 1 (or interstitial collagenase) P03956 0.63 0.17 3.2E-06 SPDEF* SAM pointed domain containing ETS transcription factor O95238 0.87 −0.14 1.3E-02 UBAC1 UBA domain containing 1 Q9BSL1 0.71 0.13 1.4E-04 TRIM33 Tripartite motif containing 33 Q9UPN9 0.84 0.13 3.0E-03 CFI Complement factor I P05156 0.65 0.13 2.9E-02 APOL6 Apolipoprotein L, 6 Q9BWW8 0.84 −0.13 4.5E-02 PPAP2A* Phosphatidic acid phosphatase type 2A (or Phospholipid phosphatase 1) O14494 0.82 0.12 9.9E-03 GRAP2 GRB2-related adaptor protein 2 O75791 0.69 0.11 3.4E-03 CRISP3* Cysteine-rich secretory protein 3 P54108 0.75 −0.10 5.5E-04 CRISP3* Cysteine-rich secretory protein 3 P54108 0.46 −0.10 1.8E-03 C6* Complement component 6 P13671 0.68 0.10 3.7E-03 Proteins are sorted based on log-fold change between SLE samples and controls. Proteins included in suggested biomarker panel are indicated by an asterisk (“*”). aProtein ID in UniProt (35). bThe Speaman’s rho correlation coefficients for screening and validation data are reported. cThe highest Bonferroni-corrected p-value among set 1 and set 2 comparing SLE and Controls is reported. Statistics NOS3, and interleukin-2 receptor subunit alpha (IL2RA) and is referred to as the IRF5 high subgroup. Levels of IRF5 in the three subgroups as well as in the two sample sets are shown in Figure 3D. associated in patients with a history of lupus nephritis, i.e., “nephritis ever” defined by ACR criteria (median signal of 1591 vs. 1409, with IQR of 613 vs. 583, and Bonferroni-adjusted Mann-Whitney U-test p-value of 0.008) and IRF5 protein levels showed a weak negative correlation to C3a plasma concentration in SLE patients (Spearman’s rho = −0.32, p < 0.0001). g Including all available clinical and serological data, considering associations comparing all three molecular subgroups (Kruskal–Wallis test), only rheumatoid factor (RF)-IgM, RF-IgG, RF-IgA, Leptin, Fibronectin, and C3a were found to be significantly different (p < 0.05) in at least one subgroup after correction for multiple testing (Supplementary Figure S-2). Molecular subgroup 1 was found to have high RF-IgM levels (Figure 4) as well as high levels of autoantibodies toward Sjögren’s Syndrom antigen A/B (SSA/SSB) (Supplementary Table S-8). This subgroup is further referred to as the RF-IgM/SSA/SSB subgroup. We also observed higher levels of RF-IgG and RF-IgA as well as higher levels of total IgG and IgM in this subgroup (Table 1, Supplementary Figure S-2). In addition, this subgroup showed higher frequency (45%) of patients with secondary Sjögren’s syndrome (sSS), as defined according to the American-European Consensus criteria (36), compared to the IRF5 high and low subgroup (both 19%) (Supplementary Table S-9). Patients in the RF-IgM/SSA/SSB subgroup showed lower frequency of nephritis (20%) compared to IRF5 low (43%) and IRF5 high (48%) subgroups. Lower ESR were reported for the IRF5 low subgroup (Table 1). The IRF5 high subgroup was slightly older compared to other subgroups, showed lower levels of C3a and increased levels of inflammatory markers e.g., TNF-α, fibrinogen and hsCRP (Table 1). Frontiers in Immunology | www.frontiersin.org SLE Molecular Subgroups Unsupervised clustering of the 281 analyzed proteins (screening data, K-means clustering) was performed to find potential molecular subgroups among the SLE patients (Figure 1D). Three distinct clusters were obtained in both experimental set 1 (Figure 3A) and set 2 (Figure 3B) and nine of the 10 proteins with the highest absolute PCA loadings were identical between the two sets. These 9 proteins were evaluated by analyzing the median protein levels and revealed concordant protein profiles between the sets (Figure 3C). This panel of biomarker candidates can be used to differentiate between suggested molecular subgroups. Molecular subgroup 1 (red, n = 51) showed higher levels of E-selectin (SELE), solute carrier family 22 (SLC22A2), Ceramide synthase 5 (CERS5) and Integrin subunit beta 1 (ITGB1, Glycoprotein IIA, CD29). Molecular subgroup 2 (green, n = 129) showed lower levels of IRF5, Ubiquitin-like protein ISG15 (ISG15), endothelial nitric oxide synthase (NOS3) and SLC22A2, and is further referred to as the IRF5 low subgroup. This subgroup was found to be similar to the control group (gray) as shown in Figure 3C. Molecular subgroup 3 (blue, n = 177) showed higher levels of IRF5, ISG15, May 2019 | Volume 10 | Article 1029 Frontiers in Immunology | www.frontiersin.org 7 Idborg et al. SLE Subgroups Differ in IRF5 FIGURE 2 | General biomarker candidates of SLE. Proteins showing the highest absolute fold change between SLE patients and controls (in both sample set 1 and 2) were (A) Interferon regulatory factor 5 (IRF5), (B) Solute carrier family 22 member 2 (SLC22A2) and (C) S100 calcium binding protein A12 (S100A12). A panel of 8 proteins, consisting of 9 antibodies proved to be the best panel for classifying SLE patients from controls. The panel of 8 proteins consist of RASD2, S100A12, SLC22A2, MMP1, CRISP3, C6, PPAP2A, and SPDEF and achieved an ROC AUC of 0.78 for the prediction of SLE patients and controls (D). FIGURE 2 | General biomarker candidates of SLE. Proteins showing the highest absolute fold change between SLE patients and controls (in both sample set 1 and 2) were (A) Interferon regulatory factor 5 (IRF5), (B) Solute carrier family 22 member 2 (SLC22A2) and (C) S100 calcium binding protein A12 (S100A12). A panel of 8 proteins, consisting of 9 antibodies proved to be the best panel for classifying SLE patients from controls. SLE Molecular Subgroups The panel of 8 proteins consist of RASD2, S100A12, SLC22A2, MMP1, CRISP3, C6, PPAP2A, and SPDEF and achieved an ROC AUC of 0.78 for the prediction of SLE patients and controls (D). CERS5 was, as mentioned, increased in the RF-IgM/SSA/SSB subgroup, and is an enzyme catalyzing the formation of C16 : 0- ceramide. We have previously quantified levels of sphingolipids in SLE (29) and data was available for a selection of patients. C16 : 0-ceramide levels were 415 ± 143 nM (mean ± SD) in RF- IgM/SSA/SSB subgroup (n = 16), 305 ± 79 nM in IRF5 low subgroup (n = 39) and 331 ± 84 nM in IRF5 high subgroup 3 (n = 44) (Supplementary Figure S-3) (Kruskal–Wallis test p = 0.02) supporting our finding of higher levels of CERS5 in RF-IgM/SSA/SSB subgroup. CERS5 was, as mentioned, increased in the RF-IgM/SSA/SSB subgroup, and is an enzyme catalyzing the formation of C16 : 0- ceramide. We have previously quantified levels of sphingolipids in SLE (29) and data was available for a selection of patients. C16 : 0-ceramide levels were 415 ± 143 nM (mean ± SD) in RF- IgM/SSA/SSB subgroup (n = 16), 305 ± 79 nM in IRF5 low subgroup (n = 39) and 331 ± 84 nM in IRF5 high subgroup 3 (n = 44) (Supplementary Figure S-3) (Kruskal–Wallis test p = 0.02) supporting our finding of higher levels of CERS5 in RF-IgM/SSA/SSB subgroup. peaks corresponding to IRF5 were detected in the blank samples and no carry-over was observed between runs. MS/MS spectra of two of the peptides detected in plasma from a SLE patient is shown in Figure 5. IRF5 could repeatedly be detected in plasma aliquots from a myositis patient using IP-MS utilizing peptide exact mass (high-resolution m/z) and retention time. Levels were close to detection limit and fragment spectra of IRF5 peptides could not always be obtained although aliquots from the same sample were analyzed. Adding the criteria of reporting fragmentation spectra of the unique peptides, IRF5 was detected in two out of three separate experiments, not detected in one SLE patient and for the second SLE patient fragment spectra could be obtained in one out of two experiments. IP-MS, as used here, is not a quantitative method and the capture of IRF5 might slightly differ between experiments and not reach detection limit. Therefore, this is not the method IRF5 in Plasma To confirm the presence of IRF5 in plasma immunoprecipitation tandem mass spectrometry (IP-MS/MS) was used. The targeted MS/MS method was optimized for four unique tryptic IRF5 peptides using recombinant IRF5 protein as a standard. No May 2019 | Volume 10 | Article 1029 Frontiers in Immunology | www.frontiersin.org 8 Idborg et al. SLE Subgroups Differ in IRF5 FIGURE 3 | SLE molecular subgroups. K-means clustering, visualized on the two first principal components (PC1 and PC2), identified three subgroups (1-red, 2-green and 3-blue) in sample set 1 (A) and set 2 (B) with a similar clustering pattern. The relative protein profiles (C) of the 9 proteins with the highest loadings in both sample sets for the RF-IgM/SSA/SSB (red, n = 51), the IRF5 low (green, n = 129) and the IRF5 high (blue, n = 177) molecular subgroups are shown and both sample set 1 (solid line) and set 2 (dashed line) shows concordant protein profiles. It is evident that the IRF5 high subgroup discriminate from the IRF5 low subgroup based on levels of IRF5, ISG15, and NOS3, while it is evident that the RF-IgM/SSA/SSB subgroup differentiate from the other two in levels of SELE, SLC22A2, CERS5, and ITGB1. Controls are included in gray for comparison but was not included in the clustering. Levels of IRF5 (D) are compared between the three molecular SLE subgroups RF-IgM/SSA/SSB (red), IRF5 low (green), and IRF5 high (blue) subgroup. FIGURE 3 | SLE molecular subgroups. K-means clustering, visualized on the two first principal components (PC1 and PC2), identified three subgroups (1-red, 2-green and 3-blue) in sample set 1 (A) and set 2 (B) with a similar clustering pattern. The relative protein profiles (C) of the 9 proteins with the highest loadings in both sample sets for the RF-IgM/SSA/SSB (red, n = 51), the IRF5 low (green, n = 129) and the IRF5 high (blue, n = 177) molecular subgroups are shown and both sample set 1 (solid line) and set 2 (dashed line) shows concordant protein profiles. It is evident that the IRF5 high subgroup discriminate from the IRF5 low subgroup based on levels of IRF5, ISG15, and NOS3, while it is evident that the RF-IgM/SSA/SSB subgroup differentiate from the other two in levels of SELE, SLC22A2, CERS5, and ITGB1. Controls are included in gray for comparison but was not included in the clustering. Frontiers in Immunology | www.frontiersin.org IRF5 in Plasma Levels of IRF5 (D) are compared between the three molecular SLE subgroups RF-IgM/SSA/SSB (red), IRF5 low (green), and IRF5 high (blue) subgroup. activity (p < 0.05) (SLE activity measure (SLAM) (15) equal or above 6) (Supplementary Figure S-4). of choice in a screening of the entire cohort comparing SLE and controls. Nevertheless, in cases where IRF5 was detected there is no doubt about the identity of IRF5 and that IRF5 is present in plasma. The IP, accurate retention times (RT) and high-resolution accurate-mass of unique IRF5 peptides and their fragment spectra, confirm the presence of IRF5 in the circulation. In addition, we developed a sandwich ELISA for detection of IRF5 in plasma. IRF5 levels were significantly higher (p = 0.014) in SLE (n = 25) compared to controls (n = 25) (Supplementary Figure S-5). However, the sensitivity of this assay was not sufficient for screening of the entire cohort since the majority of the SLE patients analyzed (56%, n = 14) report levels below quantification limit. Within this data we aimed to correlate our results with the results obtained by the suspension bead array. Excluding data outside the quantitative range of the ELISA, Spearman’s rank correlation analysis was performed on data from 11 SLE patients. A strong correlation (Spearman’s rho = 0.63, p < 0.05) and a moderate R2 of 0.36 was obtained (Supplementary Figure S-6). However, the three samples resulting in levels above the quantification range of the To further investigate the presence of IRF5 in plasma we analyzed IRF5 positive microparticles (MPs). The number of circulating MPs exposing IRF5 were significantly higher in SLE (n = 63) compared to healthy controls (n = 20) (130.5 ± 88 vs. 36.5 ± 14 MPs/µl, p < 0.0001) (Figure 6A). IRF5 positive MPs were more frequently exposed on endothelial derived MPs (CD62E+ MPs) compared to platelet and leukocyte derived MPs (p < 0.0001) (Figure 6B). Furthermore, total IRF5+ MPs (regardless of origin) were significantly higher in patients with higher disease May 2019 | Volume 10 | Article 1029 Frontiers in Immunology | www.frontiersin.org 9 Idborg et al. SLE Subgroups Differ in IRF5 (detected by a different antibody) are increased in SLE compared to controls. (detected by a different antibody) are increased in SLE compared to controls. FIGURE 4 | Serological characteristics of SLE molecular subgroups. IRF5 in Plasma The levels of RF-IgM are compared between the three molecular SLE subgroups RF-IgM/SSA/SSB (red), IRF5 low (green), and IRF5 high (blue) subgroup. We identified a weak positive association between IRF5 protein levels and the number of SLE risk alleles at one of two SNP representing the IRF5 SLE genetic association. However, this effect was not apparent when separating the data from SLE patients and control individuals, thus it could be driven by the allele frequency and protein level differences between these two groups. This indicates that the IRF5 SLE risk variants are not the sole drivers for the differences in IRF5 plasma levels that we observe. As recently discussed elsewhere (41), the contribution of IRF5 genetic risk to disease susceptibility is not known, and it is possible that IRF5 may have both a genetic and non- genetic contribution. It is an intriguing and novel finding that the IRF5 protein occurs in the circulation and that it stands out as a potential biomarker for SLE. The high IRF5 levels in the circulation may reflect increased cell death in SLE patients. However, the IRF5 levels also vary to a large extent within the group of SLE patients. In addition, SPDEF, another transcription factor, showed the opposite regulation in SLE plasma (10% decrease) and unless SPDEF is strongly down-regulated in SLE, the difference in IRF5 cannot solely be explained by increased cell death/loss during apoptotic clearance in patients. Reports of transcription factors in circulation are sparse (42, 43) and by our approach using antibodies designed to target a short linear sequence of the protein, it is not possible to determine if the protein is full-length or represents a splice variant or other modified product. There is no information about extracellular function of IRF5. However, the fact that IRF5 may be found on microparticles, known to mediate cell-cell signaling, merits further investigations. In addition, further studies are needed to investigate if the IRF5 protein is actively secreted and to study possible extracellular functions of IRF5. FIGURE 4 | Serological characteristics of SLE molecular subgroups. The levels of RF-IgM are compared between the three molecular SLE subgroups RF-IgM/SSA/SSB (red), IRF5 low (green), and IRF5 high (blue) subgroup. ELISA, showed low or medium levels of IRF5 as measured by the suspension bead array. IRF5 gene polymorphism is an established risk factor in SLE (32, 37). IRF5 in Plasma To investigate whether IRF5 levels in plasma were regulated by known SLE genetic risk variants in IRF5 we performed a protein quantitative trait locus analysis for two previously reported SNPs (32). We identified a weak additive association between IRF5 protein levels and the IRF5 SLE risk variant rs4728142 (p = 0.003, beta = 0.07) in SLE patients and controls combined, but this effect was not apparent in either group alone. There was no association between rs10488631 and IRF5 plasma protein levels (Supplementary Figures S-7, 8). Interestingly our unsupervised clustering of SLE patients demonstrate that IRF5 is characteristic for two different SLE subgroups. The IRF5 low subgroup also showed lower levels of ISG15, an ubiquitin-like protein that is conjugated to intracellular target proteins upon activation by IFN-α and IFN-β (44, 45), suggesting that this subgroup might be described as a less interferon dependent subgroup. On the other hand, the IRF5 high subgroup seems to be an interferon-driven subgroup with higher levels of IRF5 and ISG15 and one might speculate that patients in these two subgroups could respond differently to IFN- α-inhibition. Serum levels of IFN-α did not differ between IRF5 high and low subgroup and might be explained by that IFN-α is regulated by several genes and not only by IRF5. Building on these observations, we suggest stratification of patients based on plasma levels of IRF5 prior to clinical trials targeting the IFN pathway. These subgroups need to be further investigated, e.g., in the light of type I IFN blockers (46) not reaching primary endpoint. Stratification based on IRF5 levels may be more efficient, definitely less expensive and more suitable to implement in clinical routine, than to measure interferon signature on a gene level. Serum levels of IFN-α in the IRF5 high subgroup was not significantly different compared to the IRF5 low subgroup. In both subgroups 40% were defined as having detectable levels of IFN-α and the concentration (average ± SD) was 78 ± 122 pg/ml and 67 ± 149 pg/ml for the IRF5 low and IRF5 high subgroup, respectively. The number of IFN-α high patients, defined as a concentration of >100 pg/ml) was 15 in both subgroups. Frontiers in Immunology | www.frontiersin.org DISCUSSION IRF5, a transcription factor involved in regulation of interferon and cytokine production, showed the largest fold change among the differentially expressed proteins between SLE patients and controls. We also observed large variations in IRF5 levels between subgroups of SLE patients. IRF5 gene polymorphism is a well- known risk factor in SLE (38, 39) and in several other rheumatic diseases (40). IRF5 is an intracellular protein, nevertheless we detected IRF5 in plasma using affinity-based proteomics and the extracellular location was confirmed by IP-MS in a selection of plasma samples. To further illustrate the presence of IRF5 in the circulation we report that IRF5 expressing microparticles In the IRF5 high subgroup, we detected higher levels of NOS3 (endothelial (e)NOS) as compared to the IRF5 low subgroup. May 2019 | Volume 10 | Article 1029 Frontiers in Immunology | www.frontiersin.org 10 Idborg et al. SLE Subgroups Differ in IRF5 FIGURE 5 | Fragment spectra of endogenous IRF5 detected in plasma. The generated recombinant antibody (J-IRF5-5) was used to capture IRF5 by immunoprecipitation in a plasma sample from a SLE patient. We obtained fragment spectra of two unique peptides of IRF5, i.e., (A) LITVQVVPVAAR with [M+2H]2+ m/z of 633.4007 eluting at a retention time of 48.6 min and (B) FPSPEDIPSDK with [M+2H]2+ m/z of 616.29574 eluting at a retention time of 40.7 min. The retention times, the masses of the unique peptides and the fragment spectra of these peptides confirms the presence of IRF5 in this plasma sample. NOS3, an important regulator of nitric oxide (NO) production, which is essential for cardiovascular and immune functions indicate an increased risk of cardiovascular events in the IRF5 low subgroup. It is also possible that the high levels of NOS3 in the FIGURE 5 | Fragment spectra of endogenous IRF5 detected in plasma. The generated recombinant antibody (J-IRF5-5) was used to capture IRF5 by immunoprecipitation in a plasma sample from a SLE patient. We obtained fragment spectra of two unique peptides of IRF5, i.e., (A) LITVQVVPVAAR with [M+2H]2+ m/z of 633.4007 eluting at a retention time of 48.6 min and (B) FPSPEDIPSDK with [M+2H]2+ m/z of 616.29574 eluting at a retention time of 40.7 min. The retention times, the masses of the unique peptides and the fragment spectra of these peptides confirms the presence of IRF5 in this plasma sample. FIGURE 5 | Fragment spectra of endogenous IRF5 detected in plasma. DISCUSSION CCDC88A (girdin, APE), a protein important for angiogenesis (50), was also increased in the IRF5 high subgroup and decreased in the IRF5 low subgroup. Inflammatory markers were increased in this subgroup indicating that the IRF5 high subgroup is characterized by more pronounced inflammation and one may speculate that anti-inflammatory treatment is more likely to be beneficial for this subgroup of SLE patients. Although validated HPA antibodies, targeting unique peptide sequences, were used, there is still a risk that these mono-specific polyclonal antibodies give rise to unspecific signals. Adding additional antibodies to the same protein enhance the probability of detecting the correct protein (56). However, the different epitopes targeted by the additional antibodies might be subject to differences in post translational modifications or differ in affinity and might not confirm the detection although the correct protein is present. In this work we confirmed the identity of one protein (IRF5) in plasma by IP-MS using a recombinant monoclonal antibody. We were also able to confirm the increased levels of IRF5 in SLE patients compared to controls in a subset of individuals utilizing a sandwich ELISA with a complementary capturing antibody. Although we did not validate the differences in IRF5 levels in subgroups of SLE in the entire cohort, we are confident of the detection of IRF5 in plasma. g p p The RF-IgM/SSA/SSB subgroup is characterized by increased levels of SELE (endothelial cell adhesion molecule, E-selectin, CD62E, ICAM-1), ITGB1, SLC22A2, and CERS5. SELE is a cell adhesion glycoprotein on endothelium that can be stimulated by e.g., TNF-α (35, 51). ITGB1 is a cell surface receptor, which is part of the integrin family and it is important for cell adhesion (52). CERS5 synthesizes C16-ceramide and the increase in CERS5 was supported by the increase of C16- ceramide in this subgroup. Ceramides are signaling lipids involved in cell adhesion, inflammation as well as in a variety of other physiological functions (53, 54). This subgroup was also associated with higher levels of rheumatoid factor (RF) as well as higher levels of SSA/SSB antibodies. We previously reported high levels of RF-IgM (13), as well as higher levels of total IgG (55), in SLE patients with SSA/SSB antibodies. The RF-IgM/SSA/SSB subgroup share features with Sjögren’s syndrome. Diagnostic biomarkers and novel insight into possible pathogenic pathways in SLE are of great importance and we here report a panel of biomarker candidates that could differentiate between SLE and controls. DISCUSSION The generated recombinant antibody (J-IRF5-5) was used to capture IRF5 by immunoprecipitation in a plasma sample from a SLE patient. We obtained fragment spectra of two unique peptides of IRF5, i.e., (A) LITVQVVPVAAR with [M+2H]2+ m/z of 633.4007 eluting at a retention time of 48.6 min and (B) FPSPEDIPSDK with [M+2H]2+ m/z of 616.29574 eluting at a retention time of 40.7 min. The retention times, the masses of the unique peptides and the fragment spectra of these peptides confirms the presence of IRF5 in this plasma sample. NOS3, an important regulator of nitric oxide (NO) production, which is essential for cardiovascular and immune functions through regulation of vascular tone, leucocyte adhesion and platelet aggregation (47, 48). NOS3 is vasoprotective and low levels of NOS3 are related to endothelial dysfunction (49). In this context, it is difficult to dissect if the low levels of NOS3 indicate an increased risk of cardiovascular events in the IRF5 low subgroup. It is also possible that the high levels of NOS3 in the IRF5 high subgroup reflect damaged blood vessels since NOS3 is expressed in the endothelium and not expected to be increased in the circulation. In the microparticles, analyzed in another set of SLE patients, the IRF5 positive microparticles were mainly of May 2019 | Volume 10 | Article 1029 Frontiers in Immunology | www.frontiersin.org 11 Idborg et al. SLE Subgroups Differ in IRF5 FIGURE 6 | IRF5+ microparticles. Total IRF5+ MPs in SLE patients (n = 63) and healthy controls (n = 20) are shown (A). IRF5+ MPs in SLE patients were phenotyped based on cell origin (B). PMPs, platelet derived MPs; LMPs, leukocyte derived MPs; EMPs, Endothelial derived MPs. *** < 0.001 (Mann–Whitney). Data is presented as MPs/µl plasma. FIGURE 6 | IRF5+ microparticles. Total IRF5+ MPs in SLE patients (n = 63) and healthy controls (n = 20) are shown (A). IRF5+ MPs in SLE patients were phenotyped based on cell origin (B). PMPs, platelet derived MPs; LMPs, leukocyte derived MPs; EMPs, Endothelial derived MPs. *** < 0.001 (Mann–Whitney). Data is presented as MPs/µl plasma. Both the IRF5 high and the aPL+ subgroups were characterized by pronounced inflammation. Our conclusions are based on analysis of a large number of samples. However, validation in additional SLE cohorts and in other disease cohorts is needed. endothelial origin, suggestive of endothelial damage. Frontiers in Immunology | www.frontiersin.org FUNDING Swedish Research Council (grant no: 2017-02577, grant no 2018-02399, grant no 2018-02535, grant no 2018-02000); Innovative Medicines Initiative (EU/EFPIA) (ULTRA-DD grant no: 115766); Stockholm County Council (ALF grant no: 20160378, grant no. 20170038), The Swedish Rheumatism Association (grant no: R-748261, grant no: R-755861, grant no: R-753741, grant no R-850611, grant no: R-739631); King Gustaf V’s 80-year foundation (grant no: n/a); Funds from Karolinska Institutet (grant no: n/a); Swedish Society of Medicine and the Ingegerd Johansson Donation (grant nr SLS-713911, grant nr SLS-708061); This study was supported by the AstraZeneca-Karolinska Institutet Joint Research Program in Translational Science. ACKNOWLEDGMENTS We would like to thank the patients and controls for participating in the study. We are grateful to Eva Jemseby for collecting all the blood-samples and coordinating nurses, and to Sonia Möller and Birgitta Mannerstedt Fogelfors for assistance in inclusion of patients. The IRF5 expression plasmid was prepared by Protein Science Facility (PSF) at Karolinska Institutet (http:// ki.se/psf). Professor Lennart Truedsson and coworkers at Lund University are acknowledged for measuring C2 and Professor Bo Nilsson and coworkers at Uppsala University for C3a and TCC measurements. We would like to acknowledge Susanna Eketjäll and AstraZeneca for performing the cytokine screening. Ju Li (Huai’an First People’s Hospital in China) is acknowledge for performing the IRF5 ELISA. DISCUSSION Utilizing unsupervised clustering of protein profiles, three molecular subgroups were revealed and could be characterized by another set of biomarker candidates. The RF-IgM/SSA/SSB subgroup essentially reflects the autoantibody defined SSA/SSB+ subgroup, which has previously been described (13, 55). The novel finding of circulating IRF5 protein is of importance for the other two subgroups. We suggest that stratification of patients based on circulating levels of IRF5 prior to e.g., IFN modulating treatments may be a valuable strategy. Furthermore, the IRF5 high subgroup expressed multiple signs of systemic inflammation, indicating that these patients may benefit from anti-inflammatory treatment. This work adds new information to the emerging need to classify the heterogeneous sample groups within SLE. The extension of these observations indicate that subgroups might be subject to different treatment perspectives, despite similar clinical profile. In a parallel study the same proteins were investigated but the subgroups were predefined by autoantibody profile, building on previous studies and own clinical experiences (13). The SSA/SSB+ subgroup in that study consisted of 63 patients and the largest fraction (43%) was assigned to the RF-IgM/SSA/SSB subgroup in this study, while the second largest portion (32%) was found in the IRF5 high subgroup which is in line with a pronounced interferon signaling in the SSA/SSB+ subgroup. The frequency of nephritis was similar and relatively low in both RF-IgM/SSA/SSB and SSA/SSB+ subgroups (20 and 21% respectively) while higher in other subgroups (>40%). CERS5 and ITGB1 were proteins characteristic for both RF- IgM/SSA/SSB and SSA/SSB+ subgroups. The second subgroup in our previous work, i.e., an aPL+ subgroup (n = 66), was to the largest extent (58%) found in the IRF5 high subgroup in this work and only 5% overlapped with the RF-IgM/SSA/SSB subgroups. May 2019 | Volume 10 | Article 1029 Frontiers in Immunology | www.frontiersin.org 12 SLE Subgroups Differ in IRF5 Idborg et al. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.01029/full#supplementary-material AUTHOR CONTRIBUTIONS HI substantial contributions to the design of the work, data interpretation, performed IP of IRF5, and drafted the manuscript. AZ substantial contributions to the design of the work, proteomic analysis, data interpretation, and drafted the manuscript. EO performed and analyzed IP-MS experiments, and wrote sections of the manuscript. EW produced recombinant IRF5 and wrote sections of the manuscript. CP performed phage display selections of recombinant antibodies and their subsequent validation and wrote sections of the manuscript. FM analysis and interpretation of IRF5 positive MPs and wrote sections of the manuscript. AC performed analysis of lipid mediators and critically revised the manuscript. AS performed analysis of RF and critically revised the manuscript. PP and JS performed genetic data analysis and wrote sections and critically revised the manuscript. CF-C developed and supervised the ELISA analysis and critically revised the manuscript. JR interpretation of RF data and design of RF experiments and critically revised the manuscript. VO performed analysis of IFN-α and critically revised the manuscript. GG clinical evaluation of sAPS patients and critically revised the manuscript. MK clinical evaluation of sSS patients and critically revised the manuscript. AL supervision of leptin and fibronectin measurements and critically revised the manuscript. CW responsible for analysis of lipid mediators and critically revised the manuscript. AS and LR provided genetic data and critically revised the manuscript. KK responsible for MS-analysis of IRF5 and critically revised the manuscript. HP made the phage display library, supervised phage display selections and antibody validation, and wrote sections of the manuscript. SG supervision of antigen and antibody production and validation of recombinant antibodies and wrote sections of the manuscript. IG responsible for SLE cohort and clinical data and critically revised the manuscript. PN substantial contributions to the design of the work and responsible for ETHICS STATEMENT affinity proteomic platform and drafting the manuscript. ES substantial contributions to the design of the work, responsible for SLE cohort and clinical data, and drafting the manuscript. PJ substantial contributions to the design of the work, responsible for biomarker study, and drafting the manuscript. All authors contributed to manuscript revision, read and approved the final version. This study was carried out in accordance with the recommendations of The Local Ethics Committee of the Karolinska University Hospital/Karolinska Institutet in Stockholm. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by The Ethical Review Board in Stockholm (EPN Stockholm). This study was carried out in accordance with the recommendations of The Local Ethics Committee of the Karolinska University Hospital/Karolinska Institutet in Stockholm. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by The Ethical Review Board in Stockholm (EPN Stockholm). 3. Lisnevskaia L, Murphy G, Isenberg D. Systemic lupus erythematosus. Lancet. (2014) 384:1878–88. doi: 10.1016/S0140-6736(14) 60128-8 REFERENCES 3. Lisnevskaia L, Murphy G, Isenberg D. Systemic lupus erythematosus. Lancet. (2014) 384:1878–88. doi: 10.1016/S0140-6736(14) 60128-8 4. Yu C, Gershwin ME, Chang C. Diagnostic criteria for systemic lupus erythematosus: a critical review. J Autoimmun. (2014) 48–49:10–3. doi: 10.1016/j.jaut.2014.01.004 5. Sanz I. 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Copyright © 2019 Idborg, Zandian, Ossipova, Wigren, Preger, Mobarrez, Checa, Sohrabian, Pucholt, Sandling, Fernandes-Cerqueira, Rönnelid, Oke, Grosso, Kvarnström, Larsson, Wheelock, Syvänen, Rönnblom, Kultima, Persson, Gräslund, Gunnarsson, Nilsson, Svenungsson and Jakobsson. 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. 52. Shenkman B, Brill G, Solpov A, Vitkovsky Y, Kuznik B, Koltakov A, et al. CD4+ lymphocytes require platelets for adhesion to immobilized fibronectin in flow: role of β1 (CD29)-, β2 (CD18)-related integrins and non-integrin receptors. Cell Immunol. 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Mechanically interlocked functionalization of monoclonal antibodies
Nature communications
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1 Department of Molecular Medicine, Beckman Research Institute of City of Hope, 1710 Flower St., Duarte, CA 91010, USA. 2 Department of Molecular Immunology, Beckman Research Institute of City of Hope, 1710 Flower St., Duarte, CA 91010, USA. 3 Department of Surgery, Beckman Research Institute of City of Hope, 1710 Flower St., Duarte, CA 91010, USA. 4Present address: Xencor, 111W. Lemon Ave., Monrovia, CA 91016, USA. Correspondence and requests for materials should be addressed to J.C.W. (email: jcwilliams@coh.org) ARTICLE ARTICLE | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications ARTICLE The crystal structure of the original meditope in complex with the Ile83Glu anti-HER2 meditope-enabled Fab confirmed the formation of a favorable electrostatic interaction with a Glu83-Arg9 bond distance dOE2··· NH1 = 2.7 Å (Fig. 1c). As with the other meditope/meditope- enabled Fab structures, the substitution did not substantially change the overall structure of the Fab (the root-mean-square- deviation between the Fab domains of the Ile83Glu variant and the parental trastuzumab calculated over Cα is 0.28 Å). p Combining these meditope (i.e., Leu5 to diphenylalanine substitution and C-terminal extension) and meditope-enabled Fab (i.e., Ile83Glu substitution) modifications produced a dramatic increase in the affinity. The binding constant could not be accurately determined at 25 °C because the off-rate exceeded the instrument’s limits (kd < 10−5 s−1). The upper limit on the affinity, based on the calculated on-rate (ka = 3.8 × 10−5 M −1s−1 and kd = 10−5 s−1 (instrumental limit)), was 26 pM. Compared to the starting affinity, these three modifications improved the affinity by more than four orders of magnitude. To more accurately quantify the increase in affinity, we conducted SPR experiments at 37 °C. The extended, 5-diphenylalanine meditope with the Ile83Glu Fab mutation produced a final KD of 0.40 nM (Fig. 1c, lower right panel), increasing the half-life of the interaction from 16 s to almost 40 min at 37 °C. Thus, at physiological temperatures, these three modifications collectively produced a 2500-fold increase in affinity (initial KD = 1000 nM; final KD = 0.40 nM, Fig. 1c). Mechanically interlocked meditope mAb complex. Although considerably longer than the initial half-life of the meditope/ meditope-enabled Fab complex, a half-life of 40 min is short compared to the half-life of mAbs in vivo, often greater than 14 days10. We observed in the structure of the meditope/medi- tope-enabled Fab complex that the Arg8 side chain of the med- itope is partially accessible from the other side of the Fab hole (Fig. 2a). Therefore, we investigated if we could incorporate a non-natural amino acid with an orthogonal reactive group at this position, such that the side chain would extend through the hole of the meditope-enabled Fab and expose the reactive group to the other side of the Fab11,12. Then, exposing the modified meditope/ Fab complex to a steric group that specifically reacts with the modified side chain would effectively lock the meditope onto the Fab (Fig. 2b). ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 W W e recently discovered a unique peptide binding site within a hole created by the light and heavy chains of the Fab domain of cetuximab1, an anti-epidermal growth factor receptor monoclonal antibody (mAb) used clini- cally to treat head and neck and colorectal cancers (Fig. 1a). Because the position of the binding site lies within the middle of the Fab arm, we named the peptide, CQFDLSTRRLKC, that binds to this site a meditope. The residues that line the meditope binding site in the Fab are unique to cetuximab and not present in human mAbs1. Therefore, we hypothesized this site could be used as a unique ‘receptor’, not only for potentially attaching cargo2,3, but also for emerging diagnostic techniques such as pre- targeted imaging4. Showing broad applicability of this technology, we successfully grafted the meditope site onto other mAbs, including trastuzumab, an mAb used to treat human epidermal growth factor receptor 2 (HER2)-positive breast cancer1, and M5A, an anti-carcinoembryonic antigen (CEA) mAb5. We refer to mAbs onto which we have grafted the meditope site as meditope-enabled antibodies (memAbs). The affinity of the above memAbs for their cognate antigens is indistinguishable from that of the parental mAbs1,6. However, the half-life of the original meditope peptide–Fab complex is not optimal for a pre-formed memAb/drug-conjugated meditope combination to be success- fully used in vivo. Although, mAbs can circulate in the body for days to weeks, the half-life of the original meditope–Fab inter- action at 37 °C is only seconds. Herein, we introduce hydrogen bonds, increase the surface area, and eliminate strain to improve the half-life of the complex, allowing us to use click chemistry to sterically limit the dissociation of the meditope through the for- mation of a mechanical bond. We demonstrate that the mechanical bond permits the functionalization of a memAb, including the addition of fluorescent groups that permits the imaging of tumors in vivo. that the guanidinium group of Arg9 in the meditope is partially buried in a cavity lined with Ile83 of the Fab light chain. This suggested substitution with a negative charge at Ile83 would result in a favorable buried salt bridge. Mutation of Ile83 to glutamate substantially improved the affinity for the original meditope (i.e., the unmodified CQFDLSTRRLKC peptide) to 23 nM as deter- mined by SPR (Supplementary Table 1). Results Improving the affinity of the interaction. Based on previous studies on meditope/cetuximab complexes, we substituted posi- tions 3 and 5 of the meditope with different side chains and variety of strategies for cyclizing the meditope7,8. We then used surface plasmon resonance (SPR) to characterize the affinity and kinetics of binding of these variants to a trastuzumab-based, anti- HER2 memAb (Supplementary Table 1). Of the modifications tested, substitution of Leu5 with L-diphenylalanine produced a significant increase in the affinity of the meditope to the anti- HER2 memAb, from 1.2 μM to 40 nM at 25 °C. Based on the kinetic fits, this increase was largely due to a faster on-rate (Fig. 1b and Supplementary Table 1). The structure of this modified meditope bound to the Fab of the anti-HER2 memAb indicated that the diphenyl group straddles Leu115 of the Fab heavy chain (Fig. 1c and Table 1), increasing the contact area by ~30 Å2 9. Because diphenylalanine is a bulky, β-branched amino acid, part of the gain in affinity may reflect reduced entropy, which also contributes to a faster on-rate. In addition, we found that extending the C-terminus of the original meditope by four residues, Gly–Gly–Ser–Lys, to subsequently add a fluorophore through amine chemistry, also improved the affinity. These four residues, however, were disordered in the crystal structure of the extended meditope bound to the anti-HER2 memAb. Therefore, additional experiments are necessary to better understand the contribution of these residues to the increase in the overall affi- nity. A limited set of additional substitutions to the original meditope did not produce substantial gains in affinity (Supple- mentary Table 1). p g To create the mechanical bond, we added a two- or a three- subunit ethylene linker with a terminal azide to the guanidinium group of arginine, which we then incorporated into the Arg8 position of the meditope. Using SPR, we found that this 5- diphenylalanine-8-Arg-(polyethyleneglycol)2-azido-meditope bound to the Ile83Glu meditope-enabled Fab with similar affinity as the 5-diphenylalanine meditope (KD of 2.1 nM vs. 0.40 nM, respectively) but with a slower on-rate and off-rate, correspond- ing to a longer half-life (τ = 83 min at 37 °C) (Supplementary Fig. 1a). Crystal structures of the 5-diphenylalanine-8-Arg- (PEG)2- and 5-diphenylalanine-8-Arg-(PEG)3-azido-meditope bound to the Ile83Glu meditope-enabled Fab showed clear electron density of the meditope as well as the PEG-azide. ARTICLE In other words, we sought to generate a mechanical bond to sterically limit the dissociation of a functionalized meditope from the Fab (Fig. 2c)13. | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications Mechanically interlocked functionalization of monoclonal antibodies Krzysztof P. Bzymek1, James W. Puckett1, Cindy Zer1, Jun Xie1, Yuelong Ma1, Jeremy D. King1, Leah H. Goodstein1, Kendra N. Avery1,4, David Colcher 2, Gagandeep Singh3, David A. Horne1 & John C. Williams 1 Because monoclonal antibodies (mAbs) have exceptional specificity and favorable pharma- cology, substantial efforts have been made to functionalize them, either with potent cyto- toxins, biologics, radionuclides, or fluorescent groups for therapeutic benefit and/or use as theranostic agents. To exploit our recently discovered meditope–Fab interaction as an alternative means to efficiently functionalize mAbs, we used insights from the structure to enhance the affinity and lifetime of the interaction by four orders of magnitude. To further extend the lifetime of the complex, we created a mechanical bond by incorporating an azide on the meditope, threading the azide through the Fab, and using click chemistry to add a steric group. The mechanically interlocked, meditope–Fab complex retains antigen specificity and is capable of imaging tumors in mice. These studies indicate it is possible to “snap” functionality onto mAbs, opening the possibility of rapidly creating unique combinations of mAbs with an array of cytotoxins, biologics, and imaging agents. 1 Department of Molecular Medicine, Beckman Research Institute of City of Hope, 1710 Flower St., Duarte, CA 91010, USA. 2 Department of Molecular Immunology, Beckman Research Institute of City of Hope, 1710 Flower St., Duarte, CA 91010, USA. 3 Department of Surgery, Beckman Research Institute of City of Hope, 1710 Flower St., Duarte, CA 91010, USA. 4Present address: Xencor, 111W. Lemon Ave., Monrovia, CA 91016, USA. Correspondence and requests for materials should be addressed to J.C.W. (email: jcwilliams@coh.org) | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications 1 NATURE COMMUNICATIONS| (2018) 9:1580 Results Important for creating the mechanical bond, the structures y Turning to the Fab, examination of the crystal structure of the meditope bound to the Fab of the anti-HER2 memAb revealed NATURE COMMUNICATIONS| (2018) 9:1580 | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 ARTICLE indicated the azide was threaded through the Fab hole (Supplementary Fig. 1b, c and Table 1). (Supplementary Fig. 3). In control SEC experiments, the DIBO- AF647 dye alone did not co-migrate with the meditope-enabled Fab either in the absence of the azido-meditope or the presence of a non-azido-meditope. Although we have made multiple attempts at crystallizing the mechanically interlocked meditope/meditope- enabled Fab complex, the flexibility and stereochemical hetero- geneity of the system have thus far precluded us from obtaining atomic resolution data of the interlocked complex. Therefore, to provide evidence that the click reaction did in fact produce a We locked the azido-meditope onto the meditope-enabled Fab by reacting the complex with dibenzocyclooctyne-AlexaFluor647 (DIBO-AF647). We then isolated the complex by size exclusion chromatography (SEC), monitoring the absorbance at 280 nm and at 647 nm. The signal from AF647 overlapped with the 280 nm signal from the meditope-enabled Fab, suggesting that the meditope was interlocked with the meditope-enabled Fab 10 pM 100 pM LE L Ori DLE 100 nM 1 µM 1 nM Dissociation constant, KD KD = 1.0 µM KD = 39 nM KD = 20 nM KD = 0.40 nM Dissociation rate, kd (s–1) QFD I83 E83 5-Diphenylalanine 10 nM 10 µM 100 µM 6 Association rate, ka (M–1 s–1/log) 5 4 3 60 Res. units (AU) 40 20 0 Res. units (AU) 40 80 0 80 Res. units (AU) 40 0 80 Res. Discussion Finally, we used click chemistry to demonstrate the possibility of adding functionalities other than fluorophores to the complex. In one instance, we interlocked a dibenzylcyclooctyne (DBCO)- His6 moiety, which can facilitate purification of the mechanically interlocked complex from unreacted substrates (Supplementary Figs. 2 and 8d). We also conjugated the azido-meditope/ meditope-enabled Fab complex to magnetic beads conjugated with DBCO (Supplementary Fig. 9). Finally, to create a “molecular dumbbell,” we conjugated DBCO-(PEG)4-NHS to an acetylated, seven amino acid peptide with lysines at the N- and C- termini and reacted this bis-DBCO linker with the azido- meditope/meditope-enabled Fab complex. Native mass spectro- metry indicated the formation of a mechanically interlocked molecule (Fig. 2d and Supplementary Fig. 10). The combination of orthogonal conjugation chemistries (e.g., azide/alkyne11,12, tetrazine/trans-cyclooctene (TCO)14,15, etc.) and multivalent linkers opens the possibility of creating novel, bi-specific and multi-specific Fab/IgG complexes16,17. To leverage their high specificity and favorable pharmacokinetics/ pharmacodynamics, there has been and continues to be con- siderable interest to add functionality to monoclonal antibodies for therapeutic and diagnostic intent18–21. Invariably, each of these approaches involves chemical modification directly to the mAb. The approach developed here differs from these methods— the interlocked mechanical bond is sterically restricted and non- covalent by nature22. To achieve this, we needed to ensure the lifetime of the meditope-mAb complex was sufficiently long that the capping of the meditope using click chemistry would pre- dominantly occur as part of the complex. Using a rational, structural-based approach, we significantly enhanced the affinity of the meditope/meditope-enabled Fab interaction. Next, we modified the guandinium group of arginine 8 within the medi- tope to thread an azide group through the Fab hole. Using cop- per-free, click chemistry, we were able to generate several mechanically interlocked meditope/mAb complexes. Functiona- lization of the mAbs/Fabs reported here did not affect their antigen-binding properties as evidenced by SPR (Supplementary Fig. 8) and/or analytical cytometry (Fig. 3a, b and Supplementary Fig. 7b and 7c). As an example of its potential use in the clinic, we demonstrate that the interlocked azido-meditope–AF647/Ile83- Glu anti-HER2 memAb complex can be used to effectively image tumors in mice. Overall, the data presented here, to the best of our knowledge, are the first example of mechanically interlocked complex involving an antibody. Tumor imaging using the interlocked meditope IgG complex. Results units (AU) 40 0 0 400 Time (s) 800 1200 0 400 Time (s) 800 1200 0 400 Time (s) 800 1200 0 400 Time (s) 800 1200 –6 –4 –2 0 a b c RE COMMUNICATIONS| (2018) 9:1580 | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications 10 pM 100 pM LE L Ori DLE 100 nM 1 µM 1 nM Dissociation constant, KD Dissociation rate, kd (s–1) 10 nM 10 µM 100 µM 6 Association rate, ka (M–1 s–1/log) 5 4 3 –6 –4 –2 0 b b a 10 pM 100 pM LE L Ori DLE 100 nM 1 µM 1 nM Dissociation constant, KD KD = 1.0 µM KD = 39 nM Dissociation rate, kd (s–1) QFD I83 5-Diphenylalanine 10 nM 10 µM 100 µM 6 Association rate, ka (M–1 s–1/log) 5 4 3 60 Res. units (AU) 40 20 0 80 Res. units (AU) 40 0 0 400 Time (s) 800 1200 0 400 Time (s) 800 1200 –6 –4 –2 0 a b c KD = 39 nM 5-Diphenylalanine 80 Res. units (AU) 40 0 0 400 Time (s) 800 1200 KD = 1.0 µM KD = QFD I83 5-Diphenylalani 60 Res. units (AU) 40 20 0 80 Res. units (AU) 40 0 0 400 Time (s) 800 1200 0 400 Time (s) 800 c I83 c KD = 20 nM KD = 0.40 nM Res. units (AU) 40 80 0 80 Res. units (AU) 40 0 Time (s) 0 400 Time (s) 800 1200 0 400 Time (s) 800 1200 Time (s) KD = 20 nM KD = 0.40 nM Res. units (AU) 40 80 0 80 Res. units (AU) 40 0 ( ) 0 400 Time (s) 800 1200 0 400 Time (s) 800 1200 ( ) KD = 0.40 nM 80 Res. units (AU) 40 0 0 400 Time (s) 800 1200 NATURE COMMUNICATIONS| (2018) 9:1580 | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications NATURE COMMUNICATIONS| (2018) 9:1580 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 interlocked complex binds CD3-bearing Jurkat cells (Fig. 3b). Of note, Okt3 binds to human CD3 but does not cross react with murine CD3. Next, the azido-meditope–AF647/Ile83Glu anti- HER2 memAb complex or azido-meditope–AF647/Ile83Glu anti- CD3 memAb complex was injected in the tail vein of NSG female mice (approximately 9 weeks old, Jackson Laboratory) bearing BT474 HER2-overexpressing breast tumor xenografts. Fig. 1 Increasing the affinity of the meditope site. a Surface representation of an IgG with a bound meditope (yellow). Light blue indicates the light chain and white indicates the heavy chain. b Kinetics and thermodynamics of meditope and antibody modifications (n = 1). Blue points represent data collected at 25 °C. Red points represent data collected at 37 °C (see also Supplementary Table 1). DLE—5-diphenylalanine long meditope ((Ac)CQFDA (Ph)2STRRLRCGGSK) binding to Ile83Glu anti-HER2 memAb; LE—long meditope ((Ac)CQFDLSTRRLRCGGSK) binding to Ile83Glu anti-HER2 memAb; L—long meditope ((Ac)CQFDLSTRRLRCGGSK) binding to anti-HER2 memAb antibody; ori—original meditope (CQFDLSTRRLKC) binding to anti-HER2 memAb. c SPR sensograms of meditope peptide variants binding to immobilized memAb variants at 37 °C (n = 1). Top, left sensogram—CQFDLSTRRLKC meditope (QFD, original unmodified meditope) binding to anti-HER2 memAb (original, meditope-enabled anti-HER2 antibody); top, right sensogram—(Ac) CQFDA(Ph)2STRRLRCGGSK (5-diphenylalanine long meditope) binding to anti-HER2 memAb; bottom, left sensogram—QFD binding to Ile83Glu anti- HER2 memAb; bottom, right sensogram—5-diphenylalanine long meditope binding to Ile83Glu anti-HER2 memAb. Residues highlighted in green correspond to modifications in the meditope (position 5, top panel) or Fab (position 83 LC, left panels) Results The mechanically interlocked, AF647–meditope/Ile83Glu anti-HER2 memAb accumulated at the tumor within 24 h of injection and remained at 72 h albeit with less intensity (Fig. 3c, top row). There was no signal observed in three of the four mice using the azido-meditope–AF647/Ile83Glu anti-CD3 memAb control. A weak signal at the site of the tumor was observed in the fourth mouse (and in the tail), but was absent by 72 h. After the final images were collected at 72 h, the mice were euthanized and their major organs imaged. The fluorescent signal was found pre- dominantly in tumors and residual signal was observed in the stomach (GI) using azido-meditope–AF647/Ile83Glu anti-HER2 memAb (Fig. 3d). No signal was observed in the tumors in the control group. However, there was some residual signal in the stomach in the controls, similar to the experimental group. These results were consistent with the initial imaging experiments (Supplementary Fig. 11). mechanically interlocked complex, we analyzed by mass spectro- metry the mechanically interlocked, meditope/Ile83Glu anti- HER2 memAb complex under native conditions as well as the mechanically interlocked Ile83Glu meditope-enabled Fab/medi- tope complex under denaturing conditions (Fig. 2d and Supplementary Fig. 4). The calculated mass from native mass spectra of the interlocked azido-meditope–AF647/Ile83Glu anti- HER2 memAb was consistent with the formation of the mechanical bond on one or both Fab arms of the IgG. g To test the stability of the mechanically interlocked complex, we incubated the interlocked azido-meditope–AF647/Ile83Glu meditope-enabled Fab at 37 °C for 11 days and used SEC to follow the dissociation of the complex (Supplementary Fig. 5). At 37 °C, the complex was stable over 11 days, as determined by SEC as well as by flow cytometry with mechanically interlocked Ile83Glu meditope-enabled IgG complex (Supplementary Figs. 5 and 6). To show this approach is generalizable to other mAbs, we created meditope-enabled, anti-CD3 (Okt3), anti-HER2 (pertu- zumab), and anti-CEA (M5A)6 antibodies (i.e., IgG), and used the same click chemistry to mechanically interlock the azido- meditope with DIBO–AF647. Each mechanically interlocked meditope–memAb complex bound to its cellular target as determined by analytical flow cytometry, indicating the formation of the mechanically interlocked bond does not affect antigen binding in general (Supplementary Fig. 7). | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications Discussion To demonstrate a potential application of our approach, we sought to use the interlocked meditope–mAb complex to image tumors in vivo in xenograft-bearing mice. First, we show using flow cytometry that the mechanically interlocked meditope–AF647/Ile83Glu anti-HER2 memAb complex bound to HER2-overexpressing BT474 cancer cells (Fig. 3a). As a control for the animal imaging studies, we also produced the mechani- cally interlocked meditope–AF647/Ile83Glu anti-CD3 memAb and demonstrated by analytical cytometry that this mechanically In future studies, we will refine the parameters to optimize the efficiency of the interlocking reaction, including characterization of differing orthogonal chemistries (e.g., thiols and tetrazine/TCO) NATURE COMMUNICATIONS| (2018) 9:1580 | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 Table 1 Data collection and refinement statistics Table 1 Data collection and refinement statistics Apo Ile83Glu Fab (5U3D) Ile83Glu Fab+ 5- diphenylalanine long meditopea (5U5F) Ile83Glu Fab+ 5-diphenylalanine-8- arginine-(PEG)2 azido-meditopea (5U6A) Ile83Glu+ 5-diphenylalanine-8- arginine-(PEG)3 azido-meditopea (5U5M) Data collection Space group P212121 P212121 P212121 P212121 Cell dimensions a, b, c (Å) 52.85; 104.65; 116.88 53.43; 105.38; 117.00 53.25; 105.17; 117.07 52.53, 105.47, 117.13 α, β, γ (°) 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 Resolution (Å) 33.43–1.77 33.64–1.81 31.53–1.74 31.62– 1.88 (1.82–1.77)b (1.86–1.81) (1.78–1.74) (1.93–1.88) Rmrgd-F 0.075 (0.760) 0.085 (0.784) 0.062 (0.485) 0.100 (0.863) I/σ(I) 18.6 (1.88) 16.1 (1.66) 15.6 (2.24) 16.4 (2.12) Completeness (%) 99.3 (91.1) 99.4 (93.7) 95.6 (79.0) 99.9 (99.8) Redundancy 4.5 (3.2) 3.9 (2.8) 2.5 (2.0) 5.2 (5.1) Refinement Resolution (Å) 1.77 1.81 1.74 1.88 No. reflections 63,311 60,648 65,699 54,735 Rwork/Rfree 15.7/18.5 16.3/19.2 15.9/18.8 16.3/19.7 No. atoms Protein 4350 4421 4467 4328 Meditope/ligand −/8 115/8 137/8 150/8 Water 803 798 857 732 B-factors Protein 21.4 23.1 19.5 25.1 Meditope/ligand –/28.2 22.7/20.0 24.4/21.5 27.5/22.2 Water 35.7 37.2 35.2 37.6 R.m.s. deviation Bond lengths (Å) 0.007 0.007 0.007 0.007 Bond angles (°) 1.048 1.075 1.098 1.082 a-5-diphenylalanine long meditope – (Ac)CQFDA(Ph)2STRRLRCGGSK; 5-diphenylalanine-8-Arg-(PEG)2-azido-meditope – (Ac)CQFDA(Ph)2STRXLRCGGSK, where X is arginine-(PEG)2-azide; 5- diphenylalanine-8-Arg-(PEG)3-azido-meditope – (Ac)CQFDA(Ph)2STRXLRCGGSK, where X is arginine-(PEG)3-azide bAll data were collected from single crystals. Highest resolution shell is shown in parenthesis a-5-diphenylalanine long meditope – (Ac)CQFDA(Ph)2STRRLRCGGSK; 5-diphenylalanine-8-Arg-(PEG)2-azido-meditope – (Ac)CQFDA(Ph)2STRXLRCGGSK, where X is arginine-(PEG)2-azide; 5- diphenylalanine-8-Arg-(PEG)3-azido-meditope – (Ac)CQFDA(Ph)2STRXLRCGGSK, where X is arginine-(PEG)3-azide bAll data were collected from single crystals. Highest resolution shell is shown in parenthesis and steric restraints. Synthesis of azido-meditope variants. Automated peptide synthesis was used to generate 2.46 g HMP Resin-KSGGCRLR-Orn(Alloc)-TS-3,3-diphenylalanine- Synthesis of azido-meditope variants. Automated peptide synthesis was used to generate 2.46 g HMP Resin-KSGGCRLR-Orn(Alloc)-TS-3,3-diphenylalanine- Synthesis of azido-meditope variants. Automated peptide synthesis was used to generate 2.46 g HMP Resin-KSGGCRLR-Orn(Alloc)-TS-3,3-diphenylalanine- DFQC-NHAc upon drying, starting from 500 μmol HMP Resin-Lys(Boc)- NHFmoc, sourced from CS Bio. All monomers were sourced from CS Bio, except Fmoc-Orn(Alloc)-OH, which was purchased from Alfa-Aesar. DFQC NHAc upon drying, starting from 500 μmol HMP Resin Lys(Boc) NHFmoc, sourced from CS Bio. All monomers were sourced from CS Bio, except Fmoc-Orn(Alloc)-OH, which was purchased from Alfa-Aesar. p Azido-meditope variants were synthesized according to the literature23. Briefly, resin II-20 (454 μmol, Supplementary Methods) was subjected to 20% piperidine in dimethylformamide (DMF) to remove Fmoc (1 × 5 min, 1 × 20 min). The resin was rinsed 3 × 10 mL DMF, 3 × 10 mL dichloromethane (DCM), 3 × 10 mL DMF. The resin was acetylated using 10 mL 1:2:7 acetic anhydride:N,N-diisopropylethylamine (DIEA):DMF (v/v/v). The resin was again rinsed 3 × 10 mL DMF, 3 × 10 mL DCM, 3 × 10 mL methanol (MeOH). The resin was dried in vacuo, and 247.5 mg of resin (~50 μmol) was then subjected to dealloc conditions: Pd(PPh3)4 (14.5 mg, 12.5 μmol), 1.3-dimethylbarbituric acid (DMBA) (78 mg, 500 μmol), and 1:1 N- methylpyrrolidine (NMP):DCM (2 mL). The resin was copiously washed with DMF, DCM, DMF, 1 vol. 5% DIEA in DMF (by volume), 2 × 5% sodium diethyldithiocarbamate trihydrate in NMP, and then copious amounts of DMF, DCM, and MeOH (greater than 10 washed at approximately twice the volume of resin) to deswell the resin. The resin was dried in vacuo. Compound a (667 mg, 312 μmol) in 1.5 mL tetrahydrofuran (THF) and 20 μL triethylamine (TEA) was added to the dried resin. The reaction proceeded for 20 h at room temperature in a rocking, manual, solid-phase peptide synthesis vessel. The Kaiser test of a small portion of the resin was positive. Compound a23 was reconcentrated and dissolved in DCM (1.5 mL). The coupling was restarted in the presence of DCM and 30 μL TEA. After 22 h, the Kaiser test was still positive, and semi-preparative HPLC of a small fraction of material showed ~50% conversion. The resin was rinsed 2 × DMF, 2 × DCM, 2 × DMF, 2 × DCM, deswollen in MeOH, and then dried in vacuo. | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications Discussion We will also create a toolbox of multiple meditope and DBCO analogs bearing cytotoxins (e.g., Auristatin), imaging agents (e.g., 1,4,7,10-tetraazacyclododecane-1,4,7,10-tet- raacetic acid (DOTA) for radionuclide imaging studies), and other functionalities. One potential application would be the generation of bi-specific antibodies with tunable lengths between the com- plementarity determining regions of the Fabs to optimize antibody-dependent cellular cytotoxicity (ADCC), an example of which is the “molecular dumbbell”. We anticipate this toolbox will be compatible with any memAb and allow for the rapid and consistent development of multiple functional biologic agents. 150 mm, 5 µm) with a water (0.1% TFA)/acetonitrile (0.1% TFA) solvent system. Alexa647- and Cy5-labeled peptides were synthesized from Alexa647-NHS and Cy5-NHS and purified using RP-HPLC. All peptides were characterized by mass spectrometry. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 Bottom panel: mass spectrum of the Ile83Glu, meditope-enabled Fab used for the reaction above; calculated mass: 4772 bserved mass: 47730 b “Meditope bolt” R1 R2 H2N N NH O O N N N N O + – K S G G C C Q F D X S R L R Z “Nut” T b “Meditope bolt” R1 R2 H2N N NH O O N N N N O + – K S G G C C Q F D X S R L R Z “Nut” T a b c “Meditope bolt” Meditope bolt Nut R1 R1 R1 R2 R2 Meditope- enabled FAB Antigen binding sites R1 R2 H2N N NH O O N N N N O + – K S G G C C Q F D X S R L R Z “Nut” T a b a c Meditope bolt Nut R1 R1 R1 R2 R2 Meditope- enabled FAB Antigen binding sites c c Meditope bolt R1 Meditope- enabled FAB Meditope- enabled FAB d 100 151720.0 148575.0 154975.0 Relative intensity (%) 80 60 40 20 0 100 Relative intensity (%) 80 60 40 20 0 148 150 152 MW (kDa) 154 156 158 148 150 152 MW (kDa) 154 156 158 e 47730 101655 100 Relative intensity (%) 80 60 40 20 0 100 Relative intensity (%) 80 60 40 20 0 50 70 90 MW (kDa) 110 130 150 170 30 50 70 90 MW (kDa) 110 130 150 170 30 d e Fig. 2 Development of a mechanical bond to interlock the meditope to a Fab. a Back view of Ile83Glu anti-HER2 antibody Fab with (Ac)CQFDA (Ph)2STRRLKC meditope (left) and 5-diphenylalanine-8-Arg-(PEG)2-azido-meditope (right). Light blue surface is the light chain; white surface is the heavy chain. The red arrows indicate the modified residue (arginine 8, left panel) and the reactive azide (right panel). b Chemical structures of the meditope bolt (meditope-containing reactive group) and nut (reactive steric group), the sequence is represented by single letter amino acid code where X represents 5- diphenylalanine and Z represents the modified azido-arginine. R1 and R2 represent potential modification sites. c Proposed scheme for forming a mechanical bond. After binding of the azido-meditope, the reactive azide on the backside of the Fab can react with a reactive cycloalkyne to form a mechanical bond. d Native mass spectrometry analysis of the mechanically interlocked meditope–AF647/Ile83Glu anti-HER2 memAb. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 a b c d e “Meditope bolt” Meditope bolt Nut R1 100 151720.0 148575.0 47730 154975.0 101655 Relative intensity (%) 80 60 40 20 0 100 Relative intensity (%) 80 60 40 20 0 100 Relative intensity (%) 80 60 40 20 0 100 Relative intensity (%) 80 60 40 20 0 148 150 152 MW (kDa) 154 156 158 148 150 152 MW (kDa) 154 156 158 50 70 90 MW (kDa) 110 130 150 170 30 50 70 90 MW (kDa) 110 130 150 170 30 R1 R1 R2 R2 Meditope- enabled FAB Antigen binding sites R1 R2 H2N N NH O O N N N N O + – K S G G C C Q F D X S R L R Z “Nut” T ig. 2 Development of a mechanical bond to interlock the meditope to a Fab. a Back view of Ile83Glu anti-HER2 antibody Fab with (Ac)CQFDA Ph)2STRRLKC meditope (left) and 5-diphenylalanine-8-Arg-(PEG)2-azido-meditope (right). Light blue surface is the light chain; white surface is the hea hain. The red arrows indicate the modified residue (arginine 8, left panel) and the reactive azide (right panel). b Chemical structures of the meditope b meditope-containing reactive group) and nut (reactive steric group), the sequence is represented by single letter amino acid code where X represents iphenylalanine and Z represents the modified azido-arginine. R1 and R2 represent potential modification sites. c Proposed scheme for forming a mechanical bond. After binding of the azido-meditope, the reactive azide on the backside of the Fab can react with a reactive cycloalkyne to form a mechanical bond. d Native mass spectrometry analysis of the mechanically interlocked meditope–AF647/Ile83Glu anti-HER2 memAb. The difference molecular weight (3145 and 6400 Da) corresponds to 1:1 or 2:1 complex of mechanically interlocked meditope:IgG (top panel). The spectrum for unlabe gG is shown in the bottom panel. The experimentally determined mass of meditope–DIBO–AF647 is 3293.3 Da (see Supplementary Fig. 4). e Mass pectrometry analysis of “molecular dumbbell”. Top panel: mass spectrum of purified reaction product; calculated mass: 101,634 (2×47730 + 2×2178 818), observed mass: 101,655. Methods R t A Reagents. All reagents were of American Chemial Society (ACS) or higher purity and were purchased from Thermofisher or Sigma unless specified below. Cy5-DBCO, Cy5-N-hydroxysuccinimide (NHS), DBCO-(PEG)4-NHS ester and sulfo DBCO-(PEG)4-maleimide, and DBCO-magnetic beads were purchased from Click Chemistry Tools. Cell lines (BT474 and LS174T) were obtained from ATCC, validated by flow cytometry, and tested monthly to ensure they were free of mycoplasma contamination (Universal Mycoplasma Detection Kit from ATCC). Peptide synthesis. Standard solid-phase N-αFmoc chemistry was used to syn- thesize meditope derivatives on CS136XT peptide synthesizer (CS Bio). Reagent K (trifluoroacetic acid (TFA)/water/phenol/thioanisole/1,2-ethanedithiol (EDT) = 82.5:5:5:5:2.5) was used to cleave the peptides from the resin. Crude peptides were collected by precipitation with cold ether. Oxidation using 20% DMSO in ammonium acetate buffer (pH 6) was performed for disulfide cyclization. All peptides were purified using a reverse-phase high-performance liquid chromato- graphy (RP-HPLC) (Agilent 1200 system with Agilent prep-C18 column, 21.2 × 5 | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications NATURE COMMUNICATIONS| (2018) 9:1580 | DOI: 10.1038/s41467-018-03976-5| www ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 The difference in molecular weight (3145 and 6400 Da) corresponds to 1:1 or 2:1 complex of mechanically interlocked meditope:IgG (top panel). The spectrum for unlabeled IgG is shown in the bottom panel. The experimentally determined mass of meditope–DIBO–AF647 is 3293.3 Da (see Supplementary Fig. 4). e Mass spectrometry analysis of “molecular dumbbell”. Top panel: mass spectrum of purified reaction product; calculated mass: 101,634 (2×47730 + 2×2178 + 1818), observed mass: 101,655. Bottom panel: mass spectrum of the Ile83Glu, meditope-enabled Fab used for the reaction above; calculated mass: 47720, observed mass: 47730 Cys-His6 peptide was conjugated with sulfo DBCO-(PEG)4-maleimide in PBS solution at room temperature for 1 h. After purification by RP-HPLC, the product was analyzed by HPLC and mass spectrometry (MS: [M + 2 H]2+– calculated MW 885.34; observed MW 885.26), and lyophilized. Reagent K (5 mL) was used for cleavage (3 h 40 min). The material was purified by semi-preparative HPLC, and pure fractions were combined. A disulfide bond was formed by oxidative cyclization using 20 mL 2.5% NH4OAc pH 6–7 with 20% dimethylsulfoxide (DMSO). The reaction was allowed to proceed for ~24 h and then purified using semi-preparative HPLC, affording 2.6 mg azido-meditope variant a (MS: [M + H]3+ calculated: 712.0072, observed: 712.0090). Azido- meditope variant b was synthesized similarly using compound b (variant b: MS: [M + H]3+ calculated: 726.6826, observed, 726.6844). Structures of meditope variants a and b are in Supplementary Methods. Antibody expression and purification. Both original and meditope-enabled anti- HER21, M5A6, OKT3, and pertuzumab (Supplementary Methods) light and heavy chain genes were purchased from Atum in pJ609 and pJ607 expression vectors, respectively. They were sub-cloned into vector 253074 (Atum) for expression. Ile83Glu substitution on the light chain of the meditope-enabled anti-HER2 mAb to increase meditope-binding affinity was generated using standard site-directed mutagenesis and verified by DNA sequencing. Peptide conjugation. Ac-KSADASK peptide was conjugated with DBCO-(PEG)4- NHS in 100 mM NaHCO3 buffer 1 h at room temperature. After purification by RP-HPLC, the product was lyophilized and analyzed by mass spectrometry (MS: [M + 2 H]2+ - calculated MW 908.93; observed MW 908.94) and HPLC. mAbs were produced using the ExpiCHO Expression System (Gibco). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 100 Count 0 100 Count 0 100 101 102 1 nM Interlocked meditope–AF647/ α-HER2 memAb Interlocked meditope–AF647/ α-hCD3 memAb 1 nM 10 nM 100 nM 100 nM 10 nM Fluorescence AF647 (AU) Fluorescence AF647 (AU) 103 104 100 101 102 103 104 a b 100 Count 0 100 101 102 1 nM Interlocked meditope–AF647/ α-HER2 memAb 100 nM 10 nM Fluorescence AF647 (AU) 103 104 a 100 Count 0 Interlocked meditope–AF647/ α-hCD3 memAb 1 nM 10 nM 100 nM Fluorescence AF647 (AU) 04 100 101 102 103 104 b b Count Count T K S L GI Interlocked meditope–AF647/ α-HER2 memAb 24 h 2.0e9 2.0e8 RAD 1.5e9 1.0e9 5.0e8 48 h 72 h 72 h c d T K S L GI 72 h d c Interlocked meditope–AF647/ α-hCD3 memAb 2.0e9 2.0e8 RAD 1.5e9 1.0e9 5.0e8 Interlocked meditope–AF647/ α-hCD3 memAb 2.0e9 2.0e8 RAD 1.5e9 1.0e9 5.0e8 Fig. 3 In vivo imaging. a Analytical flow cytometry analysis of BT474 HER2 overexpressing breast cancer cells incubated with 1, 10, or 100 nM of Ile83Glu anti-HER2 memAb with 5-diphenylalanine meditope–AF647 at a 1:1 molar ratio (blue), Ilu83Glu anti-HER2 memAb directly labeled with AF647 (green) or with mechanically interlocked 5-diphenylalanine-8-Arg-(PEG)2-azido-meditope–DIBO–AF647 Ile83Glu anti-HER2 memAb (red). b Analytical flow cytometry analysis of PBMCs incubated with 1, 10, and 100 nM labeled antibodies. The red trace is the mechanically interlocked, 5-diphenylalanine-8-Arg- (PEG)2-azido-meditope–DIBO–AF647 Ile83Glu anti-CD3 memAb (based on Okt3). The blue traces are cells treated with the anti-CD3 memAb with pre- bound 5-diphenylalanine-meditope-Alexa-647 (e.g., non-covalent). The black trace represents untreated cells. c Xenogen images of mice bearing BT474 xenograft tumors and injected through tail vain with mechanically interlocked meditope–AF647–Ile83Glu anti-HER2 memAb top row, n = 4) or mechanically interlocked meditope–AF647–Ile83Glu anti-CD3 memAb (control, bottom row, n = 4). Images were taken 24, 48, and 72 h after injection. d Tissue distribution of mechanically interlocked AF647–meditope–Ile83Glu anti-HER2 memAb and the mechanically interlocked AF647–meditope–Ile83Glu anti-CD3 memAb 3 days post-injection: tumors (T) and organs (K kidneys, L liver, S spleen, GI gastrointestinal tract) million cells. At 18 h post-transfection, ExpiFectamine, CHO Enhancer, and ExpiCHO Feed were added to the flask and then transferred to a 32 °C incubator (Max Titer Protocol). The culture supernatant was collected at 14 days post- transfection. The mAbs were purified from the culture supernatants using Protein G agarose beads (Genscript) and further purified on a Superdex HiLoad 16/60 75 or 200 pg column (GE Healthcare). | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 The vectors were co-transfected into ExpiCHO cells at 1 µg total plasmid DNA per 6 NATURE COMMUNICATIONS| (2018) 9:1580 | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS| (2 100 Count 0 100 Count T K S L GI 0 100 101 102 1 nM Interlocked meditope–AF647/ α-HER2 memAb Interlocked meditope–AF647/ α-HER2 memAb Interlocked meditope–AF647/ α-hCD3 memAb Interlocked meditope–AF647/ α-hCD3 memAb 1 nM 10 nM 100 nM 100 nM 10 nM Fluorescence AF647 (AU) 24 h 2.0e9 2.0e9 2.0e8 2.0e8 RAD RAD 1.5e9 1.0e9 5.0e8 1.5e9 1.0e9 5.0e8 48 h 72 h 72 h Fluorescence AF647 (AU) 103 104 100 101 102 103 104 a b c d n vivo imaging. a Analytical flow cytometry analysis of BT474 HER2 overexpressing breast cancer cells incubated with 1, 10, or 100 nM of Ile83G R2 memAb with 5-diphenylalanine meditope–AF647 at a 1:1 molar ratio (blue), Ilu83Glu anti-HER2 memAb directly labeled with AF647 (green) echanically interlocked 5-diphenylalanine-8-Arg-(PEG)2-azido-meditope–DIBO–AF647 Ile83Glu anti-HER2 memAb (red). b Analytical flow try analysis of PBMCs incubated with 1, 10, and 100 nM labeled antibodies. The red trace is the mechanically interlocked, 5-diphenylalanine-8-A azido-meditope–DIBO–AF647 Ile83Glu anti-CD3 memAb (based on Okt3). The blue traces are cells treated with the anti-CD3 memAb with p 5-diphenylalanine-meditope-Alexa-647 (e.g., non-covalent). The black trace represents untreated cells. c Xenogen images of mice bearing BT4 aft tumors and injected through tail vain with mechanically interlocked meditope–AF647–Ile83Glu anti-HER2 memAb top row, n = 4) or ically interlocked meditope–AF647–Ile83Glu anti-CD3 memAb (control, bottom row, n = 4). Images were taken 24, 48, and 72 h after injection distribution of mechanically interlocked AF647–meditope–Ile83Glu anti-HER2 memAb and the mechanically interlocked AF647–meditope–Ile83G 3 memAb 3 days post-injection: tumors (T) and organs (K kidneys, L liver, S spleen, GI gastrointestinal tract) E COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 ARTICL ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 References 1. Donaldson, J. M. et al. Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies. Proc. Natl Acad. Sci. USA 110, 17456–17461 (2013). Click chemistry. In a typical reaction, IgG or Fab (0.15 mM) was incubated in 1× PBS with azido-meditope (0.9 mM) at a 2.2:1 or 1.1:1 ratio, respectively for 15 min at room temperature followed by the addition of four molar excess of reactant (DBCO-peptide, 10 mM; DIBO-Alexa647, 1.6 mM). The reaction was allowed to proceed for 2–16 h at 25 °C and the final products were separated on PD10 col- umns (GE Healthcare Life Sciences) and further purified on a Superdex 75 or 200 Increase 10/300 GL column (GE Healthcare Life Sciences). Ile83Glu memAb anti- HER2 Fab with interlocked meditope–DBCO-His6 was generated in the same manner and purified from unreacted Fab using Ni-NTA beads (Qiagen). Unreacted DBCO-His6 was removed on a Zeba Spin desalting column with 7000 MW cutoff (ThermoFisher) and analyzed by native PAGE (Supplementary Figure 2). 2. Lambert, J. M. & Morris, C. Q. Antibody-drug conjugates (ADCs) for personalized treatment of solid tumors: a review. Adv. Ther. 34, 1015–1035 (2017). 3. Leal, M. et al. Antibody-drug conjugates: an emerging modality for the treatment of cancer. Ann. N Y Acad. Sci. 1321, 41–54 (2014). 4. Meyer, J. P., Adumeau, P., Lewis, J. S. & Zeglis, B. M. Click chemistry and radiochemistry: the first 10 years. Bioconjug. Chem. 27, 2791–2807 (2016). 5. Cutrone, G., Casas-Solvas, J. M. & Vargas-Berenguel, A. Cyclodextrin- modified inorganic materials for the construction of nanocarriers. Int. J. Pharm. 531, 621–639 (2017). 6. Zer, C. et al. Engineering a high-affinity peptide binding site into the anti-CEA mAb M5A. Protein Eng. Des. Sel. 30, 409–417 (2017). y y y g Interlocked Ile83Glu anti-HER2 Fab with 5-diphenylalanine-8-Arg-(PEG)2- azido-meditope–(Cy5)2–DBCO–magnetic beads was obtained as follows: 30 µL of DBCO–magnetic beads slurry (~0.33 mM DBCO) were separated from the solution using a magnetic separation rack (NEB) and washed 4 times with 50 µl of PBS, and resuspended in 30 µL of PBS. Fab was incubated with the azido-meditope at a 1.2:1 molar ratio for 15 min. Fab/meditope complex was mixed with DBCO-magnetic beads to a final molar ratio of 1.2:1:0.9. The admixture was allowed to mix overnight at room temperature in the dark. Beads were washed 10× with 50 µL of PBS before being resuspended in 60 µL of PBS. References The presence of Fab in solution and bound to the beads was assayed by Western blot using an α-human κ light chain- HRP antibody (AbCam). Control with Fab (to account for non-specific binding of Fab to the beads) was performed in an analogous manner. 7. Bzymek, K. P., Avery, K. A., Ma, Y., Horne, D. A. & Williams, J. C. Natural and non-natural amino-acid side-chain substitutions: affinity and diffraction studies of meditope-Fab complexes. Acta Crystallogr. F Struct. Biol. Commun. 72, 820–830 (2016). 8. Bzymek, K. P., Ma, Y., Avery, K. A., Horne, D. A. & Williams, J. C. Cyclization strategies of meditopes: affinity and diffraction studies of meditope-Fab complexes. Acta Crystallogr. F Struct. Biol. Commun. 72, 434–442 (2016). strategies of meditopes: affinity and diffraction studies of meditope Fab complexes. Acta Crystallogr. F Struct. Biol. Commun. 72, 434–442 (2016). 9. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011). y g y g 10. Correia, I. R. Stability of IgG isotypes in serum. MAbs 2, 221–232 (2010). h h f b h l b 11. Shieh, P. & Bertozzi, C. R. Design strategies for bioorthogonal smart probes. Org. Biomol. Chem. 12, 9307–9320 (2014). To generate a Fab-DBCO-(PEG)4-AcKSADASK-(PEG)4-DBCO-Fab “molecular dumbbell,” pre-formed Fab-5-diphenylalanine-8-Arg-(PEG)3-azido- meditope complex (0.4 mM, 15 min at room temperature at 1:0.9 molar ratio of Fab:azido-meditope) was allowed to react overnight at room temperature with 0.5 molar equivalent of 10 mM DBCO-(PEG)4-AcKSADASK -(PEG)4-DBCO and was purified on a Superdex 75 10/300 Increase column (GE Healthcare Life Sciences) and analyzed by mass spectrometry. Mass spectrometry data was acquired using a Waters Synapt G2G mass spectrometer and MassLynx software 12. Jewett, J. C. & Bertozzi, C. R. Cu-free click cycloaddition reactions in chemical biology. Chem. Soc. Rev. 39, 1272–1279 (2010). 13. Bruns, C. J. & Stoddart, J. F. The mechanical bond: a work of art. Top. Curr. Chem. 323, 19–72 (2012). 14. Blackman, M. L., Royzen, M. & Fox, J. M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity. J. Am. Chem. Soc. 130, 13518–13519 (2008). 15. Rossin, R., van Duijnhoven, S. M., Lappchen, T., van den Bosch, S. M. & Robillard, M. S. Trans-cyclooctene tag with improved properties for tumor pretargeting with the diels-alder reaction. Mol. Pharm. 11, 3090–3096 (2014). Stability of the interlocked meditope IgG complex. References For SEC experiments, the interlocked meditope Ile83Glu anti-HER2 Fab complex (starting volume 150 μL) was dialyzed against PBS (500 mL) in a dialyzer with an MWCO 7 kDa membrane. Buffer was changed at 62, 86, 110, 134, 159, and 230 h. Samples were assayed by SEC (S75 10/300 Increase) at 0, 62, 110, 159, 232, and 278 h. p g g 16. Spiess, C., Zhai, Q. & Carter, P. J. Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol. Immunol. 67, 95–106 (2015). For flow cytometry studies, the interlocked meditope–Ile83Glu–anti-HER2 IgG was diluted in PBS to 5 μM in 20 μL. The solution was dialyzed as described above, at 37 °C on an orbital shaker for 11 days with the PBS changed on days 2, 5, and 8. Samples were withdrawn after 1, 3, and 10 days and analyzed by flow cytometry with BT474 cells6. 17. Kraeber-Bodere, F. et al. A pretargeting system for tumor PET imaging and radioimmunotherapy. Front. Pharmacol. 6, 54 (2015). 18. Schmidt, M., Toplak, A., Quaedflieg, P. J. & Nuijens, T. Enzyme-mediated ligation technologies for peptides and proteins. Curr. Opin. Chem. Biol. 38, 1–7 (2017). 19. Behrens, C. R. & Liu, B. Methods for site-specific drug conjugation to antibodies. MAbs 6, 46–53 (2014). Animal experiments. All mouse experiments were approved by City of Hope Institutional Animal Care and Use Committee (IACUC 91037). Animal handling was conducted in accordance with ethical standards and regulations by IACUC. Twenty mice were used for the study to ensure enough mice bearing tumors of similar size. Each cohort included four mice (a typical sample size was n ≥3 per cohort), no randomization or blinding was used for this study. NOD/SCID/II-2rg (NSG) female mice (approximately 9 weeks old, Jackson Laboratory) were intra- muscularly injected with Delestrogen (0.8 mg/0.25 mL, estradiol valerate) 2 days before being subcutaneously injected in the shoulder or low flank with 4 × 106 mycoplasma-negative BT474 cells suspended in 1% human serum albumin in Hanks Balanced Salt Solution (HBSS) and then mixed to a 1:1 ratio with matrigel (BD) a total volume of 200 µL. Tumor xenografts were allowed to establish for 28 days, and confirmed by palpation (100 mm3 minimum tumor size). Interlocked AF647–meditope–anti-HER2 IgG or interlocked AF647–meditope–OKT3 IgG (100 μg in 200 µL saline) was administered through tail vein injection in four mice. 20. Liu, L. Pharmacokinetics of monoclonal antibodies and Fc-fusion proteins. Received: 15 August 2017 Accepted: 27 March 2018 Analyte samples were prepared as 2-fold serial dilutions in HBS-EP+ buffer (GE Healthcare Life Sciences) and were flowed over the immobilized ligand surface at 30 µL min−1. The running buffer in all experiments was HBS-EP+ and the regeneration buffer was 10 mM glycine pH 2.0 followed by a wash with HBS-EP+. Binding constants and kinetic rate constants were calculated using the 1:1 binding model in BiaEvaluation software. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 The sized mAbs were concentrated and buffer- exchanged into PBS with Amicon Ultra-15 centrifugal filter units (Millipore) and stored at 4 °C. Protein crystallization and structure determination. Crystals of apo Ile83Glu anti-HER2 memAb and its complexes with (Ac)CQFDA(Ph)2STRRLRCGGSK, 5- diphenylalanine-8-Arg-(PEG)2-azido-meditope or 5-diphenylalanine-8-Arg- (PEG)3-azido-meditope were obtained by hanging drop method by mixing an equal volume of protein and precipitant (100 mM Tris, pH 7.2, 16–18% PEG 3350 and 150–200 mM NaCl). Apo Ile85Glu anti-HER2 memAb was mixed with equimolar concentration of Protein A and Protein L to a final con- centration of 80 μM. Peptides were added to a final ratio of 1:10 of Fab:peptide. Crystals were cryoprotected in the precipitant solution with the addition of 20% meso-erythritol and frozen in a stream of cold nitrogen (100 K). Diffraction data were collected on a Rigaku Micromax X-007 HF with RAXIS IV++ detector at 100 K, processed with XDS24 and refined with Phenix25 with iterative Fab purification. Ile83Glu anti-HER2 Fab was generated with immobilized papain (Pierce) according to manufacturer’s protocol, followed by Q-sepharose chroma- tography and SEC purification. Purity was verified by SDS-PAGE1. 7 | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications NATURE COMMUNICATIONS| (2018) 9:1580 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 model building in Coot26. Final structures have 98.4% (5UED), 99.1% (5U5F), 98.6% (5U6A), and 99.0% (5U5M) of Ramachandran favored, and no Rama- chandran outliers. Mice were imaged at 24, 48, and 72 h post-injection using a Lago system (Spectral Instruments Imaging) with 640 nm excitation and 690 nm emission filters. For image acquisition, mice were sedated with isoflurane for approximately 5 min. Mice were euthanized after the 72-h time point and tumors and major organs (liver, kidneys, spleen, and tumor) were harvested. The tumors and organs were then imaged on the Lago system using the same filter sets. Surface plasmon resonance. Kinetic binding assays of peptides and memAb Fabs were obtained by SPR analysis on a Biacore T100 instrument. Ligands (memAb variants and sHER2) were prepared in 10 mM sodium acetate pH 5.5 (GE Healthcare Life Sciences) for covalent coupling onto series S CM5 sensor chips (GE Healthcare Life Sciences) using amine coupling chemistry at densities estimated to result in Rmax values between 50 and 150 RU using the equation: Surface plasmon resonance. Kinetic binding assays of peptides and memAb Fabs were obtained by SPR analysis on a Biacore T100 instrument. Ligands (memAb variants and sHER2) were prepared in 10 mM sodium acetate pH 5.5 (GE Data availability. All protein structures (5U3D, 5U5F, 5U6A and 5U5M) were deposited in the Protein Data Bank (http://www.rcsb.org). All relevant data are available from the authors. Healthcare Life Sciences) for covalent coupling onto series S CM5 sensor chips (GE Healthcare Life Sciences) using amine coupling chemistry at densities estimated to result in Rmax values between 50 and 150 RU using the equation: RL ¼ Rmax ´ ligand MW=analyte MW ð Þ ´ 1=Sm: RL ¼ Rmax ´ ligand MW=analyte MW ð Þ ´ 1=Sm: Received: 15 August 2017 Accepted: 27 March 2018 References Protein Cell, 9, 15–32 (2017). 21. Hu, Q.-Y., Berti, F. & Adamo, R. Towards the next generation of biomedicines by site-selective conjugation. Chem. Soc. Rev. 45, 1691–1719 (2016). 22. Fang, L. et al. Mechanically bonded macromolecules. Chem. Soc. Rev. 39, 17–29 (2010). 23. van Ameijde, J. et al. Preparation of novel alkylated arginine derivatives suitable for click-cycloaddition chemistry and their incorporation into pseudosubstrate- and bisubstrate-based kinase inhibitors. Org. Biomol. Chem. 8, 1629–1639 (2010). 24. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010 25. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010). 25. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010). | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications NATURE COMMUNICATIONS| (2018) 9:1580 | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03976-5 Competing interests: J.C.W. and D.A.H. have multiple patents (issued and pending) covering the meditope technology, are co-founders, shareholders, and members of the SAB of Meditope Biosciences, Inc. However, all of the work reported here was funded by charitable contributions to City of Hope and federal grants (i.e., none of the research reported here was supported by Meditope Biosciences, Inc.). The remaining authors declare no competing interests. 26. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010). 26. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010). Author contributions J.C.W., K.P.B., J.W.P., G.S., and D.A.H. conceived the study. K.P.B., J.W.P., C.Z., J.X., Y. M., J.D.K., L.H.G, and K.N.A. generated reagents, performed the experiments, and analyzed the data. K.P.B. coordinated the data collection. D.C. oversaw tumor-imaging studies. J.C.W. and K.P.B. co-wrote the paper. All authors contributed to the review of the manuscript. Acknowledgements We gratefully acknowledge support from the Alicia and John Kruger Gift (J.C.W. and D. A.H.), the Leo and Anne Albert Charitable Trust (J.C.W. and G.S.), W.M. Keck Medical Foundation (J.C.W. and D.A.H.), the Carl and Roberta Deutsch Foundation (J.C.W. and G.S.), and awards R21 CA135216 and R21 CA174608 from the National Cancer Institute (J.C.W.). Research reported in this publication included work performed in the Animal Resource Center, X-ray Crystallography, and Mass Spectroscopy and Proteomics Cores supported by the National Cancer Institute of the National Institutes of Health under award number P30CA033572 (PI, Steven Rosen). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Finally, we thank Kurt Jenkins, Victor Kenyon, and Kaniel Cassady as well as the past and present members of the Williams and Horne groups for technical support and helpful comments, Desiree Crow from the Small Animal Imaging Core, and Roger Moore from the Mass Spectrometry Core for their technical expertise. 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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) 2018 Additional information © The Author(s) 2018 Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018 03976 5 Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-03976-5. NATURE COMMUNICATIONS| (2018) 9:1580 | DOI: 10.1038/s41467-018-03976-5| www.nature.com/naturecommunications 9
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https://refubium.fu-berlin.de/bitstream/fub188/33614/1/a-taxonomic-reassessment-of-the-genus-sticta-lichenized-ascomycota-peltigeraceae-in-the-hawaiian-archipelago.pdf
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A taxonomic reassessment of the genus<i>Sticta</i>(lichenized Ascomycota:<i>Peltigeraceae</i>) in the Hawaiian archipelago
Lichenologist/˜The œLichenologist
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Introduction The Hawaiian lichen biota has generally been presumed to contain mostly widespread species, with a comparatively low degree of endemism at around 20–30% (Smith 1993, 2013; Eldredge & Miller 1995). In contrast, endemism in vascular plants is c. 80% (Wagner et al. 1999; Evenhuis & Eldredge 2002; Wagner & Herbst 2002). However, recent molecular phylogenetic approaches indicate that many of the presumably widespread taxa in Hawaii represent distinct, putatively endemic species. Thus, in Lobariella and Pseudocyphellaria, revised phylogenetic classifications suggest putative endemism to be around 75% (Moncada et al. 2014a; Lücking et al. 2017a). The Hawaiian lichen biota has generally been presumed to contain mostly widespread species, with a comparatively low degree of endemism at around 20–30% (Smith 1993, 2013; Eldredge & Miller 1995). In contrast, endemism in vascular plants is c. 80% (Wagner et al. 1999; Evenhuis & Eldredge 2002; Wagner & Herbst 2002). However, recent molecular phylogenetic approaches indicate that many of the presumably widespread taxa in Hawaii represent distinct, putatively endemic species. Thus, in Lobariella and Pseudocyphellaria, revised phylogenetic classifications suggest putative endemism to be around 75% (Moncada et al. 2014a; Lücking et al. 2017a). g The genus Sticta is the largest genus in Peltigeraceae subfamily Lobarioideae, with 200 species currently accepted and many more recognized on the basis of molecular phylogenetic studies (Moncada et al. 2014a, 2020; Lücking et al. 2017b; Simon et al. 2018). The most recent checklist of Hawaiian lichens (Smith 2013) listed 11 names under Sticta. One of these, S. crocatoides f. sandwi- censis Zahlbr., belongs in the genus Pseudocyphellaria (Moncada et al. 2014b). The ten remaining names included seven at species level and three at the level of variety, viz. S. ambavillaria (Bory) Ach., S. cyphellulata (Müll. Arg.) Hue, S. filix (Sw.) Nyl., S. fuliginosa (Dicks.) Ach., S. plumbicolor (Zahlbr.) Zahlbr., S. tomentosa (Sw.) Ach., S. weigelii (Ach.) Vain., S. weigelii var. beauvoisii (Delise) In the current study, we present an updated taxonomic treat- ment of the genus Sticta in the Hawaiian archipelago based on these results, including a revision of numerous collections, diag- nostic descriptions and illustrations, notes on the distribution and ecology of the species, and an identification key. Abstract The taxonomy of the genus Sticta in Hawaii is reassessed, based on a separately published molecular phylogeny using the fungal barcoding marker ITS. Based on Magnusson and Zahlbruckner’s treatment from 1943 and Magnusson’s catalogue from 1955, seven species of Sticta and three infraspecific taxa had been reported from the archipelago, all widespread except the putative endemic S. plumbicolor. Here we provide a taxonomic treatment of 13 taxa, 12 species and one subspecies, distinguished in a previous phylogenetic analysis: S. acyphellata, S. andina, S. antoniana, S. emmanueliana, S. flynnii, S. fuliginosa, S. hawaiiensis, S. limbata, S. plumbicolor, S. scabrosa subsp. hawaiiensis, S. smithii, S. tomen- tosa and S. waikamoi. All taxa are described, discussed and illustrated and a dichotomous key is presented. The implications of revised species taxonomies for studies in other fields such as ecology, ecophysiology, biogeography, biochemistry, and applications such as environmental mon- itoring are discussed. We also propose a protocol to use Sticta lichens to monitor the environmental health of Hawaiian ecosystems. Key words: environmental monitoring, Kauai, Lobarioideae, Maui, Molokai, Oahu (Accepted 1 July 2020) Hue, S. weigelii var. lutescens (Taylor) H. Magn. and S. weigelii var. peruviana (Delise) Vain. Sticta beauvoisii Delise was recently reinstated at species rank (McDonald et al. 2003; Galloway 2006), whereas the correct application of the names S. weigelii var. lutescens and S. weigelii var. peruviana is unclear (Moncada et al. 2020). Thus, prior to molecular studies, the genus Sticta was represented in Hawaii by eight species, with only one, S. plumbicolor, putatively endemic, resulting in an inferred endemism of 13%. However, a recent phylogenetic revision distinguished 13 species, including seven new to science and retaining only three previously reported taxa, S. fuliginosa, S. plumbicolor and S. tomentosa, thus raising the estimate of endemism to 69% (Moncada et al. 2020). A taxonomic reassessment of the genus Sticta (lichenized Ascomycota: Peltigeraceae) in the Hawaiian archipelago Bibiana Moncada1,4, Clifford W. Smith2 and Robert Lücking3,4 1Licenciatura en Biología, Universidad Distrital Francisco José de Caldas, Cra. 4 No. 26B-54, Torre de Laboratorios, Herbario, Bogotá, Colombia; 2Department of Botany, University of Hawaii, 3190 Maile Way, St. John Hall Room #409, Honolulu, HI 96822, USA; 3Botanischer Garten und Botanisches Museum, Freie Universität Berlin, Königin-Luise-Straße 6–8, 14195 Berlin, Germany and 4Research Associate, Negaunee Integrative Research Center, The Field Museum, 1400 South Lake Shore Drive, Chicago, Illinois 60605, USA The Lichenologist (2021), 53, 117–133 doi:10.1017/S0024282920000353 Standard Paper https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at © The Author(s), 2021. Published by Cambridge University Press on behalf of the British Lichen Society. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited Author for correspondence: Robert Lücking. E-mail: r.luecking@bgbm.org The Lichenologist (2021), 53, 117–133 doi:10.1017/S0024282920000353 The Lichenologist (2021), 53, 117–133 doi:10.1017/S0024282920000353 The Lichenologist (2021), 53, 117–133 doi:10.1017/S0024282920000353 Material and Methods The underlying revised taxonomy of Hawaiian Sticta was based on a recent phylogenetic revision, which revealed recently collected Hawaiian material as belonging to 10 clades representing 13 species (Moncada et al. 2020). Based on these results, we also revised herb- arium collections mostly at HAW but also at some other herbaria (DUKE, F, NY, US) and in the Lichen Portal of the Consortium of North American Lichen Herbaria (https://lichenportal.org/cnalh), to assign specimens to the newly recognized taxa. Cite this article: Moncada B, Smith CW and Lücking R (2021) A taxonomic reassess- ment of the genus Sticta (lichenized Ascomycota: Peltigeraceae) in the Hawaiian archipel- ago. Lichenologist 53, 117–133. https://doi.org/10.1017/S0024282920000353 © The Author(s), 2021. Published by Cambridge University Press on behalf of the British Lichen Society. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. rg/10.1017/S0024282920000353 Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at 118 Bibiana Moncada et al. Bibiana Moncada et al. ZEISS Axioskop compound microscope. We employed thin-layer chromatography (TLC) according to Orange et al. (2010). ZEISS Axioskop compound microscope. We employed thin-layer chromatography (TLC) according to Orange et al. (2010). Morphological and anatomical details of the specimens were studied using a LEICA Zoom 2000 dissecting microscope and a Taxonomic Treatment Key to species of Sticta in Hawaii 1 Vegetative propagules or marginal lobules absent; apothecia present; underside whitish, with thin tomentum and minute cyphellae; Kauai, Oahu, Molokai, Maui, Hawaii (Fig. 1A–D) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sticta tomentosa Vegetative propagules or marginal lobules present; apothecia present or absent; underside variable . . . . . . . . . . . . . . . . . 2 2(1) Vegetative propagules soredia, predominantly marginal, white-grey; lobes broadly rounded; Kauai, Maui (Fig. 1E–H) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . Sticta limbata Vegetative propagules isidia or phyllidia, or marginal lobules present, concolorous with the thallus or darker; lobes variable . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3(2) Vegetative propagules predominantly laminal isidia, regularly dispersed over the entire lobe surface; lobes more or less broadly rounded . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Vegetative propagules predominantly marginal isidia, phyllidia, or with marginal lobules, if also laminal then clustered and leaving larger surface areas free; lobes irregularly truncate to elongate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4(3) Thallus extremely small (often only to 0.5 cm), pedunculate; cyphellae absent; Oahu (Fig. 1I–L) . . . . . Sticta acyphellata Thallus small to robust (1–5 cm diam.), not pedunculate; cyphellae present . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5(4) Thallus small (c. 1 cm diam.); lobes with conspicuous marginal, white cilia; cyphellae very small (to 0.2 mm diam.), rounded; Kauai, Oahu, Maui (Fig. 1M–P) . . . . . . . . . . . Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sticta hawaiiensis Thallus robust (to 5 cm diam.); lobes lacking distinct cilia; cyphellae becoming large (1–3mm diam.) and irregularly angulate; Kauai, Oahu, Maui, Hawaii (Fig. 1Q–T) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sticta fuliginosa 6(3) Thallus with somewhat large, strictly marginal lobules; apothecia rather frequent; lower tomentum rather thick but abruptly ending and leaving bare lobe margins; Kauai, Maui (Fig. 1U–X) . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sticta antoniana Thallus with small, predominantly marginal but sometimes also laminal isidia or phyllidia; apothecia rare or absent . . . . 7 7(6) Thallus very small (c. 1 cm), distinctly pedunculate, consisting of sparsely branched lobes with a basal stipe ascending from the substratum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Thallus small to robust (1–10 cm diam.), not pedunculate, with much branched lobes, overgrowing the substratum more or less horizontally . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Material and Methods 9 8(7) Cyphellae becoming rather large (1–3 mm diam.) and irregular; Kauai, Maui (Fig. 2A–D) . . . . . . . . . . . . . . Sticta flynnii Cyphellae remaining small (0.2–0.5 mm diam.) and rounded; Oahu (Fig. 2E–H) . . . . . . . . . . . . . . . . . . . . Sticta smithii 9(7) Thallus remaining small (1–3 cm diam.); lobes with distinct black, marginal cilia; Kauai, Maui (Fig. 2I–L) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sticta emmanueliana Thallus becoming robust (3–10 cm diam.); lobes lacking or with indistinct or whitish cilia . . . . . . . . . . . . . . . . . . . . . 10 10(9) Thallus bluish grey when fresh, bluish to yellowish grey when dry, with conspicuous phyllidia; underside whitish, with small, inconspicuous cyphellae; scattered apothecia usually present; Kauai, Oahu, Molokai, Maui, Hawaii (Fig. 2M–P) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sticta plumbicolor Thallus olive green to grey-brown when fresh, brownish when dry, with cylindrical to flattened isidia or small phyllidia; under- side light to dark brown, with conspicuous cyphellae contrasting with the darker tomentum . . . . . . . . . . . . . . . . . . Material and Methods 11 11(10) Lower tomentum marginally pale; vegetative propagules (phyllidia) marginal and laminal (then clustered), concolorous with thallus; lobe surface often thinly scabrous, particularly towards the tips, and also often shallowly scrobiculate-foveolate; Kauai, Oahu, Molokai, Maui, Hawaii (Fig. 2Q–T) . . . . . . . . . . . . . . . . . . . . . . . . . Sticta scabrosa subsp. hawaiiensis Lower tomentum uniformly dark; vegetative propagules (isidia or phyllidia) predominantly marginal, cylindrical, darker than the thallus; lobe surface glabrous, even to uneven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 12(11) Usually on soil between bryophytes, rarely epiphytic over bryophyte mats; lobes 4–7 mm broad; with cylindrical to somewhat flattened isidia; rhizines conspicuous; Maui, Hawaii (Fig. 2U–V) . . . . . . . . . . . . . . . . . . . . . . . . . . . Sticta waikamoi Usually epiphytic on branches, stems or tree trunks; lobes 7–15 mm broad; mostly with flattened and dorsiventral phyllidia; rhizines short; Kauai, Oahu, Maui, Hawaii (Fig. 2W–X) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sticta andina https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at Taxonomic Treatment Key to species of Sticta in Hawaii 119 The Lichenologist i Habitus of Hawaiian Sticta. A–D, S. tomentosa. E–H, S. limbata. I–L, S. acyphellata. M–P, S. hawaiiensis. Q–T, S. fuliginosa. U–X, S. antoniana. Most of these s were taken in the field, therefore no scales are added. Sticta antoniana, S. fuliginosa, S. limbata and S. tomentosa are conspicuous species, whereas S. ellata and S. hawaiiensis are comparatively small, their individual thalli usually not exceeding 1 cm. In colour online. Fig. 1. Habitus of Hawaiian Sticta. A–D, S. tomentosa. E–H, S. limbata. I–L, S. acyphellata. M–P, S. hawaiiensis. Q–T, S. fuliginosa. U–X, S. antoniana. Most of these images were taken in the field, therefore no scales are added. Sticta antoniana, S. fuliginosa, S. limbata and S. tomentosa are conspicuous species, whereas S. acyphellata and S. hawaiiensis are comparatively small, their individual thalli usually not exceeding 1 cm. In colour online. The species The species (Fig. 1I–L) (Fig. 1I–L) Sticta acyphellata Moncada & Lücking Sticta acyphellata Moncada & Lücking Primary photobiont cyanobacterial (Nostoc). Stipe present, short. Thallus monophyllous, individual thalli irregularly arranged, deli- cate, irregularly branched and dissected; lobes to 5 mm broad, spathuliform to truncate, ascending, not usually overlapping, Primary photobiont cyanobacterial (Nostoc). Stipe present, short. Thallus monophyllous, individual thalli irregularly arranged, deli- cate, irregularly branched and dissected; lobes to 5 mm broad, spathuliform to truncate, ascending, not usually overlapping, Primary photobiont cyanobacterial (Nostoc). Stipe present, short. Thallus monophyllous, individual thalli irregularly arranged, deli- cate, irregularly branched and dissected; lobes to 5 mm broad, spathuliform to truncate, ascending, not usually overlapping, Moncada et al., Pl. Fung. Syst. 65, 106 (2020); type: USA, Hawaii, Oahu, Koolau Range, Manoa Valley, 21°19ʹ55ʺN, 157°48ʹ43ʺW, 410–575 m, 2013, B. Moncada, R. Lücking & C. W. Smith 6923 (F—holotype!; B, HAW—isotypes!). https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at 120 Bibiana Moncada et al. . Habitus of Hawaiian Sticta. A–D, S. flynnii. E–H, S. smithii. I–L, S. emmanueliana. M–P, S. plumbicolor. Q–T, S. scabrosa subsp. hawaiiensis. U–V, S. waikamoi. S. andina. Most of these images were taken in the field, therefore no scales are added. Sticta andina, S. fuliginosa, S. plumbicolor, S. scabrosa subsp. hawaiien- d S. waikamoi are conspicuous to large taxa, whereas S. flynnii and S. smithii are comparatively small, their individual thalli usually not exceeding 1 cm; S. nueliana is intermediate in size. In colour online. Fig. 2. Habitus of Hawaiian Sticta. A–D, S. flynnii. E–H, S. smithii. I–L, S. emmanueliana. M–P, S. plumbicolor. Q–T, S. scabrosa subsp. hawaiiensis. U–V, S. waikamoi. W–X, S. andina. Most of these images were taken in the field, therefore no scales are added. Sticta andina, S. fuliginosa, S. plumbicolor, S. scabrosa subsp. hawaiien- sis and S. waikamoi are conspicuous to large taxa, whereas S. flynnii and S. smithii are comparatively small, their individual thalli usually not exceeding 1 cm; S. emmanueliana is intermediate in size. In colour online. branched and coralloid; arbusculae to 0.2 mm long and broad, distinctly flattened, their base lacking cyphellae, concolorous with the thallus. Lower surface uneven, forming vein-like ridges, minutely scrobiculate-foveolate between the ridges, white to cream. Primary tomentum dense but short and inconspicuous, whitish; hairs 20–70 μm long, composed of single, mostly plane to involute, their margins becoming dissected. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at Secondary chemistry. No substances detected by TLC; medulla K−. Sticta andina Moncada, Lücking & Sérusiaux Sticta andina Moncada, Lücking & Sérusiaux Moncada et al., Willdenowia (in press); type: Colombia, Bogotá, D.C., Sendero Peña Blanca, 04°26ʹ08ʺ to 04°26ʹ19ʺN, 74°08ʹ36ʺ to 74°08ʹ37ʺW, 2840–2860 m, 2015, R. Lücking (with C. Vargas) 39464 (B—holotype!; JBB—isotype!). Moncada et al., Willdenowia (in press); type: Colombia, Bogotá, D.C., Sendero Peña Blanca, 04°26ʹ08ʺ to 04°26ʹ19ʺN, 74°08ʹ36ʺ to 74°08ʹ37ʺW, 2840–2860 m, 2015, R. Lücking (with C. Vargas) 39464 (B—holotype!; JBB—isotype!). Remarks. Sticta andina was described recently to accommodate a common taxon mostly occurring in the northern Andes. It corre- sponds to the S. weigelii morphodeme but is not related to S. wei- gelii s. str., differing in the thick lower tomentum and whitish cyphellae lacking yellow pigment (Moncada et al. 2021). Sticta andina is characterized by a rather robust thallus with an olive-brown colour when fresh (yellowish brown in the herbar- ium), with predominantly marginal, rather dark, mostly flattened isidia and phyllidia. It can be distinguished from S. scabrosa subsp. hawaiiensis by the glabrous, more or less shiny lobe sur- face, by the isidia and phyllidia being darker than the thallus, and by the uniformly dark lower tomentum. Its ecology is also different, being found mostly in higher elevation rainforest, whereas S. scabrosa subsp. hawaiiensis is a weedy taxon typically occurring in lower altitude rainforest or secondary and anthropo- genic vegetation. Sticta waikamoi is quite similar to S. andina and co-occurs with it in the same habitats, but is a smaller lichen over- all, with narrower lobes forming cylindrical to somewhat flattened isidia and conspicuous rhizines; in addition, it often also grows on the ground. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at Sticta acyphellata Moncada & Lücking Upper sur- face even, dark bluish grey when fresh, bluish grey when dry, glab- rous, without papillae or pruina, with white maculae forming a reticulate pattern. Isidia frequent, marginal and laminal, 0.1–0.2 mm long and 0.05–0.1 mm broad, dark grey, shiny, flattened and somewhat imbricate, becoming somewhat arbuscular, 121 The Lichenologist unbranched, cylindrical, colourless hyphae. Secondary tomentum absent. Cyphellae absent. per cm2, rounded, plane, erumpent to prominent, white, strongly contrasting with the dark tomentum; pore 0.3–1(–1.8) mm diam.; cells of basal membrane lacking papillae. yp Apothecia not observed. yp Apothecia not observed. g Apothecia not rare, often on thalli lacking phyllidia, mostly submarginal, dispersed, sessile to substipitate with a pronounced invagination on the underside, 2–4 mm diam., 0.5–0.6 mm high; disc orange-brown; proper margin verrucose to crenulate, some- times thinly pilose when young, dark brown. Excipulum 100– 150 μm broad. Hymenium 115–155 μm high; epithecium 2.5–5 μm thick, orange-brown. Ascospores fusiform, 1–3-septate, 27– 38 × 5.5–7.5 μm. Secondary chemistry. No substances detected by TLC; medulla K−. Secondary chemistry. No substances detected by TLC; medulla K−. Secondary chemistry. No substances detected by TLC; medulla K−. Secondary chemistry. No substances detected by TLC; medulla K−. Distribution and ecology. This species is thus far known only from the island of Oahu, probably overlooked elsewhere. It is dif- ficult to detect in the field due to its diminutive size. It is found in liverwort carpets, such as Odontoschisma spp., on the lower trunk and exposed roots, generally no higher than 1.5 m above ground, in open, non-native, mature rainforest understory at lower eleva- tions (410–575 m). The only identified phorophytes to date are Elaeocarpus bifidus and Cordyline fruticosa (as C. terminalis). Secondary chemistry. No substances detected by TLC; medulla and basal membrane of cyphellae K+ yellowish. Distribution and ecology. Sticta andina is apparently native to the Neotropics and found most frequently in the northern Andes (Moncada et al. 2021). In Hawaii, this species is known from Hawaii, Kauai, Maui and Oahu, with many collections ori- ginating from introduced conifer forest on Maui. A lack of devi- ation in the ITS sequences compared to the main haplotype found in the northern Andes suggests the records from Hawaii to be the result of recent, perhaps anthropogenic long-distance dispersal (Moncada et al. 2020). In the northern Andes, Sticta andina is an upper montane to alpine taxon, typically confined to undis- turbed forest and shrubby habitats, where it grows on trees and shrubs, rarely on rock. In Hawaii it was found in partially dis- turbed native rainforest and gulches of adjacent upland dry forest, between 1200 and 2400 m, with most collections originating from between 1500 and 2000 m. Reported phorophytes mostly include Cheirodendron and Metrosideros, but also Coprosoma (montana), Leptocophylla, Myrsine, Sophora (chrysophylla) and Vaccinium. Remarks. This species is unique within the genus due to its diminutive size and the complete lack of cyphellae. It provides a contrary example of what is seen in Lobaria anomala (Brodo & Ahti) T. Sprib. & McCune and L. anthraspis (Ach.) T. Sprib. & McCune. Although producing pseudocyphellae, these two species form part of Lobaria s. str., which otherwise lacks lower surface pores (McCune et al. 2014; Cornejo & Scheidegger 2015). Additional specimens examined. USA: Hawaii: Oahu, Koolau Mountains, Tantalus, 467 m, 1977, G. Y. Daida 503 (HAW); Oahu, Koolau Range, Manoa Valley, Manoa Cliffs Trail, 21° 19ʹ55ʺN, 157°48ʹ43ʺW, 410–575 m, 2013, B. Moncada, R. Lücking & C. W. Smith 6918b (B), 6920 (B, F). (Fig. 2W & X) Primary photobiont cyanobacterial (Nostoc). Stipe absent. Thallus monophyllous, forming suborbicular rosettes or becoming irregu- lar, to 15 cm across, mostly anisotomously branched; lobes 7–15 mm broad, elongate to flabellate, ±horizontal, adjacent to imbri- cate, involute to slightly canaliculate, their margins entire to sinuose or shallowly crenate. Upper surface smooth to rugose or shallowly scrobiculate, olive when fresh, brownish when dry, glab- rous, with or without scattered papillae, without pruina, with indistinct, cream maculae; marginal cilia not differentiated but lower tomentum sometimes projecting beyond the margins to resemble short, brown-black, agglutinate to fasciculate cilia. Phyllidia and flattened isidia present, predominantly marginal, to 1 mm long and broad, rather dark grey-brown, somewhat shiny, branched and becoming coralloid to palmate with a basal stipe with diminutive cyphellae. Lower surface uneven to undu- late, dark brown to blackish. Primary tomentum thick and dense to the lobe margins, spongy, forming dark brown to black- ish brown, arachnoid tufts of fasciculate hyphae; hairs 200–1000 μm long, in fascicles of 12–20, partly branched, agglutinate, api- cally intertwined, brown, cylindrical hyphae with free apices. Secondary tomentum discernible only in microscopic sections; hairs 10–35 μm long, composed of single, branched, moniliform hyphae with free apices. Rhizines present but short, scattered towards the thallus centre. Cyphellae rather dense, (20–)60–100 Specimens examined. USA: Hawaii: Hawaii, Mauna Loa, Pohakuloa training area, 1585 m, 1977, P. K. Higashino 327 (HAW); Kauai, West Kauai, Koke’e State Park, Pihea Trail, 22° 08ʹ51ʺN, 159°37ʹ53ʺW, 1250–1350 m, 2013, B. Moncada, R. Lücking & T. Flynn 7047 (B, PTBG); Maui, East Maui, Kipahulu Forest Reserve, 1385 m, 1976, P. K. Higashino & G. Mizuno 2034 (HAW); Maui, East Maui, Makawao Forest Reserve, 1800 m, 1975, C. W. Smith 1922 (HAW); Maui, East Maui, Haleakalā oi.org/10.1017/S0024282920000353 ore. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at 122 Bibiana Moncada et al. Volcano, 1890–1950 m, 1981, C. W. Smith 5074 (HAW); Maui, East Maui, Haleakalā Volcano, Haleakalā National Park, 2100 m, 1975, C. W. Smith 2105, 2164, 2168 (HAW); Maui, East Maui, Haleakalā Volcano, Haleakalā National Park, Halemauu Trail, 2400 m, 1975, C. W. Smith 2075 (HAW); Maui, East Maui, Haleakalā Volcano, Lower Waikamoi Preserve, 20° 48ʹ23ʺN, 156°15ʹ19ʺW, 1200–1300 m, 2013, B. Moncada, R. Lücking & P. Bily 6951 (F); Maui, East Maui, Haleakalā Volcano, Upper Waikamoi Preserve, 20°46ʹ07ʺN, 156°14ʹ17ʺW, 1800–2100 m, 2013, B. Moncada, R. Lücking & P. (Fig. 2W & X) Thomas 6983 (B), 6984 (F), 6997 (B, F); Maui, Olinda, Koolau Forest Reserve North Haleakala, 910 m, 1992, R. Rosentreter 8108 (SRP); Maui, Kipahulu Valley, Haleakalā National Park, 2100 m, 1984, A. C. Medeiros & D. Miranda 322 (HAW); Maui, Paliku, 2130 m, 1976, C. W. Smith 3091 (HAW); Oahu, Waianae Mountain Range, Mt Kaala, 1212 m, 1980, G. Y. Daida 628 (HAW); Oahu, Gully en route from Kuiki to Paliku, 2130 m, 1975, C. W. Smith 2220 (HAW). Remarks. Sticta antoniana belongs to the S. tomentosa complex and cannot be phylogenetically separated from the latter on ITS sequences alone (Moncada et al. 2020). Due to its distinctive morphology, differing from typical S. tomentosa by the highly dis- sected lobe margins and the thick lower tomentum that leaves an abrupt bare margin, it is considered a separate species. Highly dis- parate, discrete morphologies in phylogenetically closely related lineages that may not be resolvable with standard barcoding mar- kers are not rare in island biota, also being found in vascular plants (Baldwin et al. 1991; Baldwin & Sandersson 1998; Carlquist et al. 2003). In lichens, similar phenomena were also demonstrated for Hawaiian Lobariella and Pseudocyphellaria (Moncada et al. 2014b; Lücking et al. 2017a). The view that S. antoniana is a distinctive lineage not resolved by the ITS barcod- ing marker, and not a habitat-induced morph, is supported by the notion that the S. antoniana morphodeme is not known in popu- lations of S. tomentosa outside Hawaii. A recent example demonstrating a case where the ITS barcod- ing marker does not resolve two closely related species is the pair Neuropogon antarcticus (Du Rietz) I. M. Lamb versus Neuropogon aurantiacoater (Jacq.) I. M. Lamb, as shown by RADseq data (Grewe et al. 2018). The latter is a promising method to test cases where ITS exhibits limits of resolution, such as in S. antoni- ana versus S. tomentosa. Sticta antoniana Moncada & Lücking Moncada et al., Pl. Fung. Syst. 65, 108 (2020); type: USA, Hawaii, Kauai, West Kauai, Koke’e State Park, Pihea Trail, 22°08ʹ51ʺN, 159°37′53′′W, 1250–1350 m, 2013, B. Moncada, R. Lücking & T. Flynn 7053 (F—holotype!; B, HAW—isotypes!). Additional specimens examined. USA: Hawaii: Maui, East Maui, Haleakalā Volcano, Lower Waikamoi Preserve, 20°48′23′′N, 156° 15′19′′W, 1200–1300 m, 2013, B. Moncada, R. Lücking & P. Bily 6947 (B, F, HAW), 6948 (B, F); Kauai, West Kauai, Koke’e State Park, Pihea Trail, 22°08′51′′N, 159°37′53′′W, 1250– 1350 m, 2013, B. Moncada, R. Lücking & T. Flynn 7045a (F), 7065 (F). (Fig. 1U–X) Primary photobiont cyanobacterial (Nostoc). Stipe absent. Thallus monophyllous, irregularly orbicular, to 10 cm across, frequently anisotomously branched; lobes to 7 mm broad, flabellate to trun- cate, ascending, not usually overlapping, their margins dissected into elongate lobules. Upper surface even, olive green when fresh, brownish grey when dry, glabrous, without papillae or pruina, with or without scattered, indistinct maculae. Marginal lobules abundant, much branched, 1–3(–5) mm long and 0.5–1 mm broad, concolorous with the thallus. Lower surface slightly uneven, whitish. Primary tomentum thick and dense, forming light grey-brown, arachnoid tufts of fasciculate hyphae except for a 2–5 mm broad marginal zone abruptly bare of tomentum; hairs 500–1500 μm long, formed of densely entangled, mostly unbranched, pale brown, cylindrical hyphae with free apices. Secondary tomentum inconspicuous; hairs scattered, 20–30 μm long, comprising single, unbranched, cylindrical, colourless hyphae. Cyphellae dense, 21–60 per cm2, rounded, plane, immersed, white; pore 0.1–0.3 mm diam.; cells of basal membrane irregularly bulging but lacking distinct papillae. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at Sticta emmanueliana Moncada, Lücking & Lumbsch Moncada et al., Pl. Fung. Syst. 65, 109 (2020); type: USA, Hawaii, Kauai, West Kauai, Koke’e State Park, Pihea Trail, 22°08′51′′N, 159°37′53′′W, 1250–1350 m, 2013, B. Moncada, R. Lücking & T. Flynn 7058 (F—holotype!; B, HAW—isotypes!). (Fig. 2I–L) Primary photobiont cyanobacterial (Nostoc). Stipe present, short and rather broad. Thallus mono- to polyphyllous, with indivi- duals irregularly arranged, delicate, irregularly branched; lobes to 4 mm broad, irregularly elongate to tapering or truncate, hori- zontal to ascending, not usually overlapping, plane to slightly involute, their margins not thickened. Upper surface even, olive-brown to dark brown when fresh, brownish grey when dry, glabrous, without papillae or pruina, with scattered, white maculae; marginal cilia present, formed by submarginal tufts of primary tomentum that become visible beyond the margins, con- spicuous particularly in younger lobes, evanescent in old lobes, brownish black, 0.1–0.4 mm long. Isidia present, predominantly marginal, 0.1–0.3 mm long and 0.05–0.1 mm broad, darker grey- brown, shiny, cylindrical, branched and becoming coralloid to arbuscular; arbusculae to 0.5 mm long and broad, their base lack- ing cyphellae. Lower surface uneven, mostly dark brown but mar- ginally becoming white to cream. Primary tomentum becoming thin towards the margins, except for the cilia, otherwise forming loosely to densely arranged, brown tufts of fasciculate hyphae, much shorter and lighter than the cilia; hairs 100–200 μm long, Apothecia common, laminal, sessile, 0.7–1.5 mm diam., ini- tially zeorine with hairy margins but mature apothecia biatorine, with remnants of thallus layer basally and with scattered marginal hairs; disc dark reddish brown; margin crenulate, cream to pale orange. Ascospores oblong-fusiform, 3-septate, 40–50 × 6–7 μm. Secondary chemistry. No substances detected by TLC; medulla K−, cyphellae K−. Distribution and ecology. This species is thus far known only from the islands of Maui and Kauai where it is apparently restricted to more or less undisturbed or little disturbed montane rainforest at mid elevations (between 1200 and 1350 m). It grows on shaded bark of unidentified phorophyte trees. 123 The Lichenologist in fascicles of 10–20, mostly unbranched but loosely agglutinate, cylindrical hyphae with free apices, usually brownish. Secondary tomentum developed to the margins, thin, pubescent, whitish to light brownish; hairs 25–100 μm long, comprising single, mostly unbranched, cylindrical hyphae with free apices, somewhat pale brown. Cyphellae dense, 41–80 per cm2, rounded, immersed-erumpent, white, appearing pruinose, with cream to light brown margins bare of tomentum; pore 0.1–0.2 mm diam. towards the margins, 0.2–0.5 mm diam. Sticta emmanueliana Moncada, Lücking & Lumbsch towards the centre; cells of basal membrane irregularly bulging but without distinct papillae. abundant, mostly marginal but also laminal, isidia 0.1–0.2 mm long and 0.05–0.1 mm broad, darker brown, shiny, cylindrical to somewhat flattened, arbuscular, branched and becoming coral- loid; arbusculae to 2 mm long and broad, their base lacking cyphellae. Lower surface uneven, forming shallow, vein-like ridges, white to cream. Primary tomentum absent. Secondary tomentum developed to the margins, thin, pubescent, white; hairs 20–50 μm long, comprising single, mostly unbranched, cylindrical hyphae with free apices. Cyphellae scattered, 1–20 per cm2 towards the thallus centre and 21–40 per cm2 towards the margin, rounded to irregular or becoming angular, plane, immersed, white, appearing pruinose; pore 0.5–1 mm diam. towards the margins, 1–3 mm across towards the centre; cells of basal mem- brane irregularly bulging but without distinct papillae. h b d p p Apothecia not observed. Secondary chemistry. No substances detected by TLC; medulla K−to K+ pale yellow. g y g g Apothecia not observed. Secondary chemistry. No substances detected by TLC; medulla and membrane of cyphellae K−. Secondary chemistry. No substances detected by TLC; medulla and membrane of cyphellae K−. Distribution and ecology. This species was found on the islands of Maui and Kauai, at altitudes between 1200 and 1800 m, chiefly at the same localities as Sticta antoniana. Like that species, it also occurred in rather undisturbed montane rainforest at mid to higher elevations, growing on shaded tree bark including branches, for example on Myrsine, usually associated with bryo- phyte mats. Distribution and ecology. This species has been found at the type locality on Kauai and one additional site on Maui. It was found in more or less undisturbed montane rainforest at mid to high eleva- tions (between 1250 and 2300 m) on shaded tree bark associated with bryophytes. Given that it is a rather small species, it is likely to have been overlooked and might be more common. Remarks. On account of its marginal isidia and black cilia, Sticta emmanueliana comes morphologically close to Sticta cometiella Vain., described from Mexico. The latter appears to be strictly neotropical and is only very distantly related, clustering in a different clade of the global Sticta phylogeny (Moncada et al. 2020). It also frequently produces laminal isidia. The Australasian S. duplolimbata (Hue) Vain. is also similar (Galloway 1998, 2001) but likewise only distantly related (Moncada et al. 2020). Sticta flynnii Moncada & Lücking Sticta fuliginosa (Hoffm.) Ach. Moncada et al., Pl. Fung. Syst. 65, 110 (2020); type: USA, Hawaii, Kauai, West Kauai, Koke’e State Park, Pihea Trail, 22°08′51′′N, 159°37′53′′W, 1250–1350 m, 2013, B. Moncada, R. Lücking & T. Flynn 7055 (F—holotype!). Meth. Lichenum, 280 (1803).—Lobaria fuliginosa Hoffm., Deutschl. Flora 2, 109 (1796); type: United Kingdom, Wales, Cader Idris, Icon. in Dillenius, Historia Muscorum, tab. 26, fig. 100A (1742; lectotype fide Laundon (1984), 218–219); corre- sponding specimen from hb. Dillenius (OXF—epitype fide Jørgensen & Tønsberg (2007), 146). Meth. Lichenum, 280 (1803).—Lobaria fuliginosa Hoffm., Deutschl. Flora 2, 109 (1796); type: United Kingdom, Wales, Cader Idris, Icon. in Dillenius, Historia Muscorum, tab. 26, fig. 100A (1742; lectotype fide Laundon (1984), 218–219); corre- sponding specimen from hb. Dillenius (OXF—epitype fide Jørgensen & Tønsberg (2007), 146). (Fig. 2A–D) Primary photobiont cyanobacterial (Nostoc). Stipe present, short and broad. Thallus mono- to polyphyllous, with one to few indi- viduals that are irregularly arranged, delicate, unbranched to sparsely branched, becoming apically dissected; lobes to 7 mm broad, suborbicular to reniform or truncate, ascending, not usu- ally overlapping, plane to involute, their margins becoming coarsely dissected. Upper surface even, olive green when fresh, bluish grey when dry, glabrous, with or without scattered papillae, without pruina, with scattered, white maculae. Isidia present, Sticta emmanueliana Moncada, Lücking & Lumbsch Within Hawaiian Sticta, the species is best recognized by its dark marginal cilia. Remarks. Together with Sticta acyphellata and S. smithii, S. flyn- nii is one of three Hawaiian species with small, caulescent, isidiate thalli. Sticta acyphellata is distinguished by the complete lack of cyphellae, whereas S. smithii differs in the small cyphellae and thicker tomentum. Several other, non-Hawaiian taxa share a cau- lescent, isidiate thallus, including S. brevipes (Müll. Arg.) Zahlbr., S. cyphellulata (Müll. Arg.) Hue, S. hypochra Vain., S. longipes (Müll. Arg.) Malme and S. marginifera Mont. (Galloway 1994a, 1998). These are all phylogenetically distinct (Moncada et al. 2020) and differ in their more robust thalli, as well as in other details. Notably, even the larger species do not appear to produce cyphellae as large as those found in S. flynnii. Additional specimens examined. USA: Hawaii: Maui, East Maui, Haleakalā Volcano, Lower Waikamoi Preserve, 20°48′23′′N, 156° 15′19′′W, 1200–1300 m, 2013, B. Moncada, R. Lücking & P. Bily 6949 (F), 6954 (B, F, HAW), 6955 (F); Maui, East Maui, Makawao Forest Reserve, 1800 m, 1975, C. W. Smith 1922 (HAW); Kauai, West Kauai, Koke’e State Park, Pihea Trail, 22° 08′51′′N, 159°37′53′′W, 1250–1350 m, 2013, B. Moncada, R. Lücking & T. Flynn 7045a (F), 7064 (F). Additional specimens examined. USA: Hawaii: Maui, East Maui, Haleakalā Volcano, Lower Waikamoi Preserve, 20°48′23′′N, 156° 15′19′′W, 1200–1300 m, 2013, B. Moncada, R. Lücking & P. Bily 6949 (F), 6954 (B, F, HAW), 6955 (F); Maui, East Maui, Makawao Forest Reserve, 1800 m, 1975, C. W. Smith 1922 (HAW); Kauai, West Kauai, Koke’e State Park, Pihea Trail, 22° 08′51′′N, 159°37′53′′W, 1250–1350 m, 2013, B. Moncada, R. Lücking & T. Flynn 7045a (F), 7064 (F). Additional specimens examined. USA: Hawaii: Maui, East Maui, Haleakalā Volcano, Haleakalā National Park, 2233 m, 1975, C. W. Smith 1754 (HAW); Kauai, West Kauai, Koke’e State Park, Pihea Trail, 22°08′51′′N, 159°37′53′′W, 1250–1350 m, 2013, B. Moncada, R. Lücking & T. Flynn 7059 (HAW). Sticta flynnii Moncada & Lücking Sticta flynnii Moncada & Lücking https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at (Fig. 1Q–T) Primary photobiont cyanobacterial (Nostoc). Stipe absent. Thallus mono- to polyphyllous; individuals robust, unbranched to sparsely branched; lobes 5–8(–12) cm broad, suborbicular, ascending, imbricate, plane to slightly revolute, margins entire to irregular. Upper surface uneven to shallowly scrobiculate, dark brown-grey when fresh, greyish brown when dry, glabrous, 124 Bibiana Moncada et al. Koke’e State Park, Kalua Puhi Trail, 1070 m, 1985, W. A. Weber & D. Randolph L-77946 (US); Kauai, West Kauai, Koke’e State Park, Mohihi Trail, 467 m, 1979, G. Y. Daida 267 (HAW); Kauai, West Kauai, Koke’e State Park, Pihea Trail, 22° 08′51′′N, 159°37′53′′W, 1250–1350 m, 2013, B. Moncada, R. Lücking & T. Flynn 7043 (B), 7052 (F); Kauai, West Kauai, Waimea Canyon, Iliau Nature Loop & Kukui Trail at Kokee Road, 22°03′04′′N, 159°39′36′′W, 900 m, 2013, B. Moncada, R. Lücking & T. Flynn 7023 (B, F), 7026 (B, F, HAW); Kauai, Alakai Swamp, C. W. Smith 6179 (HAW); Maui, East Maui, Haleakalā National Park, 2070 m, 1977, C. W. Smith 3343 (HAW); Maui, Auahi Forest Reserve, 760 m, 1992, P. DePriest 8213 (US); Maui, East Maui, Haleakalā Volcano, 1890–1950 m, 1981, C. W. Smith 5072 (HAW); Maui, East Maui, Haleakalā Volcano, Haleakalā National Park, 2233 m, 1975, C. W. Smith 1762 (HAW); Maui, East Maui, Haleakalā Volcano, Lower Waikamoi Preserve, 20°48′23′′N, 156° 15′19′′W, 1200–1300 m, 2013, B. Moncada, R. Lücking & P. Bily 6945 (F); Maui, East Maui, Haleakalā Volcano, Upper Waikamoi Preserve, 20° 46′07′′N, 156°14′17′′W, 1800–2100 m, 2013, B. Moncada, R. Lücking & P. Thomas 6978 (B, F, HAW), 6979 (F), 6982 (HAW), 6988 (B, F), 6990 (F), 6994 (F), 6998 (HAW); Maui, East Maui, Kipahulu Forest Reserve, 1160 m, 1976, P. K. Higashino & G. Mizuno 2551 (HAW); Maui, Olinda, Koolau Forest Reserve, 20°49′23′′N, 156°16′12′′W, 910 m, 1992, R. Rosentreter 8085, 8087, 8101 (SRP); Oahu, Honolulu, Ho’omaluhia Botanical Garden, 21°23′08′′N, 157°48′16′′W, 1991, S. C. Tucker 30518 (LSU); Oahu, Honolulu, Wahiawa Botanical Garden, 21°25′N, 158°00′W, 1991, S. C. Tucker 30485 (LSU); Oahu, Waahila Ridge, 275 m, 1979, G. Y. Daida 251 (HAW); Oahu, Waianae Mountains, Honouliuli Forest Reserve, 945 m, 1978, C. W. Smith 4112 (HAW); Oahu, Waianae Mountains, Kaua Trail, 945 m, 1978, G. Y. Daida 203 (HAW). with or without scattered papillae, without pruina, with or with- out indistinct maculae. Isidia present, laminal, to 0.5 mm long and broad, dark grey to brownish grey, shiny, cylindrical to some- what flattened, branched and becoming coralloid. Sticta hawaiiensis Moncada & Lücking Sticta hawaiiensis Moncada & Lücking Moncada et al., Pl. Fung. Syst. 65, 111 (2020); type: USA, Hawaii, Kauai, West Kauai, Koke’e State Park, Pihea Trail, 22°08′51′′N, 159°37′53′′W, 1250–1350 m, 2013, B. Moncada, R. Lücking & T. Flynn 7048 (F—holotype!; HAW—isotype!). Remarks. The name Sticta fuliginosa has traditionally been applied to all forms with more or less broad, rounded lobes with laminal isidia (Joshi & Awasthi 1982; Swinscow & Krog 1988; Galloway 1994a, 2001, 2007; Galloway et al. 1995; Büdel et al. 2000; Brodo et al. 2001; Farkas 2003; Galloway & Thomas 2004; Jørgensen & Tønsberg 2007; Makryi 2008; Smith et al. 2009). Molecular phylogenetic data, however, have demonstrated that this morphodeme corresponds to more than a dozen, mostly unrelated species (Moncada et al. 2014a, 2015, 2020; Magain & Sérusiaux 2015). Surprisingly, even in a narrow sense supported by molecular data, S. fuliginosa s. str. remains the most wide- spread species of the genus. Morphologically, it is best character- ized (and separated from similar species) by the rather robust thalli, the glabrous lobe surface, the cylindrical isidia, the whitish underside with rather large, plane cyphellae that often become angular in outline, and the lack of papillae on the cells of the membrane of the cyphellae. The only similar species in Hawaii is S. hawaiiensis, which differs in the much smaller thalli and lobes, the distinct, white, marginal cilia, and the numerous papil- lae on the membrane of the cyphellae. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at (Fig. 1Q–T) Lower surface slightly uneven, white to cream. Primary tomentum rather thin and becoming thinner along the margins, whitish to pale brown- ish, forming somewhat scattered to more densely arranged tufts of fasciculate hyphae; hairs 100–200 μm long, in fascicles of 10–20, mostly unbranched but agglutinate, cylindrical hyphae with free apices. Secondary tomentum not discernible. Cyphellae numerous, 20–60 per cm2, rounded to usually angular, plane, immersed, white to cream; pore 0.5–3(–5) mm diam.; cells of basal mem- brane without papillae. Apothecia not observed. Secondary chemistry. No substances detected by TLC; medulla K−, membrane of cyphellae K−. Distribution and ecology. Sticta fuliginosa in the strict sense is a subcosmopolitan species, with specimens confirmed through sequence data known from most regions of the world, including Hawaii (Magain & Sérusiaux 2015; Moncada et al. 2020). Although exhibiting little genetic variation, there is some subtle geographical structure in the ITS sequence data, suggesting a very recent, rapid, yet natural expansion of this taxon. In Hawaii, it is usually epiphytic in more or less undisturbed rainfor- est habitats and gulches in adjacent dry forest. It tolerates a certain degree of disturbance and may also be found in secondary or anthropogenic vegetation, as records from introduced trees in botanical gardens demonstrate. Together with S. tomentosa, S. fuliginosa is the most common and widespread Sticta species in Hawaii, with a well-documented, broad altitudinal range between under 500 to over 2300 m. Identified phorophytes include indi- genous species of Cordyline ( fruticosa), Dodonaea (viscosa), Leptecophylla, Metrosideros (collina), Rubus (hawaiiensis), and Sophora (chrysophylla). Sticta hawaiiensis Moncada & Lücking (Fig. 1M–P) Primary photobiont cyanobacterial (Nostoc). Stipe indistinct or absent. Thallus mono- to polyphyllous, with one to few indivi- duals that are irregularly arranged, delicate, unbranched; lobes 0.5–1 cm broad, suborbicular, ascending, not usually overlapping, plane to revolute, margins entire to irregular. Upper surface irregularly verruculose-rugose, dark grey when fresh, dark bluish grey when dry, thinly whitish tomentose, with or without scat- tered papillae, without pruina, without distinct maculae; marginal cilia abundant and distinct, particularly in younger thalli, white, 0.2–0.7 mm long; hairs of upper tomentum 10–20 μm long, of single, branched, moniliform hyphae with free apices. Isidia pre- sent, laminal, to 0.2 mm long and broad, dark grey to brownish grey, shiny, cylindrical, branched and becoming somewhat coral- loid. Lower surface slightly uneven, white to cream. Primary tomentum developed except along the margins, forming some- what scattered to more densely arranged, mottled brown tufts of fasciculate hyphae; hairs 100–150 μm long, in fascicles of 10–20, mostly unbranched but agglutinate, cylindrical hyphae with free apices. Secondary tomentum developed to the margins, Specimens examined. USA: Hawaii: Hawaii, Mauna Loa, Pohakuloa training area, 1585 m, 1977, P. K. Higashino 525 (HAW); Hawaii, Puu Huluhulu Saddle Road, 19°51′20′′N, 155° 08′56′′W, 2365 m, 1992, W. L. Culberson 22164 (DUKE); Kauai, The Lichenologist 125 Secondary chemistry. No substances detected by TLC; medulla K−, membrane of cyphellae K−. Secondary chemistry. No substances detected by TLC; medulla K−, membrane of cyphellae K−. thin, pubescent, white; hairs 10–15 μm long, comprising single, branched, strongly moniliform hyphae with globose cells and free apices. Cyphellae scattered, 1–20 per cm2, rounded, plane, immersed to becoming erumpent, white; pore 0.1–0.2 mm diam.; cells of basal membrane irregularly bulging, with numer- ous tiny papillae per cell. Distribution and ecology. Similar to Sticta fuliginosa, S. limbata is a subcosmopolitan species with specimens confirmed through sequence data, known from many regions of the world including Hawaii (Moncada et al. 2020). Sticta limbata usually grows epi- phytically on trunks and branches in undisturbed habitats. It is generally much rarer than S. fuliginosa, although the known col- lections indicate a similarly broad altitudinal range between 900 and 2100 m. Apothecia not observed. Secondary chemistry. No substances detected by TLC; medulla K−, membrane of cyphellae K−. Secondary chemistry. No substances detected by TLC; medulla K−, membrane of cyphellae K−. Distribution and ecology. The ecology of this putatively endemic species is rather similar to that of Sticta antoniana and S. Sticta limbata (Sm.) Ach. Primary photobiont cyanobacterial (Nostoc). Stipe absent or indis- tinct (in young thalli). Thallus monophyllous, irregularly orbicu- lar to palmate, to 5 cm across, anisotomously to polytomously branched; lobes 3–6 mm broad, flabellate to truncate, horizontal, not usually overlapping, their margins entire to slightly crenate. Upper surface even, bluish grey when fresh, bluish to yellowish grey when dry, glabrous, without papillae or pruina, with scat- tered, whitish maculae, with marginal, fasciculate, white cilia. Phyllidia present, predominantly marginal but frequently also extending onto the lamina, to 0.7 mm long and broad, dark bluish grey, darker than the thallus, branched and forming coralloid to palmate clusters with a basal, flattened stipe lacking cyphellae. Lower surface uneven to ridged-scrobiculate, white to pale cream (becoming yellow-orange in the herbarium). Primary tomentum thin, sparse towards the margins, fasciculate, whitish to pale yellowish grey; hairs 70–250 μm long, in fascicles of 10– 20, mostly unbranched but agglutinate, colourless, cylindrical hyphae with free apices. Secondary tomentum thinly arachnoid, whitish; hairs scattered, 10–20 μm long, comprising single, unbranched, moniliform, colourless hyphae. Cyphellae scattered, 20–40(–60) per cm2, rounded to somewhat irregular in outline, plane, immersed-erumpent, white; pore 0.1–0.3 mm diam.; cells of basal membrane with 2–4 papillae per cell. Meth. Lichenum, 280 (1803).—Lichen limbatus Sm. in Smith & Sowerby, Engl. Bot., 16, tab. 1104 (1803); type: United Kingdom, Wales, Cardiganshire, Haford, J. E. Smith s. n. (BM- BM000763531—lectotype!). https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at (Fig. 1M–P) emma- nueliana, as it was found chiefly at the same localities on the islands of Maui and Kauai, between 1200 and 1300 m. It occurs in rather undisturbed montane forest at mid elevations, on shaded tree bark over and between bryophyte mats. However, an older collection was identified from Oahu, in non-indigenous rainforest at lower altitude. Remarks. The formation of soredia is remarkably rare in the genus Sticta. Even so, sorediate forms traditionally identified with the name S. limbata represent several unrelated species (Moncada et al. 2014a). Sticta limbata s. str. is characterized by suborbicular lobes with a whitish underside and cyphellae that become rather large and angular, similar to those of the closely related S. fuliginosa. In Hawaii, the species is unmistakable. Specimens examined. USA: Hawaii: Kauai, West Kauai, Waimea Canyon, Iliau Nature Loop & Kukui Trail at Kokee Road, 22° 03′04′′N, 159°39′36′′W, 900 m, 2013, B. Moncada, R. Lücking & T. Flynn 7024 (B, F); Maui, East Maui, Haleakalā Volcano, Upper Waikamoi Preserve, 20°46′07′′N, 156°14′17′′W, 1800– 2100 m, 2013, B. Moncada, R. Lücking & P. Thomas 6995 (HAW), 6999 (F). Remarks. This species belongs to the apparently pantropical Sticta ciliata complex which appears to contain several recently- diverged, phenotypically cryptic lineages (Magain & Sérusiaux 2015; Mercado-Díaz et al. 2020; Moncada et al. 2020). Species of this clade resemble S. fuliginosa with respect to their rounded lobes with laminal isidia but are much smaller overall and are consistently set apart by the conspicuous, white marginal cilia. Sticta plumbicolor (Zahlbr.) Zahlbr. Additional specimens examined. USA: Hawaii: Oahu, Koolau Mountains, Tantalus, 467 m, 1977, G. Y. Daida 520 (HAW); Maui, East Maui, Haleakalā Volcano, Lower Waikamoi Preserve, 20°48′23′′N, 156°15′19′′W, 1200–1300 m, 2013, B. Moncada, R. Lücking & P. Bily 6953 (B, F, HAW). Cat. Lich. Univers. 3, 398 (1925).—Stictina plumbicolor Zahlbr., Annls Mycol. 1, 356 (1903); type: USA, Hawaii, Molokai, 760 m, on shrubs, D. D. Baldwin 17 (W—holotype!). (Fig. 2M–P) rg/10.1017/S0024282920000353 Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at (Fig. 2M–P) (Fig. 2M–P) Sticta limbata (Sm.) Ach. (Fig. 2Q–T) (Fig. 2Q–T) pale orange. Ascospores oblong-fusiform, (1–)3-septate, 30–40 × 7–8 μm. pale orange. Ascospores oblong-fusiform, (1–)3-septate, 30–40 × 7–8 μm. Primary photobiont cyanobacterial (Nostoc). Stipe absent. Thallus monophyllous, forming suborbicular rosettes or becoming irregu- lar, to 20 cm across, anisotomously to polytomously branched; lobes 5–10 mm broad, flabellate, more or less horizontal, imbri- cate, undulate to slightly canaliculate, their margins entire to shal- lowly crenate. Upper surface uneven to foveolate-pitted, olive when fresh, greyish brown when dry, glabrous, with or without scattered papillae bearing tiny trichomes, without pruina but especially towards the margins thinly scabrous, with indistinct, cream maculae; rarely with marginal, pale to golden brown cilia. Phyllidia present, predominantly marginal but frequently also extending onto the lamina, to 0.5 mm long and broad, grey- brown, branched and becoming coralloid to palmate with a basal, flattened stipe lacking cyphellae. Lower surface uneven to undulate, dark brown to blackish. Primary tomentum dense but becoming sparse towards the lobe margins, spongy, forming pale to dark grey-brown, arachnoid tufts of fasciculate hyphae; hairs 200–1000 μm long, in fascicles of 6–12, partly branched, agglutinate, apically intertwined, pale to light brown, cylindrical hyphae with free apices. Secondary tomentum appressed, arach- noid; hairs 15–25 μm long, composed of single, branched, monili- form hyphae with free apices. Rhizines sparse. Cyphellae rather dense, 20–60 per cm2, rounded, plane, erumpent to prominent, cream; pore 0.5–1.2(–1.8) mm diam.; cells of basal membrane lacking papillae. Secondary chemistry. No substances detected by TLC; medulla K−, cyphellae K−. Distribution and ecology. Sticta plumbicolor is known with cer- tainty only from Hawaii. Reports from other regions, such as Jamaica (Minter et al. 2001; three collections housed at DUKE: 1967, C. Racine s. n.; 1968, W. L. Culberson & C. F. Culberson 13860, 13884), are misidentifications probably pertaining to S. filicinella (Nyl.) Zahlbr. In Hawaii, the species is found on all major islands (Kauai, Oahu, Molokai, Maui, Hawaii), generally in more or less undisturbed rainforests at low to mid elevation (between 75 and 640 m; a single outlier is from 2100 m). Notably, the closely related S. tomentosa, which cannot be sepa- rated using the ITS barcoding marker, is mostly found between 1000 and 2200 m (see below). Sticta plumbicolor grows mainly on tree trunks and branches, rarely on other substrata, often asso- ciated with bryophytes; identified phorophytes include Cordyline ( fruticosa), Metrosideros, Myrsine and Psidium (cattleianum). Remarks. (Fig. 1E–H) Primary photobiont cyanobacterial (Nostoc). Stipe absent. Thallus mono- to more rarely polyphyllous; individuals small, unbranched to sparsely branched; lobes 1–3 cm broad, suborbicu- lar, ascending, imbricate, plane, their margins entire to irregular. Upper surface uneven, brown-grey when fresh, greyish brown when dry, glabrous, with or without scattered papillae, without pruina, with or without indistinct maculae. Soredia present, mar- ginal, forming labriform soralia, white-grey. Lower surface slightly uneven, white to cream. Primary tomentum rather thin and becoming thinner along the margins, whitish to pale brownish, forming somewhat scattered to more densely arranged tufts of fas- ciculate hyphae; hairs 100–150 μm long, in fascicles of 10–20, mostly unbranched, agglutinate, cylindrical hyphae with free api- ces. Secondary tomentum not discernible. Cyphellae numerous, 20–60 per cm2, rounded to usually angular, plane, immersed, white to cream; pore 0.5–3 mm diam.; cells of basal membrane without papillae. p p p Apothecia present, submarginal, sessile, 0.8–1.5 mm diam., biatorine, glabrous or rarely with scattered marginal hairs; disc reddish brown; margin smooth to minutely crenulate, cream to p p Apothecia not observed. Bibiana Moncada et al. 126 Bibiana Moncada et al. (Fig. 2Q–T) Prior to our molecular phylogenetic revision of Hawaiian Sticta (Moncada et al. 2020), S. plumbicolor was consid- ered to be the only potentially endemic taxon, bearing in mind that reports from Jamaica were erroneous (see above). To our sur- prise, S. plumbicolor proved to be phylogenetically indistinguish- able from S. tomentosa based on the ITS barcoding marker. However, for the time being it is retained as a separate taxon, given the discrete morphological differences, providing a similar case to that of S. antoniana (see discussion above). Sticta plumbi- color is smaller and more delicate than S. tomentosa and forms abundant marginal and partly laminal clusters of conspicuous phyllidia. This morphology is not known from any collection out- side Hawaii, while S. tomentosa in its typical form is a pantropical taxon. The most similar species appears to be the neotropical S. filicinella which produces delicate, cylindrical isidia instead of robust phyllidia. g p p Apothecia not observed. Apothecia not observed. Secondary chemistry. No substances detected by TLC; medulla and basal membrane of cyphellae K+ (pale) ochraceous(-yellow). Distribution and ecology. The typical subspecies, Sticta scabrosa Moncada et al. subsp. scabrosa, is widely distributed in the Neotropics, whereas subsp. hawaiiensis is known only from Hawaii (Moncada et al. 2020, 2021). Both subspecies share the same ecology, being found in more tropical climates than most other Sticta species and often in somewhat exposed microsites in disturbed or secondary and anthropogenic habitats, sometimes with a weedy character. The taxon typically grows on tree trunks but is also found occasionally on rocks and other substrata. Most specimens were collected below 1000 m altitude, more rarely up to 1350 m and exceptionally (two collections) between 1800 and 2130 m. The broad range of reported phorophytes includes vari- ous native and introduced taxa: Araucaria, Casuarina, Cocos (nucifera), Cordyline ( fruticosa), Eucalyptus, Eugenia (cumini), Leptecophylla (tameiameiae), Litchi (chinensis), Metrosideros (col- lina, tremuloides), Myrsine, Osteomeles (anthyllidifolia), Pisonia, Sapindus, Schinus (terebinthifolius), Stachytarpheta ( jamaicensis) and Trema (micrantha). Specimens examined. USA: Hawaii: Hawaii, east coast, Honolii area, 1977, P. K. Higashino & J. Green 324 (HAW); Maui, East Maui, Haleakalā National Park, Paliku cabin, 2100 m, 1975, C. W. Smith 2131 (HAW); Molokai, Wailau Valley, 150 m, 1977, L. Stemmermann 2062 (HAW); Oahu, Ewa District, Ewa Forest Reserve, 1975, D. Vitt 14519 (MIN); Oahu, Honolulu, Manoa Valley, Manoa Falls Trail, 75 m, 1992, B. D. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at (Fig. 2Q–T) 127 The Lichenologist 6922 (B, F, HAW), 6924 (B, F), 6919b (B, F); Oahu, Niu Ridge, 290 m, 1980, G. Y. Daida 428 (HAW); Oahu, Upper Manoa Valley, 1978, S. Conant 304 (HAW); Oahu, Waahila Ridge, 275 m, 1979, G. Y. Daida 250 (HAW); Oahu, Waialae Iki Ridge, 1983, W. Char 312 (HAW); Oahu, Waianae Mountains, Honouliuli Forest Reserve, 945 m, 1978, C. W. Smith 4129 (HAW); Oahu, Waianae Mountains, Makua Keaau Forest Reserve, 250 m, 1975, E. Funk 302 (HAW); Oahu, Waianae Mountains, Mokuleia Forest Reserve, 550–670 m, 1977, L. Stemmermann 319 (HAW); Oahu, Waianae Range, Land of 10 000 Snails, 730 m, 2011, C. W. Smith s. n. (HAW); Oahu, Waianae Range, Makahela Valley, 1975, L. Yoshida 318 (HAW); Oahu, Kahuku, 275 m, 1979, E. Funk 311 (HAW); Oahu, summit of Waikane Trail, 395 m, 1977, G. Y. Daida 115 (HAW); Oahu, Tantalus, 460 m, 1979, K. Clark 4 (HAW); Oahu, Waahila Ridge Trail, 1979, C. S. Futa 47 (HAW). distinguished by the paler lower tomentum, the phyllidia being frequently laminal and concolorous with the thallus, the opaque lobe surface that becomes thinly scabrous towards the apices, and the sometimes scrobiculate-foveolate lobe tips. It also differs ecologically in its weedy character, being found mostly in some- what exposed situations at lower altitudes. Specimens previously identified under the name S. beauvoisii (Elix & McCarthy 1998, 2008; Benner & Vitousek 2012; Smith 2013) largely correspond to this taxon. Additional specimens examined. USA: Hawaii: Hawaii, Black Sand Beach, 1980, G. Y. Daida 594 (HAW); Hawaii, Hilo, Rainbow Falls, 70 m, 1976, C. W. Smith 2460 (HAW); Hawaii, North Kohala District, Hawi, 305 m, 1978, C. W. Smith 4366 (HAW); Kauai, South Kauai, Hawaii Agro Forestry Products Agro Forestry Demonstration plot, 21°57′49′′N, 159°24′56′′W, 140 m, 2013, B. Moncada, R. Lücking & T. Flynn 7076 (F, PTBG), 7078 (F); Kauai, South Kauai, Kahili Adventist School near Kahili Mountain Park, 21°57′49′′N, 159°29′03′′W, 275 m, 2013, B. Moncada, R. Lücking & T. Flynn 7069 (B, PTBG), 7070 (B, F), 7071 (PTBG), 7075 (B); Kauai, South Kauai, Kahili Ridge Trail, 21°57′52′′N, 159°29′29′′W, 300–450 m, 2013, B. Moncada, R. Lücking & T. Flynn 7072 (F), 7074 (F); Kauai, West Kauai, Koke’e State Park, Pihea Trail, 22°08′51′′N, 159° 37′53′′W, 1250–1350 m, 2013, B. Moncada, R. Lücking & T. (Fig. 2Q–T) Ryan 29039 (ASU); Oahu, Koolau Mountains, ridge from Tantalus to Puu Konahuanui, 1970, C. W. Smith 130c (HAW); Oahu, Koolau Mountains, Tantalus, Manoa Cliffs Trail, 467 m, 1977, G. Y. Daida 463 (HAW); Oahu, Koolau Range, Manoa Valley, Manoa Cliffs, 21°19′55′′N, 157°48′43′′W, 410–575 m, 2013, B. Moncada, R. Lücking & C. W. Smith 6910 (B, F, HAW), 6918a (F), 6925 (B); Oahu, Waianae Mountains, Makaleha Valley, Mokuleia Forest Reserve, 400 m, 1975, C. W. Smith 1717 (HAW). Specimens examined. USA: Hawaii: Hawaii, east coast, Honolii area, 1977, P. K. Higashino & J. Green 324 (HAW); Maui, East Maui, Haleakalā National Park, Paliku cabin, 2100 m, 1975, C. W. Smith 2131 (HAW); Molokai, Wailau Valley, 150 m, 1977, L. Stemmermann 2062 (HAW); Oahu, Ewa District, Ewa Forest Reserve, 1975, D. Vitt 14519 (MIN); Oahu, Honolulu, Manoa Valley, Manoa Falls Trail, 75 m, 1992, B. D. Ryan 29039 (ASU); Oahu, Koolau Mountains, ridge from Tantalus to Puu Konahuanui, 1970, C. W. Smith 130c (HAW); Oahu, Koolau Mountains, Tantalus, Manoa Cliffs Trail, 467 m, 1977, G. Y. Daida 463 (HAW); Oahu, Koolau Range, Manoa Valley, Manoa Cliffs, 21°19′55′′N, 157°48′43′′W, 410–575 m, 2013, B. Moncada, R. Lücking & C. W. Smith 6910 (B, F, HAW), 6918a (F), 6925 (B); Oahu, Waianae Mountains, Makaleha Valley, Mokuleia Forest Reserve, 400 m, 1975, C. W. Smith 1717 (HAW). Remarks. Sticta scabrosa is another recently established species of the S. weigelii morphodeme (Moncada et al. 2021). It is only dis- tantly related to S. weigelii and differs in several important fea- tures: the opaque, apically thinly scabrous lobe surface, the flattened phyllidia that are concolorous with the thallus, the rather thick, paler lower tomentum, and the lack of a yellow pigment in the cyphellae. The Hawaiian material differs consistently from neotropical populations in two substitutions in the ITS region and was formally recognized as a subspecies (Moncada et al. 2021). The only similar taxa in Hawaii are S. andina and S. wai- kamoi, from which S. scabrosa subsp. hawaiiensis can be Sticta scabrosa subsp. hawaiiensis Moncada, Lücking & C.W. Sm. Sticta scabrosa subsp. hawaiiensis Moncada, Lücking & C.W. Sm. Moncada et al., Willdenowia (in press); type: USA, Hawaii, Oahu, Koolau Range, Manoa Valley, Manoa Cliffs Trail, 21°19′55′′N, 157°48′43′′W, 410–575 m, 2013, B. Moncada et al. 6915 (BISH —holotype!; B, F—isotypes!). (Fig. 2Q–T) Flynn 7051 (F, HAW), 7054 (B, F, PTBG), 7056 (PTBG), 7057 (F), 7060 (B), 7063 (F); Kauai, West Kauai, Waimea Canyon, Iliau Nature Loop & Kukui Trail at Kokee Road, 22° 03′04′′N, 159°39′36′′W, 900 m, 2013, B. Moncada, R. Lücking & T. Flynn 7025 (B, F, PTBG); Kauai, Koke’e State Park, Mohihi Trail, 1220 m, 1979, G. Y. Daida 287 (HAW); Kauai, Lawai Cemetery, 1978, G. Y. Daida 126 (HAW); Kauai, Na Pali, Kona Forest Reserve, 1035 m, 1975, P. K. Higashino 1018 (HAW); Kauai, Wahiawa Bog, 707 m, 1979, G. Y. Daida 293, 295 (HAW); Maui, East Maui, Makawao Forest Reserve, 1800 m, 1975, C. W. Smith 1924 (HAW); Maui, East Maui, Haleakalā Volcano, Haleakalā National Park, 2130 m, 1977, C. W. Smith 3256 (HAW); Maui, East Maui, Manawainui Valley, 1976, P. K. Higashino & G. Mizuno 2304 (HAW); Maui, East Maui, East Maui Irrigation (EMI), 20°53′06′′N, 156°12′29′′W, 225 m, 2013, B. Moncada & R. Lücking 6934 (B, F, HAW), 6935 (B, F), 6938 (F, HAW); Maui, East Maui, Hana Highway, Nua‘ailua Bay scenic lookout, 20°51′33′′N, 156°09′13′′W, 100 m, 2013, B. Moncada & R. Lücking 6937 (B, F, HAW); Maui, East Maui, Kipahulu Forest Reserve, 1160 m, 1976, P. K. Higashino & G. Mizuno 2588 (HAW); Maui, Kipahulu Valley, 520 m, 1980, G. Y. Daida 759 (HAW); Maui, West Maui Mountains, Maunalei Arboretum, 20°58′46′′N, 156°37′14′′W, 400 m, 2013, B. Moncada, R. Lücking & P. Thomas 7013 (F), 7014 (F), 7015 (B, F, HAW), 7018 (F), 7019 (HAW), 7020 (B, F, HAW); Molokai, Pakuwoaku Ridge, 150 m, 1977, L. Stemmermann 2048 (HAW); Molokai, Wailau Valley, 1975, P. K. Higashino 315 (HAW); Molokai, Kalaupapa Cliff Trail, 21°10′40′′N, 157° 00′13′′W, 30–470 m, 1993, C. W. Smith s. n. (HAW); Oahu, Koolau Mountains, Kahuku Forest Reserve, 275 m, 1975, C. W. Smith 1648 (HAW); Oahu, Koolau Mountains, Likeke Trail, 300 m, 1975, C. W. Smith 1448 (HAW); Oahu, Koolau Mountains, Manoa Valley, Manoa Falls Trail, 1975, P. K. Higashino 160 (HAW); Oahu, Koolau Range, Manoa Valley, Manoa Cliffs Trail, 21°19′55′′N, 157°48′43′′W, 410–575 m, 2013, B. Moncada, R. Lücking & C. W. Smith 6911 (F, HAW), 6912 (F), 6914 (F), 6917 (F, HAW), 6919 (B, F, HAW), Sticta smithii Moncada & Lücking Sticta smithii Moncada & Lücking Sticta smithii Moncada & Lücking Moncada et al., Pl. Fung. Syst. 65, 113 (2020); type: USA, Hawaii, Oahu, Koolau Range, Manoa Valley, Manoa Cliffs Trail, 21° 19′55′′N, 157°48′43′′W, 410–575 m, 2013, B. Moncada, R. Lücking & C. W. Smith 6916 (F—holotype!; B, HAW—isotypes!). https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at (Fig. 2E–H) Primary photobiont cyanobacterial (Nostoc). Stipe present, short. Thallus mono- to polyphyllous, with one to few individuals that are irregularly arranged, delicate, irregularly branched and dis- sected; lobes to 0.5 mm broad, spathuliform to truncate, ascend- ing, not usually overlapping, plane to involute, their margins becoming strongly dissected. Upper surface even, olive-grey when fresh, bluish grey when dry, glabrous, without papillae or pruina, with scattered, white maculae. Isidia present, predomin- antly marginal, 0.1–0.2 mm long and 0.05–0.1 mm broad, darker grey-brown, shiny, somewhat flattened, arbuscular, much branched and becoming coralloid; arbusculae to 1 mm long and broad, distinctly flattened, base lacking cyphellae. Lower surface uneven, forming shallow, vein-like ridges, white to cream, with pale orange streaks towards the centre. Primary tomentum dense but short, becoming even shorter towards the margins, whitish; hairs 50–100 μm long, of single to somewhat agglutinate, mostly unbranched, cylindrical, colourless hyphae with free apices. Secondary tomentum absent. Cyphellae rather dense, 21–40 per cm2, rounded to somewhat irregular, plane, immersed, white; pore 0.2–0.5 mm diam.; cells of basal membrane irregularly bulging, each cell with 1–3 elongated papillae, therefore appearing thorny. Apothecia not observed. Secondary chemistry. No substances detected by TLC; medulla K−, membrane of cyphellae K−. Distribution and ecology. Sticta smithii is known from a partially disturbed secondary rainforest on the densely populated and strongly altered island of Oahu, growing in shaded conditions on tree trunks between bryophytes, at c. 500 m altitude. It was previously also collected on Maui, on Haleakalā Volcano, at an altitude of nearly 2000 m. This suggests that this small species has been overlooked and might be more common. Remarks. Sticta smithii is similar to S. flynnii in that both have small, caulescent, isidiate thalli. The main difference between 128 Bibiana Moncada et al. these two species is the size of the cyphellae: they remain small and rounded in S. smithii but become rather large and irregular in S. flynnii. vegetative propagules or marginal lobules. Most similar is S. antoniana, but in that taxon, the lobe margins are deeply dis- sected into narrow lobules and the underside features a thick, grey-brown tomentum abruptly ending short of the margins and leaving the margins entirely bare. Additional specimen examined. USA: Hawaii: Maui, East Maui, Haleakalā Volcano, 1890–1950 m, 1981, C. W. Smith 5073 (HAW). Additional specimen examined. USA: Hawaii: Maui, East Maui, Haleakalā Volcano, 1890–1950 m, 1981, C. W. Smith 5073 (HAW). Specimens examined. Sticta tomentosa (Sw.) Ach. Meth. Lichenum, 279 (1803).—Lichen tomentosus Sw., Prodr., 147 (1788).—Lobaria tomentosa (Sw.) Räuschel, Nomenclat. Bot. 3, 330 (1797).—Stictina tomentosa (Sw.) Nyl., Syn. Meth Lich. 1 (2), 343 (1860).—Dystictina tomentosa (Sw.) Clem., Gen. Fung., 175 (1909); type: Jamaica, unknown locality, Swartz s. n. (STB sheet 44, lectotype! fide Galloway (1994b), 47). (Fig. 1A–D) Primary photobiont cyanobacterial (Nostoc). Stipe absent. Thallus monophyllous, irregularly orbicular to palmate, to 5 cm across, anisotomously to polytomously branched; lobes 7–15 mm broad, flabellate to truncate, horizontal to ascending, not usually overlapping, their margins entire to slightly crenate. Upper surface even, bluish grey when fresh, bluish to yellowish grey when dry, glabrous, without papillae or pruina, with scattered, whitish maculae, with marginal, fasciculate, white cilia. Isidia and soralia not observed. Lower surface uneven to ridged-scrobiculate, white to pale cream. Primary tomentum thin, sparse towards the mar- gins, fasciculate to somewhat spongy towards the centre, whitish to pale grey; hairs 70–300(–500) μm long, in fascicles of 10–20, mostly unbranched but agglutinate, colourless, cylindrical hyphae with free apices. Secondary tomentum thinly arachnoid, whitish; hairs scattered, 10–25 μm long, composed of single, unbranched, moniliform, colourless hyphae. Cyphellae dense, (20–)40–60(–100) per cm2, rounded, plane, immersed-erumpent, white; pore 0.1–0.3(–0.5) mm diam.; cells of basal membrane with 2–4 papillae per cell. p Apothecia common, submarginal, sessile, 1–2 mm diam., bia- torine, glabrous or with scattered marginal hairs; disc reddish brown; margin crenulate, cream. Ascospores oblong-fusiform, 1–3-septate, 30–45 × 6–10 μm. Secondary chemistry. No substances detected by TLC; medulla K−, cyphellae K−. Secondary chemistry. No substances detected by TLC; medulla K−, cyphellae K−. Distribution and ecology. On the basis of molecular data, Sticta tomentosa is demonstrably a pantropical species (Moncada et al. 2020). It is found in more or less undisturbed, mid- to high- elevation rainforest habitats. In Hawaii it has been collected in numerous localities on all major islands, at altitudes between 500 and 2200 m but mostly above 1000 m. It typically grows on bark and is often associated with bryophytes, with the following identified phorophyte species: Broussasia (arguta), Dodonaea, Geranium (multiflorum), Metrosideros (collina, tremuloides), Myrsine and Pinus. Sticta waikamoi Moncada & Lücking https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at Sticta waikamoi Moncada & Lücking Moncada et al., Pl. Fung. Syst. 65, 114 (2020); type: USA, Hawaii, Maui, East Maui, Haleakalā Volcano, Upper Waikamoi Preserve, 20°46′07′′N, 156°14′17′′W, 1800–2100 m, 2013, B. Moncada, R. Lücking & P. Thomas 7000 (F—holotype!; B, HAW—isotypes!). (Fig. 2U & V) (Fig. 2U & V) (Fig. 2E–H) USA: Hawaii: Hawaii, Olaa Forest Tract, 1150 m, 1979, P. J. Burton 428 (HAW); Kauai, West Kauai, Koke’e State Park, Pihea Trail, 22°08′51′′N, 159°37′53′′W, 1250– 1350 m, 2013, B. Moncada, R. Lücking & T. Flynn 7044 (F), 7050 (HAW), 7061 (B), 7062 (F), 7066 (F); Kauai, Kalalau Lookout area, 1220 m, 1979, G. Y. Daida 297, 304 (HAW); Kauai, Wahiawa Bog, 707 m, 1979, G. Y. Daida 293 (HAW); Maui, East Maui, Haleakalā National Park, 1920 m, 1977, L. Stemmermann 2224 (HAW); Maui, East Maui, Haleakalā National Park, 1982, A. Medeiros 313 (HAW); Maui, East Maui, Haleakalā National Park, Kipahulu Valley, 2100 m, 1984, A. Medeiros & D Miranda 314 (HAW); Maui, East Maui, Haleakalā National Park, Paliku, C. W. Smith 2163, 3098 (HAW); Maui, East Maui, Kipahulu Forest Reserve, 1995 m, 1976, P. K. Higashino & G. Mizuno 326, 1719 (HAW); Maui, East Maui, Wainapanapa, 2060 m, 1977, L. Stemmermann 2450 (HAW); Maui, East Maui, Haleakalā Volcano, Haleakalā National Park, Paliku, 1950 m, 1975, W. J. Hoe 3761 (HAW); Maui, East Maui, Haleakalā Volcano, Lower Waikamoi Preserve, 20°48′23′′N, 156°15′19′′W, 1200–1300 m, 2013, B. Moncada, R. Lücking & P. Bily 6946 (F), 6950 (B, F); Maui, East Maui, Haleakalā Volcano, Upper Waikamoi Preserve, 20°46′07′′N, 156°14′17′′W, 1800–2100 m, 2013, B. Moncada, R. Lücking & P. Thomas 6977 (F, HAW), 6980 (B), 6981 (F), 6989 (F), 6991 (B, F, HAW), 6992 (B), 6993 (F), 6996 (B, F, HAW), 7001 (HAW); Maui, Kipahulu Valley, 520 m, 1980, G. Y. Daida 756 (HAW); Maui, Olinda, Koolau Forest Reserve, 20°49′23′′N, 156° 16′12′′W, 910 m, 1992, R. Rosentreter 8086b, 8094, 8107 (SRP); Maui, Olinda Koolan Forest Reserve, 20°49′40′′N, 156°14′10′′W, 1280 m, 1992, T. H. Nash III 42205 (ASU, MIN, WIS); Maui, Waikamoi Nature Conservancy Reserve, 20°46′12′′N, 156° 14′48′′W, 2073 m, 1992, R. Rosentreter 8129b (F, SRP); Molokai, Molokai Forest Reserve, 1975, P. K. Higashino 460 (HAW); Oahu, Koolau Mountains, 1970, C. W. Smith 130f (HAW); Oahu, Waianae Mountains, 1220 m, on scrub Metrosideros, 1985, C. W. Smith 8421 (HAW); Oahu, Waianae Mountain Range, Kaua Trail, 945 m, 1978, G. Y. Daida 201 (HAW); Oahu, Kaala, C. W. Smith 1742 (HAW); Oahu, Waahila Ridge Trail, 1979, C. S. Futa 12 (HAW). Sticta tomentosa (Sw.) Ach. (Fig. 2U & V) Primary photobiont cyanobacterial (Nostoc). Stipe absent. Thallus monophyllous, irregularly orbicular, to 7 cm across, anisoto- mously branched; lobes 4–7 mm broad, elongate to narrowly fla- bellate, more or less horizontal, not usually overlapping, involute to shallowly canaliculate, their margins entire to broadly crenu- late. Upper surface even, olive-grey to grey-brown when fresh, light yellowish to greyish brown when dry, glabrous, without Remarks. Sticta tomentosa is characterized by the typically bluish grey thallus with ciliate margins, the numerous apothecia, and the white underside with a very thin tomentum and tiny, often barely discernible cyphellae (Galloway 1994b; Galloway & Thomas 2004; Moncada 2012). In Hawaii, it is the only species that reproduces via apothecia and ascospores only and that does not form 129 The Lichenologist Fig. 3. Correspondence of identifications of Sticta lichens in HAW under the previous (above, purple) and the revised taxonomic concept (below, orange). The size of the circles is proportional to the number of specimens corresponding to a given name. The number of specimens corresponding to a particular ‘pair’ of identi- fications ‘before’ vs. ‘after’ is also indicated. In colour online. The Lichenologist 12 Fig. 3. Correspondence of identifications of Sticta lichens in HAW under the previous (above, purple) and the revised taxonomic concept (below, orange). The size of the circles is proportional to the number of specimens corresponding to a given name. The number of specimens corresponding to a particular ‘pair’ of identi- fications ‘before’ vs. ‘after’ is also indicated. In colour online. Remarks. Together with Sticta andina, S. waikamoi corresponds best to what has been traditionally identified with the name S. weigelii. However, the latter is a neotropical taxon and not directly related (Moncada et al. 2020). Sticta weigelii s. str. also deviates in its thinner lower tomentum and the often yellow cyphellae (Galloway 2006; Moncada 2012). Sticta andina differs in the gen- erally narrower lobes and lack of rhizines and typically grows epi- phytically. Sticta waikamoi is closely related to S. rhizinata but differs in several substitutions in the ITS barcoding marker and is much smaller overall, with less conspicuous rhizines (Moncada & Lücking 2012; Moncada et al. 2020). papillae or pruina, without distinct maculae; marginal cilia not differentiated but lower tomentum often projecting beyond the margins to resemble short, brown-black cilia. Isidia present, pre- dominantly marginal, to 0.5 mm long and broad, grey-brown, somewhat shiny, usually somewhat flattened, branched and becoming coralloid. (Fig. 2U & V) Lower surface slightly uneven, dark brown. Primary tomentum thick and dense, forming dark brown, arach- noid tufts of fasciculate hyphae; hairs 100–300 μm long, in fasci- cles of 10–20, mostly unbranched but strongly agglutinate, dark brown, cylindrical hyphae with free apices. Secondary tomentum not discernible except in microscopic sections; hairs 20–30 μm long, comprising single, branched, weakly moniliform hyphae with somewhat inflated cells and free apices. Rhizines present, scattered towards the thallus centre. Cyphellae rather dense, 21– 60 per cm2, rounded, plane, immersed to becoming erumpent, white to cream, strongly contrasting with the dark tomentum; pore 0.3–1(–1.5) mm diam.; cells of basal membrane irregularly bulging but lacking papillae. Additional specimens examined. USA: Hawaii: Hawaii, Mauna Loa, Pohakuloa training area, 1585 m, 1977, P. K. Higashino 323 (HAW); Maui, East Maui, Makawao Forest Reserve, 1800 m, 1975, C. W. Smith 1950a (HAW); Maui, Haleakalā, N slope, Frisbee meadow above Poouli Flats, 2195 m, 1987, C. W. Smith 8455 (HAW). Additional specimens examined. USA: Hawaii: Hawaii, Mauna Loa, Pohakuloa training area, 1585 m, 1977, P. K. Higashino 323 (HAW); Maui, East Maui, Makawao Forest Reserve, 1800 m, 1975, C. W. Smith 1950a (HAW); Maui, Haleakalā, N slope, Frisbee meadow above Poouli Flats, 2195 m, 1987, C. W. Smith 8455 (HAW). Apothecia not observed. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at rg/10.1017/S0024282920000353 Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at Discussion As outlined above and by Moncada et al. (2021), eight species of Sticta had previously been listed for Hawaii: S. ambavillaria, S. beauvoisii, S. cyphellulata, S. filix, S. fuliginosa, S. plumbicolor, S. tomentosa and S. weigelii (Elix & McCarthy 1998, 2008; Smith 2013). One additional species, S. limbata, was listed in an ecophy- siological study (Benner & Vitousek 2012). Of these, only three widespread taxa (S. fuliginosa, S. limbata, S. tomentosa) and one putatively endemic species (S. plumbicolor) could be confirmed as a result of a molecular phylogenetic revision (Moncada et al. 2021). Of the 13 species now recognized, one was a new record (S. andina) and eight were taxa new to science (seven species and one subspecies). Thus, the overlap in names recorded for Hawaii before and after this revision is only 38%. This sub- stantial change in the taxonomic delimitation of Hawaiian Sticta has implications for other studies of these lichens, including their potential applications, for example as environmental monitors. These examples show that revised species taxonomies also challenge the results of studies in ecology, ecophysiology, biogeog- raphy, biochemistry and other fields of research, a largely over- looked problem. Due to the frequent lack of voucher citations, correct identification of taxa used in such studies can often not be ascertained. It is therefore of the utmost importance that such studies deposit voucher material in public herbaria in the same way as taxonomic works, preferably with accessible digital information including specimen imagery. Collaboration with experts or DNA barcoding may improve the taxonomic compo- nent of such studies (e.g. Paudel et al. 2008; Plaza et al. 2014; Bajpai et al. 2015; Xu et al. 2016), but this does not replace proper deposition and citation of voucher material. DNA barcoding in particular is also often misinterpreted. In a study on physiological and genetic effects of chromium on thalli of Pyxine cocoes in India (Bajpai et al. 2015), the authors used ITS barcodes to support the identification of their material. However, our own revision of ITS sequences available for P. cocoes revealed that it forms three dis- tinct clades: one neotropical clade (KX512936), sister to a clade including a subclade formed by two specimens from Singapore For example, Czeczuga et al. (1997) analyzed carotenoid con- tent in various lichens including S. weigelii from Hawaii. Revised taxonomic concept and excluded names Secondary chemistry. No substances detected by TLC; medulla K−to slowly K+ faintly yellowish, cyphellae K+ slowly ochraceous. Several names previously reported in the genus Sticta for Hawaii must be excluded from the Hawaiian lichen biota, including at least one name from digital online repositories. We also per- formed an analysis of the specimens housed in HAW to see how previously applied names correspond to the revised tax- onomy (Fig. 3). Thus, the two common species confirmed to be present in Hawaii, S. fuliginosa and S. tomentosa, were largely identified correctly but some specimens also corresponded to other taxa, including S. antoniana and S. scabrosa var. hawaiiensis in the case of S. tomentosa and S. hawaiiensis and S. scabrosa var. hawaiiensis in the case of S. fuliginosa. Specimens previously Distribution and ecology. Sticta waikamoi is so far known only from a small number of collections, at mid to high altitudes between 1585 and 2195 m, including mixed conifer forest on the island of Maui. While this type of forest conveys the impres- sion of being rather undisturbed, it is not native to Hawaii, since all conifers have been introduced to the archipelago. While the type was growing on the ground over bryophytes, the species may occasionally also be epiphytic, then usually over bryophyte mats. Bibiana Moncada et al. 130 identified as S. weigelii represented no less than seven different taxa, mostly S. andina and S. scabrosa subsp. hawaiiensis. Sticta plumbicolor was mostly correctly identified but some specimens corresponded to S. andina and to the newly recognized S. flynnii and S. smithii (Fig. 3). H. Magn., P. xanthosticta (Pers.) Moncada & Lücking, and Sticta weigelii. While the taxonomy of Pseudocyphellaria followed the earlier revision by Moncada et al. (2014a), the material iden- tified as S. weigelii is likely to correspond to S. scabrosa subsp. hawaiiensis, because the Hawaiian lichens in that study had been collected at low altitudes (0–500 m). identified as S. weigelii represented no less than seven different taxa, mostly S. andina and S. scabrosa subsp. hawaiiensis. Sticta plumbicolor was mostly correctly identified but some specimens corresponded to S. andina and to the newly recognized S. flynnii and S. smithii (Fig. 3). ( ) Benner et al. (2007), Benner (2011) and Benner & Vitousek (2012) investigated the correlation between phosphorus, nitrogen and cyanolichens in Hawaiian forests, including experimental P fertilization. Revised taxonomic concept and excluded names Lobarioid species included in these studies were Crocodia aurata (Ach.) Link (as Pseudocyphellaria), Lobariella crenulata (Hook.) Yoshim., P. crocata (L.) Vain., Sticta beauvoisii, S. fuliginosa, S. limbata, S. tomentosa and S. weigelii. The presence of L. crenulata in Hawaii has not been confirmed by molecular data and the studied material may represent the newly recognized L. sandwicensis Lücking et al. (Lücking et al. 2017a). Crocodia aurata indeed occurs in Hawaii (Moncada et al. 2014a). However, Lobariella and Crocodia are chlorolichens and it is unclear why they were classified as cyanolichens, with rather high rates of N-fixation (Benner & Vitousek 2012), raising doubt about the underlying species identifications. Pseudocyphellaria crocata s. str. is absent from the Hawaiian archipelago and the material studied by Benner et al. (2007) and Benner & Vitousek (2012) refers to one or several newly distinguished taxa (Moncada et al. 2014a). In the absence of voucher material, it is impossible to ascertain which species were studied. In the case of the five representatives of Sticta, the material identified as S. beauvoisii is likely to represent S. scabrosa subsp. hawaiiensis, whereas S. weigelii could well refer to S. andina. The identifica- tions of the other three taxa (S. fuliginosa, S. limbata, S. tomen- tosa) appear to be correct. Notably, Benner & Vitousek (2012) found that the forest plot fertilized with P showed an increased abundance of lobarioid macrolichens, including species of Sticta. Between the two species of Sticta specifically tested for N-fixation, S. tomentosa showed higher N-fixation in the non- fertilized control plot, whereas for S. beauvoisii (= S. scabrosa subsp. hawaiiensis) it was higher in the P-fertilized plot. This result can be viewed in a new light, given the finding that among Hawaiian Sticta, S. tomentosa is largely confined to undisturbed forests (see also Smith 1991), whereas S. scabrosa subsp. hawaiiensis is frequent in disturbed and anthropogenic vegetation. Sticta ambavillaria (Bory) Ach. — Not present in Hawaii; speci- mens identified with this name represent S. antoniana (Moncada et al. 2020). Sticta babingtonii D. J. Galloway — The name of this green-algal species was applied to an unpublished herbarium collection from Maui (Olinda, Koolau Forest Reserve North Haleakala, 910 m, 1992, R. Rosentreter 8108 (SRP)), reported in the Lichen Portal of the Consortium of North American Lichen Herbaria (CNALH): [https://lichenportal.org/cnalh/collec- tions/individual/index.php?occid=1577932&clid=0]. Revision of a digital image of the specimen revealed it to be S. andina. Sticta cyphellulata (Müll. Revised taxonomic concept and excluded names Arg.) Hue — Not present in Hawaii; specimens identified with this name represent S. smithii and probably also S. flynnii (Moncada et al. 2020). p y f y Sticta filix (Sw.) Nyl. — Not present in Hawaii; an erroneous report of uncertain origin (Moncada et al. 2020). report of uncertain origin (Moncada et al. 2020). Sticta weigelii (Ach.) Vain. — Not present in Hawaii; specimens identified with this name represent S. andina, S. scabrosa subsp. hawaiiensis and S. waikamoi (Moncada et al. 2020). Sticta beauvoisii Delise; S. weigelii var. beauvoisii (Delise) Hue — Not present in Hawaii; specimens identified with this name represent S. scabrosa subsp. hawaiiensis (Moncada et al. 2020). Sticta weigelii var. lutescens (Taylor) H. Magn. — Not present in Hawaii; specimens identified with this name probably represent S. scabrosa subsp. hawaiiensis (Moncada et al. 2020). Sticta weigelii var. peruviana (Delise) Vain. — Not present in Hawaii; specimens identified with this name probably represent S. scabrosa subsp. hawaiiensis (Moncada et al. 2020). Sticta weigelii (Ach.) Vain. — Not present in Hawaii; specimens identified with this name represent S. andina, S. scabrosa subsp. hawaiiensis and S. waikamoi (Moncada et al. 2020). Sticta beauvoisii Delise; S. weigelii var. beauvoisii (Delise) Hue — Not present in Hawaii; specimens identified with this name represent S. scabrosa subsp. hawaiiensis (Moncada et al. 2020). Sticta weigelii var. lutescens (Taylor) H. Magn. — Not present in Hawaii; specimens identified with this name probably represent S. scabrosa subsp. hawaiiensis (Moncada et al. 2020). Sticta beauvoisii Delise; S. weigelii var. beauvoisii (Delise) Hue — Not present in Hawaii; specimens identified with this name represent S. scabrosa subsp. hawaiiensis (Moncada et al. 2020). Sticta weigelii var. lutescens (Taylor) H. Magn. — Not present in Hawaii; specimens identified with this name probably represent S. scabrosa subsp. hawaiiensis (Moncada et al. 2020). g p ( ) p Hawaii; specimens identified with this name probably represent S. scabrosa subsp. hawaiiensis (Moncada et al. 2020). https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at Discussion Conceivably, a next step following these results would be a con- servation assessment of Sticta lichens in Hawaii. However, since this is the first study that offers an accurate taxonomic treatment of the species, we consider it premature to carry out such an assess- ment with the current data. Instead, we hope this treatment encourages researchers to study the genus in more detail and with quantitative approaches, including the protocol proposed above. The small species especially need to be looked for more extensively, since these are likely to have been overlooked in the past or misinterpreted as young specimens not worth collection. It is therefore difficult to state whether these species are genuinely rare. Among the conspicuous taxa, S. fuliginosa and S. scabrosa subsp. hawaiiensis are certainly of the least concern, as they seem to tolerate various degrees of disturbance. Also S. tomentosa, although mostly found in undisturbed forest, appears to be abun- dant where forest vegetation is properly protected. and China (AF540540, KY611874), and another subclade formed by the Indian specimens from the above study (KF691782, KF691783, KF691784, KF691785, KF691786, KF691787). Based on the underlying topology and the large number of substitutions in the ITS barcoding marker, these clades represent three different species. Yet, even with these data to hand, the authors did not provide an adjusted taxonomy and identified their material uncritically with the name P. cocoes. y Another important aspect of revised and refined species taxon- omies is the use of lichens in environmental monitoring. In a study of the Sticta filix group in New Zealand, Ranft et al. (2018) showed that two previously unrecognized species in this group had a higher fidelity for undisturbed forest vegetation than the two species previously distinguished based on a broader approach to species delimitation. Given the particular situation in Hawaii as a threatened island biota (Kirch 1982; Stone & Scott 1985; Stone et al. 1992; Sakai et al. 2002; Department of Land and Natural Resources 2007; US Fish and Wildlife Service 2010; Wood 2012), the disparate ecology of Hawaiian Sticta enables the use of these lichens to monitor ecosystem health. This approach is further facilitated by the possibility of identifying the currently recognized taxa in the field. Nine of the species now recognized appear to be confined to closed or more or less undisturbed forest (Table 1). Discussion No vou- cher information was given but the locality data (Oahu, Melaleuca) indicate that the studied material probably represented S. scabrosa subsp. hawaiiensis, which is common in that area. Hagiwara (2016) and Hagiwara et al. (2016) analyzed antioxidant and antitumoral properties in a range of lichens collected in Hawaii and Iceland, including Pseudocyphellaria hawaiiensis 131 The Lichenologist Table 1. Proposed environmental scores (1 = least disturbed, 5 = most disturbed) for currently distinguished species of Hawaiian Sticta to be used in a simple monitoring protocol to assess ecosystem health. on relative species abundance at a given monitoring site: E =  Ei × Ai Species Preferred vegetation and disturbance level Score S. andina undisturbed rainforest 1 S. antoniana undisturbed rainforest 1 S. emmanueliana undisturbed rainforest 1 S. flynnii undisturbed rainforest 1 S. hawaiiensis undisturbed rainforest 1 S. limbata undisturbed rainforest 1 S. plumbicolor undisturbed rainforest 1 S. tomentosa undisturbed rainforest 1 S. waikamoi undisturbed rainforest 1 S. acyphellata partly disturbed or secondary old-growth rainforest 2 S. smithii partly disturbed or secondary old-growth rainforest 2 S. fuliginosa closed rainforest to partially disturbed and exposed vegetation 3 S. scabrosa subsp. hawaiiensis disturbed and anthropogenic, more or less exposed sites 5 where Ei = individual species score (see Table 1) and Ai = individ- ual relative species abundance (e.g. using a grid system or other sampling design). For example, a site with S. scabrosa subsp. hawaiiensis (90% abundance) and S. fuliginosa (10%) would give a weighted score of E = 5 × 0.9 + 3 × 0.1 = 4.8 (highly dis- turbed). In contrast, a site with S. andina (30%), S. flynnii (5%), S. fuliginosa (10%), S. smithii (5%) and S. tomentosa (50%) would result in a weighted score of E = 1 × 0.3 + 1 × 0.05 + 3 × 0.1 + 2 × 0.05 + 1 × 0.5 = 1.25 (less disturbed). Traditional taxonomy of the genus Sticta in Hawaii would not have allowed such an approach, as for instance the ecologically disparate S. andina and S. scabrosa subsp. hawaiiensis or S. hawaiiensis and S. fuliginosa had been subsumed under the names S. weigelii and S. fuliginosa, respectively. This impact of accurate taxonomy on the use of lichens as biomonitors was also discussed in a study of the S. filix group in New Zealand (Ranft et al. 2018). Author ORCIDs. Bibiana Moncada, 0000-0001-9984-2918; Robert Lücking, 0000-0002-3431-4636. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at Discussion As ‘undisturbed’ one may thereby also classify non-native old-growth forest such as the introduced conifer forest on Maui. On the other side of the spectrum is the widespread and somewhat weedy S. scabrosa subsp. hawaiiensis. Sticta fuliginosa is somewhat intermediate in its ecological requirements, being found in undisturbed forest but also dis- turbed sites. These observations would allow the establishment of a protocol using Sticta species along a disturbance gradient (Table 1) (1 = least disturbed, 5 = most disturbed), using an abundance-weighted environmental monitoring score E based Acknowledgements. Funding for this study was provided by the National Science Foundation (NSF) to The Field Museum: ‘ATM– Assembling a Taxonomic Monograph: The Lichen Family Graphidaceae’ (DEB-1025861; PI HTL, CoPI RL) and ‘Collaborative Research: Evolution, Diversification, and Conservation of a Megadiverse Flagship Lichen Genus’ (DEB-1354884; PI HTL, CoPI RL). Further financial support came from The Field Museum’s Women’s Board Field Dreams program 2011, through gifts by the Robert Thomas Bobins Foundation through Mrs Virginia Bobins (Chicago), Mr and Mrs John Borland Jr. (Chicago), Mrs Peggy Carr (Chicago) and Mrs Sue Dickes (Winnetka). Patrick Bily (The Nature Conservancy Hawaii), Timothy Flynn (National Tropical Botanical Garden), Daniel Pomaika’i (Maui Soil and Water Conservation Districts, Maunalei Arboretum) and Philip Thomas (Research Corporation of the University of Hawaii and the Hawaiian Ecosystems at Risk Project) assisted with fieldwork on Maui and Kauai. Jill Kajikawa-Kent (University of Hawaii) and Rae Matthews and the Clark family (National Tropical Botanical Garden) also provided logistical support. The Hawaii Department of Land and Natural Resources, Divisions of Forestry and Wildlife and Division of State Parks, provided collecting and research permits, and Chelsea Carineo, Wendee Kokubun, Ryan Peralta, Patrick Porter, Matthew Rittenhouse and Lancede Silva are thanked for pro- cessing the corresponding requests. Roger Rosentreter kindly provided infor- mation on and digital images of Hawaiian specimens identified as Sticta babingtonii. Author ORCIDs. Bibiana Moncada, 0000-0001-9984-2918; Robert Lücking, 0000-0002-3431-4636. Author ORCIDs. Bibiana Moncada, 0000-0001-9984-2918; Robert Lücking, 0000-0002-3431-4636. Bibiana Moncada et al. 132 Sonoran Desert Region, Vol. 2. Tempe, Arizona: Lichens Unlimited, Arizona State University, pp. 513–524. Sonoran Desert Region, Vol. 2. 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Honolulu: University of Hawai‘i Press and Bishop Museum Press. Simon A, Goffinet B, Magain N and Sérusiaux E (2018) High diversity, high insular endemism and recent origin in the lichen genus Sticta (lichenized Ascomycota, Peltigerales) in Madagascar and the Mascarenes. Molecular Phylogenetics and Evolution 122, 15–28. Wood KR (2012) Possible extinctions, rediscoveries, and new plant records within the Hawaiian Islands. Bishop Museum Occasional Papers 113, 91–102. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000353 Downloaded from https://www.cambridge.org/core. Charité Universitätsmedizin Berlin, on 18 Jan 2022 at 10:18:56, subject to the Cambridge Core terms of use, available at Discussion Smith CW (1991) Lichen conservation in Hawaii. In Galloway DJ (ed.), Tropical Lichens: Their Systematics, Conservation, and Ecology. The Systematics Association Special Volume No. 43. Oxford: Clarendon Press, pp. 35–45. 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A Constituição da Equipe Terapêutica- O Processo de Formação do Terapeuta Sistêmico de Família
Revista Brasileira de Terapia Familiar
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1 Graduada em psicologia pela UFRJ, psicoterapeuta familiar, Doutora em Educação pela UFMG, Membro da ATF, Membro colaboradora CEFAI- Centro de Estudo da Família, Adolescência a Infân- cia. Professora da UEMG/unidade Carangola. Resumo 2REMHWLYRGHVVHWH[WRpIRPHQWDUXPDUHÀH[mRDFHUFDGHFRPRYDLVHFRQV WLWXLQGRRVHQWLPHQWRGHHTXLSHWHUDSrXWLFDWRPDQGRFRPRIRFRDVUHODo}HVHV WDEHOHFLGDVHPXPJUXSRTXHSDUWLFLSDGRFXUVRGHIRUPDomRGHWHUDSHXWDVGH IDPtOLDTXHWHPFRPRSHUVSHFWLYDR&RQVWUXFLRQLVPR6RFLDO$QRomRGHHTXLSH WHUDSrXWLFD HVWi DVVRFLDGD DR VHQWLPHQWR FROHWLYR GH SHUWHQFLPHQWR DR JUXSR FRQ¿DQoDHUHVSHLWRQRo}HVTXHLQWHJUDPXPFRQMXQWRGHDWLWXGHVRULHQWDGDVTXH VHFRQ¿JXUDPHPXPDH[SHUWLVHSHVVRDOHJUXSDOTXHFRQMXJDGDDRUHSHUWyULR teórico e de práticas integradas, permite com que o aluno se sinta encorajado a GDULQtFLRDRDWHQGLPHQWRWHUDSrXWLFRjVIDPtOLDV1HVVDWHVVLWXUDSULPHLUDPHQWH delineamos os princípios epistemológicos do Construcionismo Social como cená ULRTXHFRORFDHPHYLGrQFLDRVHQWLGRGDVH[SHULrQFLDVSHVVRDLVHVHFRDGXQDP FRPDDSUHQGL]DJHPFRODERUDWLYD(PVHJXLGDRIRFRUHFDLVREUHRVDVSHFWRV UHIHUHQWHVDRFXUVRGHIRUPDomRGRWHUDSHXWDFRPrQIDVHSDUDRHQIRTXHTXH projeta a construção do que denominamos como sentimento de equipe, cujos ele mentos emergem por meio da mudança de postura, pela escuta sensível e pela demonstração de um comprometimento responsável e ético com o outro. Palavras chave: Formação do terapeuta; Equipe terapêutica; Práticas co- laborativas $&RQVWLWXLomRGD(TXLSH7HUDSrXWLFD 2SURFHVVRGHIRUPDomRGRWHUDSHXWDVLVWrPLFRGHIDPtOLD Antônia Simone Gomes1 $EVWUDFW 7KHSXUSRVHRIWKLVWH[WLVWRIRVWHUDUHÀHFWLRQDERXWKRZWKHIHHOLQJRIDWKH UDSHXWLFWHDPLVEHFRPLQJIRFXVLQJRQWKHUHODWLRQVKLSVHVWDEOLVKHGLQDJURXS WKDWSDUWLFLSDWHVLQWKHWUDLQLQJFRXUVHRIIDPLO\WKHUDSLVWVZKRVHSHUVSHFWLYHLV Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 38 A Constituição da Equipe Terapêutica – A. S. Gomes 6RFLDO&RQVWUXFWLRQLVP7KHQRWLRQRIWKHUDSHXWLFWHDPLVDVVRFLDWHGZLWKWKHFRO OHFWLYHIHHOLQJRIEHORQJLQJWRWKHJURXSWUXVWDQGUHVSHFWQRWLRQVWKDWLQWHJUDWH DVHWRIRULHQWHGDWWLWXGHVWKDWDUHFRQ¿JXUHGLQSHUVRQDODQGJURXSH[SHUWLVH WKDW FRPELQHG ZLWK WKH WKHRUHWLFDO UHSHUWRLUH DQG LQWHJUDWHG SUDFWLFHV DOORZV WKHVWXGHQWIHHOVHQFRXUDJHGWRLQLWLDWHWKHUDSHXWLFFDUHIRUWKHIDPLOLHV,QWKLV FRQWH[WZH¿UVWRXWOLQHWKHHSLVWHPRORJLFDOSULQFLSOHVRI6RFLDO&RQVWUXFWLRQLVP DVDVHWWLQJWKDWKLJKOLJKWVWKHPHDQLQJRISHUVRQDOH[SHULHQFHVDQGLVFRQVLV WHQWZLWKFROODERUDWLYHOHDUQLQJ7KHQWKHIRFXVLVRQWKHDVSHFWVUHODWHGWRWKH WUDLQLQJFRXUVHRIWKHWKHUDSLVWZLWKHPSKDVLVRQWKHDSSURDFKWKDWSURMHFWVWKH FRQVWUXFWLRQRIZKDWZHFDOOWKHIHHOLQJRIWHDPZKRVHHOHPHQWVHPHUJHWKURXJK WKHFKDQJHRISRVWXUHVHQVLWLYHOLVWHQLQJDQGWKHGHPRQVWUDWLRQRIDUHVSRQVLEOH and ethical commitment to the other. .H\ZRUGV Therapist training; Therapeutic team; Collaborative practices Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) Introdução Pensar os contornos que envolvem a noção de equipe terapêutica dentro de um curso de formação de terapeutas, requer estarmos atentos às dinâmicas de interação, às trocas relacionais e aos diálogos que acontecem entre os pares. No presente texto, estamos tratando do curso de formação de terapeutas de família ministrado pelo CEFAI, Centro de Estudo da Família, Adolescência e Infância, ins- tituição de atendimento clínico e de formação de terapeutas que se dedica desde jUHÀH[mRWHyULFRHSUiWLFDQRDWHQGLPHQWRGHIDPtOLDVFULDQoDHDGROHVFHQWHV O curso de Formação em Terapia de Família e Casal, em linhas gerais, tem o propósito de formalizar uma proposta de consolidação contextual e relacional SDUDRDWHQGLPHQWRjVIDPtOLDV(VWHFXUVRSRGHVHUGH¿QLGRFRPRXPHVSDoR de orientação demarcado pela criação de um contexto conversacional que se es- WDEHOHFHSRUPHLRGHGLiORJRVUHÀH[LYRVTXHVHFRQVWLWXHPHQTXDQWRSURFHVVR colaborativo de saberes e fazeres (McNamee, 2014). Para Ladvocat e Ricci (2009), a formação do terapeuta: “é vista como um processo de aprendizagem em que de um lado está quem fornece o instrumento SDUDDFRQVWUXomRGHXPQRYRSDSHOHIXQomRSUR¿VVLRQDOHGHRXWURDTXHOH que está buscando o desenvolvimento do referido papel.” (p.464). Para essas autoras, o processo de formação deve apoiar-se tanto em um constructo teórico sólido como também deve incluir um trabalho pessoal de cunho terapêutico. O curso de formação de terapeutas constitui-se em aulas quinzenais em um percurso de dois anos e meio, sendo que, no primeiro ano de estudo os alunos dedicam-se às leituras indicadas com objetivo de proporcionar um repertório teó- Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 39 A Constituição da Equipe Terapêutica – A. S. Gomes rico-prático que permite que se apropriem dos conceitos que embasam a compre- ensão da Teoria Geral dos Sistemas (Bertalanffy, 1967), articulados às discussões que envolvem os conceitos da psicodinâmica, da cibernética e da comunicação. A proposta do curso subjaz na lógica de pensar relacional, em que o cami- nho trilhado se sustenta na mudança de paradigma que nos convida a sair de uma postura cartesiana para perceber a família sob a égide do paradigma da complexidade (Morin, 1996). Nessa perspectiva, o curso de formação passa a ser considerado como um processo de construção social de caráter dialético, em que a linguagem e os processos conversacionais podem ser entendidos como instrumentos constitutivos de subjetividades, o que equivale a um espaço de criação/recriação/transformação do sujeito. $VHJXQGDHWDSDGRFXUVRGHIRUPDomRFRQ¿JXUDVHGHIRUPDPDLVFRQWXQ- dente pelo início da prática do atendimento clínico, momento em que o trabalho em equipe se torna um instrumento fundamental no processo de formação do te- rapeuta. Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) Introdução Esse espaço de interações e produção de conhecimento é marcado por um sentimento de natureza coletiva que que irá funcionar como um suporte para os alunos/terapeutas que iniciam a função de atendimento clínico às famílias. Aqui, a noção de equipe terapêutica está associada a esse sentimento coletivo GHSHUWHQFLPHQWRDRJUXSRFRQ¿DQoDHUHVSHLWRSHORRXWURQRo}HVTXHLQWHJUDP XPFRQMXQWRGHDWLWXGHVRULHQWDGDVTXHFRQ¿JXUDPVHHPXPDH[SHUWLVHSHVVRDOH grupal que, conjugada ao repertório teórico e de práticas integradas, permite com que o aluno em processo de formação, se sinta encorajado a dar início ao atendi- mento terapêutico às famílias inscritas no programa de atendimento clínico-social. Ao tratar das questões referentes à construção de uma equipe terapêuti- ca no contexto de um curso de formação, deparamo-nos com pontos bási- cos que se referem tanto aos conhecimentos produzidos e que são constru- ídos a partir do aprofundamento teórico, quanto às competências sociais e humanísticas que potencializam um espaço de criação/transformação. Nessa tessitura, os princípios epistemológicos do Construtivismo Social (Vygotsky, 1984; Castanõn, 2009, 2004), fundamentam os modelos sociocultu- rais que formulam um cenário que coloca em evidência o sentido das experiên- cias pessoais. Ao mesmo tempo adotamos uma abordagem relacional sistêmica na vertente do Construcionismo Social (Gerken & Gergen, 2010; Gergen, 2009), DTXLHQWHQGLGRFRPRXPSUHVVXSRVWR¿ORVy¿FR $QGHUVRQ FXMRVVLJQL¿- cados são gerados nas relações a partir das produções discursivas. Estas são SURGXo}HVOLQJXtVWLFDVQHJRFLDGDVGHQWURGHFRQWH[WRVHVSHFt¿FRVFRPDLQWHQ- ção de comunicar ou expressar algo. No curso de formação em Terapia de Família busca-se com que a prática se sustente nos aportes do Construcionismo Social associados aos princípios da Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 40 A Constituição da Equipe Terapêutica – A. S. Gomes 7HRULD6LVWrPLFDRTXHVLJQL¿FDDWXDUFRQVRDQWHXPDDERUGDJHPQDTXDORIRFR está direcionado para o dinamismo discursivo dos acontecimentos, à imprevisibi- lidade no controle das situações, para o sujeito em sua auto-referência, eviden- ciando que não existe realidade independente de um observador. Nesse âmbito, SDVVDVHDUHFRQKHFHUXPPXQGRHPSURFHVVRGHWRUQDUVHRTXHVLJQL¿FDTXH o “dinamismo dos acontecimentos corresponde a uma realidade compartilhada DSDUWLUGDVSUySULDVH[SHULrQFLDVLQGLYLGXDLVHDVVLPFRQ¿JXUDDGLPHQVmRGRV espaços consensuais de intersubjetividades” (Vasconcellos, 2002, p. 124). 7HRULD6LVWrPLFDRTXHVLJQL¿FDDWXDUFRQVRDQWHXPDDERUGDJHPQDTXDORIRFR está direcionado para o dinamismo discursivo dos acontecimentos, à imprevisibi- lidade no controle das situações, para o sujeito em sua auto-referência, eviden- ciando que não existe realidade independente de um observador. Nesse âmbito, SDVVDVHDUHFRQKHFHUXPPXQGRHPSURFHVVRGHWRUQDUVHRTXHVLJQL¿FDTXH o “dinamismo dos acontecimentos corresponde a uma realidade compartilhada DSDUWLUGDVSUySULDVH[SHULrQFLDVLQGLYLGXDLVHDVVLPFRQ¿JXUDDGLPHQVmRGRV espaços consensuais de intersubjetividades” (Vasconcellos, 2002, p. 124). Introdução É importante salientar que o processo de formação de terapeutas sistêmicos de família tendo em uma capacitação teórico-prática, leva em consideração não só os princípios básicos de uma epistemologia que coloca o mundo como uma cons- trução, cujos aportes se encontram no Construcionismo Social (Gerken & Ger- gen, 2010; Gergen, 2009; Japur, 2007), como busca assegurar um trabalho que se sustenta em dinâmicas de natureza colaborativa (Anderson, 2011; Grandesso, 2000). A proposta colaborativa pressupõe práticas que se alinham aos princípios YDORUDWLYRVGDFRQ¿DQoDFRPSDUWLOKDGDQRUHVSHLWRP~WXRQRGLiORJRUHÀH[LYR na alteridade e na disponibilidade em relação ao outro, o que pode ser traduzido pelo crescente sentimento de pertencimento ao grupo e na responsabilidade ética. 2FRQKHFLPHQWRSURGX]LGRQHVVHHVSDoRLQWHUDFLRQDOHQYROYHHLQÀXHQFLD WDQWRDVSURIHVVRUDVIRUPDGRUDVTXDQWRRVDOXQRVVHQGRFULDGRXPÀX[RFRQ- versacional de trocas de experiências de natureza colaborativa que passa a afe- tar as histórias de cada participante. As práticas conversacionais apresentam-se como instrumentos de transformação e atribuição de sentidos e constituem-se em recursos potentes no processo de formação, tão diferente do modelo adotado por outras abordagens, a exemplo da Psicanálise. Com base nesses princípios, HVVHDUWLJRWHPFRPRREMHWLYRIRPHQWDUXPDUHÀH[mRDFHUFDGHFRPRYDLVH constituindo o sentimento de equipe terapêutica, tomando como foco as relações estabelecidas no grupo de formação de terapeutas, que tem como perspectiva o Construcionismo Social e o aprendizado colaborativo. Nesse caminho, primeiramente, delineamos os aportes teóricos do Cons- trucionismo Social seguindo os contornos das práticas colaborativas, para em seguida jogar luz sobre alguns aspectos referentes ao curso de formação de terapeuta e assim descrever pontos que ajudam a pensar como se dá a constru- ção de uma equipe terapêutica. Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 2&RQVWUXFLRQLVPR6RFLDO O Construcionismo Social desponta nas últimas décadas como um processo de construção de conhecimento e proposta epistemológica que coloca em des- Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 41 A Constituição da Equipe Terapêutica – A. S. Gomes taque os processos relacionais e discursivos, o que na perspectiva de McNamee (2003) e Gergen (2009), surge como sendo um conhecimento que se instala no interior dos processos de intercâmbio social que ³RFXSDVHGHH[SOLFDURVSURFHV VRVSHORVTXDLVDVSHVVRDVGHVFUHYHPH[SOLFDPRXGHDOJXPDIRUPDGmRFRQWD GRPXQGRHPTXHYLYHP´(p.23). Para esses autores, a epistemologia dialógica do Construcionismo Social se perspectiva na criação de práticas conversacionais em que os conhecimentos passam a ser compartilhados como produtos de sentidos. Esses aspectos constituem-se a partir de um conjunto de princípios e práticas que derivam do conceito de responsabilidade relacional, envolvendo os processos in- terativos como produtores de sentido, gerando ações no mundo. O Construcionismo Social focaliza as práticas discursivas como base de qualquer conhecimento e considera os processos microssociais, defendendo a compreensão humana a partir da esfera relacional em que sentidos e signi- ¿FDGRVVmRSURGX]LGRVRTXHQRHQIRTXHGH*HUJHQ  FRUUHVSRQGHj “construção de sentidos geradas nas práticas sociais cotidianas” (p.26). Esse movimento se dá sob a égide de uma ação conjunta (Grandesso, 2000) e dialéti- ca, em que “o sujeito afeta a realidade social na qual está inserido e passa a ser afetado pelas mudanças efetivadas nesse contexto, caracterizando assim, um processo social e linguístico” (p.66). O Construcionismo Social focaliza as práticas discursivas como base de qualquer conhecimento e considera os processos microssociais, defendendo a compreensão humana a partir da esfera relacional em que sentidos e signi- ¿FDGRVVmRSURGX]LGRVRTXHQRHQIRTXHGH*HUJHQ  FRUUHVSRQGHj “construção de sentidos geradas nas práticas sociais cotidianas” (p.26). Esse movimento se dá sob a égide de uma ação conjunta (Grandesso, 2000) e dialéti- ca, em que “o sujeito afeta a realidade social na qual está inserido e passa a ser afetado pelas mudanças efetivadas nesse contexto, caracterizando assim, um processo social e linguístico” (p.66). Sob esta ótica, a linguagem pode ser considerada uma construção social, fruto de negociação de sentidos onde são ressaltadas as produções discursivas compartilhadas por um determinado grupo, em um determinado contexto, que legitimam os saberes e os fazeres do grupo. Dizer que a linguagem é uma prá- tica social é considerar que ela está para além da representação da realidade, é dizer que a linguagem constrói a realidade. Para o discurso Construcionista Social, a linguagem é um elemento central na constituição do sujeito. Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 2&RQVWUXFLRQLVPR6RFLDO Essa pers- pectiva nos anuncia que é na linguagem que o sujeito estrutura suas experiên- cias com/do o mundo e de nós mesmos. Para Burr (In CorradI-Webster, 2014) a linguagem é constituinte do que somos. A linguagem auxilia a organizar nossas experiências. O discurso Construcionista Social convida a pensar que as características TXHLGHQWL¿FDPRVQRVFRPSRUWDPHQWRVGDVSHVVRDVVmRFRQVWUXtGDVFRPRXVR da linguagem. Nessa proposição, Anderson (2011) aponta o deslocamento do conceito de linguagem, que passa de um papel representacional da realidade, como é entendida no paradigma tradicional da ciência, para um lugar de centra- lidade no processo relacional, situado na produção de sentidos que considera os discursos como ação conjunta tendo o poder de construir/reconstruir pessoas e relações. Sob esse enfoque, a linguagem deixa de ser entendida como um meio Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 42 A Constituição da Equipe Terapêutica – A. S. Gomes para representar a realidade e passa a ser considerada como um instrumento, o que Ibañez-Gracia (2004) sistematiza como a capacidade da linguagem não só em “fazer pensamento” como também de “fazer realidades”. Considerando o espaço conversacional como uma “arena de negociações”, local onde emergem experiências que pontuam as diferenças resultando em dis- FXUVRVTXHSRVVLELOLWDPRFRPSDUWLOKDPHQWRGHQRYRVVLJQL¿FDGRVHQWHQGHPRV o espaço da formação do terapeuta como um espaço em que ecoam múltiplas vozes e sobressaem as singularidades que ganham convergências e divergên- cias no/pelo diálogo. Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) $DERUGDJHPFRODERUDWLYD $DERUGDJHPFRODERUDWLYDSRGHVHUFRPSUHHQGLGDFRPRXPDSRVLomR¿OR- Vy¿FDTXHGHPDUFDXPDDomRFRPSDUWLOKDGDVLJQL¿FDGDSHORGLiORJRFROHWLYR cujos pressupostos encontram-se assentados em um conjunto de princípios de natureza relacional, que “pressupõe uma parceria na qual a sabedoria, conheci- mentos e costumes dos membros de uma comunidade de aprendizagem local, são reconhecidos, acessados e utilizados” (Anderson, 2011, p.36). Nessa abor- dagem os diálogos são considerados recursos linguísticos que colocam em cena o intercâmbio de ideias, a exploração de pensamentos, a expressão de opiniões e os sentimentos, com a intenção de entendimento mútuo. Na conversação dia- lógica os participantes envolvem-se uns com os outros e o conteúdo conversa- FLRQDO¿FDVXMHLWRjLQWHUDomRHLQWHUSUHWDomR 0DWWRVGH%ULWR *HUPDQR  Podemos então dizer, que o processo dialógico colaborativo, passa a ser exercitado como um convite à diversidade em que as diferentes formas de se entender e de perceber a realidade são expostas. Trabalhar nessa direção, per- mite o aprimoramento de um nível de interesse pelo ponto de vista do outro, FDPLQKDQGRSDUDDFRQVROLGDomRGHXPDPELHQWHGHFRQ¿DQoDHSHUWHQFLPHQWR A abordagem colaborativa vem sendo compartilhada por vários autores (An- derson & Goolishian,1998; Anderson, 2011) como um processo que opera sob formas diferenciadas de trocas e interações uns com os outros, de modo que to- dos contribuem na produção de uma nova compreensão do processo. Anderson e Goolishian (1998, p.36) partem do pressuposto que “as pessoas vivem e com- preendem seu viver por meio de realidades narrativas construídas socialmente, que conferem sentido e organização à sua experiência”. Como nos assegura Anderson (2011), este processo acontece em um movi- mento de mão dupla: o que está sendo aprendido é transformado e as pessoas que estão envolvidas em todo o aprendizado também são transformadas. Tra- Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 43 A Constituição da Equipe Terapêutica – A. S. Gomes ta-se de uma atividade dialógica de natureza coletiva que envolve intercâmbios de ideias, trocas de experiências, opiniões e sentimentos, através do quais o produto resultante dessas interações será algo novo. Para Anderson (2011), Os parceiros colaborativos, em suas trocas dinâmicas, geram conheci- PHQWRVHRXWUDVLQRYDo}HVPXLWRPDLVFULDWLYDVDEXQGDQWHVHHVSHFt¿FDV ao contexto e necessidades locais do que qualquer membro da parceria poderia alcançar sozinho. Esse pressuposto baseia-se na suposição de que o conhecimento é uma construção em comunidade, criado no intercâmbio social, não na interação instrucional (p.36- 37). Quando se trata do processo de formação do terapeuta, pode-se dizer que a postura de natureza colaborativa se constitui na construção de um espaço de trocas e circularidades de saberes em que especialistas e alunos são afetados por essa ação. $DERUGDJHPFRODERUDWLYD O educador reconhece e valoriza a fala dos participantes de for- PDLQFRQGLFLRQDORTXHLQFRUUHDVVLPQDFULDomRGHXPDPELHQWHGHFRQ¿DQoD HPTXHWRGRVSDUWLFLSDPGRSURFHVVRGHWUDQVIRUPDomR8PLQÀXHQFLDRRXWUR em um processo de construção subjetiva e de co-responsabilidades. Estabe- lece-se assim, um sistema linguístico dialógico focado na relação, na postura FUtWLFDHUHÀH[LYD 3HUHLUD 5LJXHWWL $OLQJXDJHPTXHFLUFXODHQWUHRV participantes se efetiva como instrumento/ferramenta e veículo de construção de VLJQL¿FDGRVFDUDFWHUL]DQGRHVVHFRQWH[WRFRPRXPHVSDoRGHDSUHQGL]DJHP Segundo Anderson e London (2012, p.23), esse tipo de ambiente de aprendi- zado colaborativo requer valores e atitudes particulares do educador em relação a: a) a natureza transformadora do diálogo e da colaboração E FRQ¿DQoDHVHJXUDQoDQDVFRPSHWrQFLDVHQRMXOJDPHQWRGHFDGDPHP- bro quanto; à sua vida diária e futura, e quanto ao que é crucial para elas; c) o conhecimento e as experiências que os alunos trazem considerados como valiosos e necessários como os que os professores trazem; G rQIDVHQDUHÀH[LYLGDGHHDXWRUHIHUrQFLDQDFRQVWUXomRGRFRQKHFLPHQWR E FRQ¿DQoDHVHJXUDQoDQDVFRPSHWrQFLDVHQRMXOJDPHQWRGHFDGDPHP- bro quanto; à sua vida diária e futura, e quanto ao que é crucial para elas; c) o conhecimento e as experiências que os alunos trazem considerados como valiosos e necessários como os que os professores trazem; G rQIDVHQDUHÀH[LYLGDGHHDXWRUHIHUrQFLDQDFRQVWUXomRGRFRQKHFLPHQWR Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) A Constituição da Equipe Terapêutica – A. S. Gomes No que tange à utilização da sala de espelho como recurso didático, Cruz, Vicente e Pereira (2014) consideram uma ferramenta privilegiada no processo de orientação, uma vez que possibilita tanto a observação da atuação daqueles pro- ¿VVLRQDLVTXHHstão no campo, como permite acompanhar o discurso real explici- tado pelas famílias no setting terapêutico. Adotar essa metodologia no processo de formação, como nos indicam os autores, representa uma “forma generosa dos mestres abrirem as portas e permitir que sentemos na sala de atendimento, o que FRQ¿JXUDDVVLPXPDPXGDQoDQDVUHODo}HVGHHQVLQRDSUHQGL]DJHPWUDGLFLRQDLV para outras mais democráticas” (Cruz, Vicente & Pereira, 2014, p.310). Quanto ao modelo de atendimento temos: a pré-sessão que pode ser enten- dida como aquele momento de conversação que antecipa o atendimento à família e se constitui como um espaço de conexão entre as terapeutas de campo, as professoras formadoras e os demais participantes da equipe. As práticas colabo- rativas, aí encaminhadas, em geral se apresentam como alternativas úteis para o desenvolvimento da sessão terapêutica e servem como um aquecimento conver- sacional que favorece a atenção para os acontecimentos que envolvem a família/ casal. A pré-sessão é também um espaço de fortalecimento dos vínculos entre as WHUDSHXWDVTXHHVWmRDWXDQGRQRFDPSRHVSDoRGHUHÀH[mRHSUREOHPDWL]DomR das situações referentes à família. Nesse momento se estabelece uma interlo- cução entre os terapeutas de campo e os demais do grupo, em que as múltiplas vozes têm o poder de construir caminhos possíveis para atuação junto à família. 2PRPHQWRGDVHVVmRSURSULDPHQWHGLWRVHFRQ¿JXUDSHODHQWUDGDGDGX- pla de terapeutas no setting terapêutico enquanto os demais participantes per- manecem acompanhando a sessão por trás do espelho unilateral. A conexão HQWUHDVSDUWHVVHIRUWDOHFHQDPHGLGDHPTXH¿FDHVWDEHOHFLGRTXHRVTXH estão atrás do espelho podem intervir na sessão por meio do interfone, caso seja necessário. Essa possibilidade de participação conjunta opera como ego auxiliar e representa um elemento positivo de natureza colaborativa. Nesse enquadre, pode-se dizer então, que é estabelecida uma conexão entre os que estão em FDPSRHRJUXSRREVHUYDGRU(VVDFRQH[mRDFRQWHFHWDQWRSHODFRQ¿DQoDGH- positada uns nos outros, quanto pelo respeito às diferenças, no que se refere ao estilo próprio e pessoal de condução do percurso da terapia. É aqui que o sentimento de pertencimento ao grupo se faz mais presente e a relação colabo- rativa constitui o elo de ligação entre as partes. Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 2FRQWH[WRGHDWHQGLPHQWRFOtQLFR Voltando-se o olhar para o contexto de formação, tomamos como fonte de ob- servação os atendimentos clínicos à família e casal que acontecem como procedi- mento da prática. Os atendimentos ocorrem na sala do espelho seguindo o modelo de orientação cuja matriz se sustenta nos aportes da Escola de Milão. Essa proposta de atuação se estrutura segundo quatro momentos distintos: a pré-sessão, a ses- são, a inter-sessão e a pós sessão (Palazzoli, Boscolo, Cecchin & Prata, 1982). Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 44 A Constituição da Equipe Terapêutica – A. S. Gomes A Constituição da Equipe Terapêutica – A. S. Gomes Entendemos então, que o lugar do terapeuta vai sendo produzido pela conexão e sintonia com os participantes do grupo como um processo de co-construção no contexto conversacional cola- borativo em que sobressai a capacidade inventiva de cada um em criar um canal de comunicação consigo próprio, com o grupo e com a família. O momento da inter-sessão é marcado pelo encontro dos terapeutas de Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 45 A Constituição da Equipe Terapêutica – A. S. Gomes campo com a equipe que permanece atrás do espelho para formularem uma bre- YHFRQYHUVDSDUDGH¿QLUHPMXQWRVRVUXPRVGRWpUPLQRGDVHVVmR'HSRLVGHVVH encontro, há o retorno à campo para proceder à devolução, que pode acontecer SRUPHLRGHLQWHUYHQo}HVHUHÀH[}HVUHDOL]DGDVSHOD dupla de terapeutas, pode VHUSURSRVWDXPDGLQkPLFDYLYHQFLDORXDLQGDVHU¿QDOL]DGDVREDIRUPDGHHTXL- SHUHÀH[LYDXPUHFXUVRTXHVHFRQVWLWXLVHJXQGRRPRGHORWHUDSrXWLFREDVHDGR QRVSURFHVVRVUHÀH[LYRVGH$QGHUVHQ  HPTXHRVWHUDSHXWDVREVHUYDGR- UHVRIHUHFHPjIDPtOLDXPDUHÀH[mRDFHUFDGRTXHREVHUYDUDPHRXVHQWLUDP com os discursos produzidos na sessão. Nesse contexto a linguagem age como LQVWUXPHQWRSURSLFLDGRUGHUHÀH[mRHGHVHQFDGHDGRUGHVHQWLPHQWRVHHPRo}HV que fazem ressonâncias com as histórias de vida de cada um dos participantes que se encontram na sala atrás do espelho. Quanto ao quarto momento destinado à conversação da equipe sobre o atendimento à família, a pós-sessão, constitui-se como uma prática privilegiada na formação do terapeuta, uma vez que nesse contexto, busca-se relacionar os conteúdos que emergiram da prática aos conhecimentos teóricos. A conversação colaborativa ganha tessitura à medida que são compartilhadas as percepções e os sentimentos que emergiram ao longo da sessão de terapia. Podemos dizer que nesse momento, o sentimento de equipe se materializa no exercício de en- trega, de escuta e de disponibilidade em relação ao outro. A prática conversacio- QDOGLDOyJLFDWRUQDVHXPLQVWUXPHQWRGHIRUPDomRHPTXHpSRVVtYHOLGHQWL¿FDU os sentidos atribuídos aos discursos de cada participante, entendendo que essa construção se dá a partir de como cada um é afetado pelo outro, seja pela história do cliente, pela fala do professor formador ou pela ação do colega que está em campo. Vê-se, nesse percurso o delineamento de uma identidade grupal. Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) Postura de acolhimento Olhando sob a perspectiva do curso de formação, vivenciar a postura de DFROKLPHQWRVLJQL¿FDH[SHULHQFLDUXPDSRVLomRGHDOWHULGDGHHPUHODomRDRRX- tro, o que implica criar um espaço interacional de aceitação e escuta interessa- da. Acolher o outro em sua singularidade corresponde em imprimir um clima no qual se aceita a diferença e os pontos de vista distintos, sabendo-se sentir con- ¿UPDGRHDFHLWRSHORTXHpLQFRQGLFLRQDOPHQWH,VVRHQYROYHWDQWRXPDHVFXWD sensível e atenta sobre o ponto de vista apresentado, como supõe a valorização da sua expertise e de seu estilo próprio. 1RJUXSRGHIRUPDomRDSRVWXUDGHDFROKLPHQWRUHÀHWHRDPDGXUHFLPHQWR HPRFLRQDOGRJUXSRRTXHVLJQL¿FDXPLPEULFDGRSURFHVVRGHFRQVWUXomRFROH- tiva que convida os participantes a se tornarem protagonistas desta construção. (QWmRFRPRVHFRQVWLWXLDHTXLSHWHUDSrXWLFD" É importante considerar que o sentimento de equipe se dá passo a passo, ao longo das vivências coletivas e grupais em que são experimentadas postu- ras mais colaborativas, mais comprometidas, autônomas e responsáveis, o que permite escutar as diferentes vozes, possibilitando que cada participante (re) co- QKHoDVHXHVWLORHVHDSURSULHGHUHFXUVRVVLJQL¿FDWLYRVSDUDDWXDomRQDSUiWLFD Para nós, dimensionar como se constitui uma equipe terapêutica se torna possível pela observação de mudança de postura dos alunos. As inter-relações HDVWURFDVFRQYHUVDFLRQDLVVmRVLJQL¿FDGDVFRPRIRQWHGHWUDQVIRUPDomRe VLJQL¿FDWLYDDPXGDQoDGHFRPSRUWDPHQWRIUHQWHDRVQRYRVFRQWH[WRVFRQYHU- sacionais, como também se fortalece a escuta sensível. A realidade discursiva se apresenta de forma responsável e ética. Aos poucos, o grupo vai se consti- Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 46 A Constituição da Equipe Terapêutica – A. S. Gomes tuindo a partir das/nas diferenças, na diversidade, no jeito próprio de cada um se manifestar em seu processo de subjetivação. O compartilhamento de ideias, o GLiORJRUHÀH[LYRHSUREOHPDWL]DGRURexercício de escuta e o limite como forma GHFRQWHQomRGRÀX[RGHLGeias, emergem como elementos que se conjugam na constituição do sentimento de equipe terapêutica. Podemos dizer que estamos diante de um paradoxo, pois é na diferença, QDVVLQJXODULGDGHVHDSDUWLUGDVHVSHFL¿FLGDGHVTXHRJUXSRVHXQHHFRQVWL- tui-se como grupo identitário. Nesse movimento o grupo cresce e amadurece. A participação de cada um ganha versão de comprometimento relacional em que RVSURFHVVRVFRQYHUVDFLRQDLVVmRVLJQL¿FDGRVFRPDFRQWULEXLomRGHWRGRVRV sujeitos: a expertise de cada um é valorizada no coletivo. Passamos a apresentar quatro práticas através das quais nos foi possível perceber indícios de como se dá a constituição do sentimento de equipe. Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) (VSDoRGHHVFXWDFROHWLYD 'LiORJRVUHÀH[LYRVHRHVSDoRGHHVFXWDVHQVtYHOVmRDVSHFWRVIXQGDQWHV no processo de formação do terapeuta, na medida em que se ancora no re- conhecimento do outro. Como nos aponta Camargo Borges, (2010), o diálogo torna-se um recurso importante na promoção da dialética dos saberes, e como tal a escuta coletiva permite que os processos interativos se tornem mais dinâmi- cos. A escuta aqui é potencializada como forma de reconhecimento e validação. Anderson e Goolishian (1994) referem-se à essa postura, como um instrumento terapêutico a serviço de uma investigação curiosa que se apoia na singularida- de e na novidade. A escuta curiosa e atenta se apresenta pelo posicionamento Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 47 A Constituição da Equipe Terapêutica – A. S. Gomes YiOLGRGDKLVWyULDSHVVRDODRPHVPRWHPSRHPTXHDSRVWDQRGHVD¿RGRLQYHV- timento em uma postura responsável co-construída. A escuta coletiva estimula e encoraja os alunos a se expressarem, pois representa uma forma de elaboração de seus conhecimentos. Isso equivale à criação de um ambiente de respeito, pertencimento e co-participação. No contexto de formação do terapeuta, a escuta coletiva corresponde à postura de atenção e respeito em relação ao outro, o que sugere uma posição SDXWDGDQDFRQVWUXomRGHVDEHUHVTXHpFRQ¿JXUDGRQDUHODomRHVWDEHOHFLGD por meio dos vínculos. 3UiWLFDVUHÀH[LYDV eLPSRUWDQWHFRQVLGHUDUTXHDRDVVXPLUXPDSRVWXUDUHÀH[LYDHGHSUREOH- PDWL]DomRVLJQL¿FDLUDOpPGDDSUHQGL]DJHPGHWpFQLFDVHGRXVRGHUHFXUVRVWH- UDSrXWLFRV6LJQL¿FDFRPRQRVDSRQWD3DNPDQ  GHVHQYROYHUXPDSRVWXUD política transformadora que toma o diálogo e as práticas conversacionais como elementos/ferramentas de transformação. 1RFXUVRGHIRUPDomRGR&()$,RSURFHVVRGHUHÀH[mRGLDOyJLFDVHDSUH- senta como um recurso para a mudança de ponto de vista do âmbito individual para uma perspectiva social e interacional. Nessa abordagem são privilegiadas DVDWLYLGDGHVHPTXHVHSURS}HXPDSRVWXUDUHÀH[LYDHGHFXULRVLGDGHTXHVXV- cita uma forma colaborativa e dialógica de aprender.2VVLJQL¿FDGRVHPHUJHP do discurso, das conversações e provoca reações emocionais nos participantes. As atitudes dentro do grupo expressam a capacidade de raciocinar e se emocio- QDU7UDEDOKDUQDSHUVSHFWLYDUHÀH[LYDLQFOXLDGRWDUDVIHUUDPHQWDVOLQJXtVWLFDV como elemento mediador facilitador de contextos colaborativos em que a lingua- gem ganha centralidade e se constitui como instrumento de transformação. $VUHVVRQkQFLDV A imersão na orientação de base colaborativa valoriza uma forma diferen- te de escuta trazendo à tona as vozes internas invisíveis tornando-as visíveis. A ressonância é um mecanismo relacionado às conversações internas que o terapeuta tem consigo mesmo, sobre os sentimentos e emoções geradas pelo cliente durante a terapia. Segundo Elkain (1990) as pessoas envolvidas no aten- dimento da família podem experimentar um sentimento que tem uma conexão com os fatos e emoções que emergem na/da terapia. No grupo de terapeutas em formação, as ressonâncias sLJQL¿FDPDSRVVLELOL- dade de conexão com a história do outro, sintonizada com uma atitude orientada Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) 48 A Constituição da Equipe Terapêutica – A. S. Gomes para criar uma abertura para a mudança. Aqui, estamos falando da possibilidade de criação/ recriação que se constitui no espaço da emoção, da interação signi- ¿FDWLYDTXHVHSRWHQFLDOL]DHPXPID]HUWHUDSrXWLFRTXHVHGLVWDQFLDGRXVRGH técnicas refratárias, de intervenções engessadas e de posturas pré-concebidas. Tomando como inspiração a perspectiva de base colaborativa, estar sensí- vel às ressonâncias que aparecem no grupo permite uma atuação menos hierár- TXLFDTXHUHVSHLWDRVVLJQL¿FDGRVFRQVWUXtGRVSRUFDGDSDUWLFLSDQWHHPVLQWRQLD com a sua própria história. Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) &RQVLGHUDo}HV¿QDLV $OJXQVSRQWRVVH¿]HUDPPDUFDQWHVQRSHUFXUVRGDIRUPDomRHTXHIHFXQ- daram um olhar mais aprofundado trazido pelo paradigma relacional. Destaca- mos a forma como o sentimento de equipe vai se constituindo como postura GHFRFRQVWUXomRQDVUHODo}HV6LQWRQL]DGDFRPDVUHÀH[}HVGH-DSXU   segundo o qual o outro não é considerado uma experiência exterior, mas um co -autor na construção de nós mesmos, me reporto, mais uma vez à condição de UHÀH[LYLGDGHGLDOyJLFDTXHVXS}HXPDSRVWXUDFRODERUDWLYDTXHYDORUL]DRSRQWR de vista e o jeito de ser de cada participante. $VSUiWLFDVUHÀH[LYDVRDFROKLPHQWRDHVFXWDVHQVtYHOHDVUHVVRQkQFLDV se destacam como recursos privilegiados no processo de formação e como tal, sobressaem como aspectos constitutivos do sentimento de equipe terapêutica. Foi possível dimensionar o sentimento de equipe por entre os alunos, à medida que a mudança de postura se fez pela escuta sensível, pelas diferentes formas de socialização e pelo comprometimento com o outro. Um ponto considerado importante no processo de formação como terapeuta familiar diz respeito ao autoconhecimento e a ética relacional baseada na compreensão do “outro”. Nesse processo conversacional dialógico são privilegiadas as trocas entre os SDUHVDVQHJRFLDo}HVGHVLJQL¿FDGRVDpWLFDUHVSRQVLYDRGLiORJRUHÀH[LYR e crítico. As práticas desenvolvidas ao longo do curso visam trabalhar as diferenças e evidenciar as singularidades. Essas ações tomam como base o sentimento GHSHUWHQFLPHQWRDRJUXSRHRVHQWLGRGHFRQ¿DQoDRTXHUHTXHUXPH[HUFtFLR de entrega dentro dos princípios de alteridade com base no acolhimento e na escuta. Esses são elementos de base na construção de uma equipe terapêutica É importante sinalizar que no processo de formação, não se trabalha com interpretações, avaliações ou julgamento sobre as condutas. O contexto da for- mação propiciou a aceitação da diversidade marcada pela existência da diferen- ça, a marca da subjetividade de cada um. As práticas desenvolvidas ao longo do curso visam trabalhar as diferenças e evidenciar as singularidades. Essas ações tomam como base o sentimento GHSHUWHQFLPHQWRDRJUXSRHRVHQWLGRGHFRQ¿DQoDRTXHUHTXHUXPH[HUFtFLR de entrega dentro dos princípios de alteridade com base no acolhimento e na escuta. Esses são elementos de base na construção de uma equipe terapêutica É importante sinalizar que no processo de formação, não se trabalha com interpretações, avaliações ou julgamento sobre as condutas. O contexto da for- mação propiciou a aceitação da diversidade marcada pela existência da diferen- ça, a marca da subjetividade de cada um. 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Revista Brasileira de Terapia Familiar, 7(1), agosto, 2018 (38-51) antoniasimone.gomes@gmail.com antoniasimone.gomes@gmail.com Enviado em 01/03/2018 1ª revisão em 08/03/2018 2ª revisão em 25/05/2018 3ª revisão em 19/06/2018 Aceito em 19/06/2018 Enviado em 01/03/2018 1ª revisão em 08/03/2018 2ª revisão em 25/05/2018 3ª revisão em 19/06/2018 Aceito em 19/06/2018 51
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EGR1 Transcription Factor is a Multifaceted Regulator of Matrix Production in Tendons and Other Connective Tissues
International journal of molecular sciences
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To cite this version: Emmanuelle Havis, Delphine Duprez. EGR1 Transcription Factor is a Multifaceted Regulator of Matrix Production in Tendons and Other Connective Tissues. International Journal of Molecular Sciences, 2020, 21, ￿10.3390/ijms21051664￿. ￿hal-03011223￿ Distributed under a Creative Commons Attribution 4.0 International License Emmanuelle Havis and Delphine Duprez * Laboratoire de Biologie du Développement, UMR7622, Institut Biologie Paris Seine, Sorbonne Université,  CNRS, Inserm U1156, F75005 Paris, France  *  Correspondence: delphine.duprez@upmc.fr  Received: 5 February 2020; Accepted: 25 February 2020; Published: 28 February 2020 Laboratoire de Biologie du Développement, UMR7622, Institut Biologie Paris Seine, Sorbonne Université,  CNRS, Inserm U1156, F75005 Paris, France  *  Correspondence: delphine.duprez@upmc.fr  R i d 5 F b 2020 A t d 25 F b 2020 P bli h d 28 F b 2020 Received: 5 February 2020; Accepted: 25 February 2020; Published: 28 February 2020 Received: 5 February 2020; Accepted: 25 February 2020; Published: 28 February 2020 Abstract: Although the transcription factor EGR1 is known as NGF1‐A, TIS8, Krox24, zif/268, and  ZENK, it still has many fewer names than biological functions. A broad range of signals induce Egr1  gene expression via numerous regulatory elements identified in the Egr1 promoter. EGR1 is also the  target of multiple post‐translational modifications, which modulate EGR1 transcriptional activity. Despite the myriad regulators of Egr1 transcription and translation, and the numerous biological  functions  identified  for  EGR1,  the  literature  reveals  a  recurring  theme  of  EGR1  transcriptional  activity in connective tissues, regulating genes related to the extracellular matrix. Egr1 is expressed  in  different  connective  tissues,  such  as  tendon  (a  dense  connective  tissue),  cartilage  and  bone  (supportive connective tissues), and adipose tissue (a loose connective tissue). Egr1 is involved in  the development, homeostasis, and healing processes of these tissues, mainly via the regulation of  extracellular matrix. In addition, Egr1 is often involved in the abnormal production of extracellular  matrix in fibrotic conditions, and Egr1 deletion is seen as a target for therapeutic strategies to fight  fibrotic conditions. This generic EGR1 function in matrix regulation has little‐explored implications  but is potentially important for tendon repair. Keywords: connective tissues; tendon; adipose tissue; extracellular matrix; collagen; EGR1 HAL Id: hal-03011223 https://hal.science/hal-03011223v1 Submitted on 18 Nov 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1. Tendon is a Proper Dense Regular Connective Tissue Connective tissues support and link organs, and are composed of specialized fibroblasts derived  from mesenchymal stem cells. A feature of connective tissues is the presence of extracellular matrix  conferring  specific  biomechanical  properties  and  functions,  reviewed  in  [1]. Connective  tissues  include  proper and supportive  connective tissues, such as cartilage  and  bone. Proper connective  tissues are further divided into two types: dense and soft/loose, reviewed in [2]. The dense connective  tissues  can  also  be  divided  into  two  subtypes:  (1)  the  regular  connective  tissue,  which  refers  to  tendons/ligaments  built  with  regular  collagen  fibers  and  (2)  the  irregular  connective  tissues  embedding organs, composed of irregular collagen fibers, such as skeletal muscle connective tissue,  pericardium, or peritoneum. Adipose tissue is a proper loose connective tissue mainly composed of  adipocytes,  included  in  a  disorganized  network  of  collagen  fibers. Connective  tissues  contain  fibroblasts described as interstitial cells producing extracellular matrix. However, fibroblasts display  remarkable  heterogeneity  of  molecular  signatures  and  phenotypes  across  connective  tissues,  reviewed in [3] and fibroblast populations are still not well characterized. Fibroblast deregulation  leads to fibrosis, assessed by excessive deposition and anarchic organization of extracellular matrix  (ECM). www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 1664; doi:10.3390/ijms21051664 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 1664 2 of 26 Tendon is a proper dense regular connective tissue that links muscle to bone and is involved in  the transmission of forces generated by muscle contraction to bone. Tendon is a key component of  the musculo‐skeletal system, which allows body movement. Although tendon stem cells have been  identified [4], the molecular identity of tendon cells is not well defined and the understanding of  tendon biology lags behind that of other organs. Researchers in the tendon field eagerly await the  analysis of tendon transcriptomic single‐cell data to characterize tendon cell propulations. The main  structural and functional component of the tendon is type I collagen. However, the presence of type  I collagen is not specific to tendon since it is expressed in many other connective tissues such as bone  and adipose tissue. Tendon specificity is given by the spatial organization of type I collagen fibrils  paralel to the tendon axis. A multitude of matrix molecules are involved in collagen fibrillogenesis  leading to the specific spatial organization of type I collagen in tendons, reviewed in [5,6]. 1. Tendon is a Proper Dense Regular Connective Tissue To date,  the bHLH transcription factor Scleraxis (SCX) and the transmembrane protein tenomodulin (TNMD)  are  recognized  as  matrix  regulators  in  tendons  during  development,  homeostasis,  and  repair,  as  reviewed in [7] for SCX and in [8] for TNMD. SCX is recognized to regulate Tnmd expression [9,10]. However,  these  two  genes  are  not  fully  specific  to  tendons,  since  they  are  expressed  in  other  connective  tissues. Scx  is  also  expressed  in  heart  valves  [11],  muscle  connective  tissue  [12],  and  fibroblasts of kidney, testis, and lung [13]. In addition to being expressed in tendon, Tnmd is also  expressed  in  dermis,  brain,  and  adipose  tissue,  reviewed  in  [8]. In  addition  to  SCX,  two  other  transcription factors have been shown to positively regulate the expression of Col1a and Col1a2 genes  and  type  I  fibril  organization  in  tendons:  the  homeobox  Mohawk  (MKX)  and  the  zinc  finger  transcription  factor  Early  growth  response  gene  1  (EGR1). However,  Mkx  and  Egr1  are  also  not  specific to tendon, since they display numerous expression sites and have been shown to be involved  in mutiple biological processes, reviewed in [14,15]. In  this  review,  we  will  focus  on  the  EGR1  transcription  factor  and  its  generic  function  as  a  regulator  of  gene  transcription  of  extracellular  matrix  components  in  tendon,  bone,  and  adipose  tissue (Figure 1), both in physiological and pathological conditions. Figure 1. Schematic representation of two connective tissues: a dense regular connective tissue, the  tendon (A), and a loose connective tissue, the adipose tissue (B). Left panels represent tendon (A) and  adipose tissue (B). Arrows in left panels indicate the section levels in each tissue. Right panels show  the cellular composition on sections of tendon (A) and adipose tissue (B). Figure 1. Schematic representation of two connective tissues: a dense regular connective tissue, the  tendon (A), and a loose connective tissue, the adipose tissue (B). Left panels represent tendon (A) and  adipose tissue (B). Arrows in left panels indicate the section levels in each tissue. Right panels show  the cellular composition on sections of tendon (A) and adipose tissue (B). Int. J. Mol. Sci. 2020, 21, 1664 3 of 26 2.1. Multiple Names for a Single Gene The  Egr1  (Early  growth  response  1)  gene  was  first  identified  as  NGFI‐A  (Nerve  Growth  Factor  Induced‐A) because of its activation by NGF (nerve growth factor) in the neuronal rat cell line PC12  [16]. It was subsequently described in four different laboratories as a rapidly and transiently activated  gene in various fibroblast cell lines. Serum addition or mitogen treatment with the tumor promoter  TPA (tetradecanoyl phorbol acetate) on mouse fibroblasts led to a rapid and strong induction of an  early  growth  response  gene  then  named  Egr1  [17,18]. Egr1  was  also  named  TIS8  (TPA  Inducible  Sequence 8), because of its activation following TPA treatment in a murine 3T3 cell line [19]. After the  identification of a zinc‐finger‐region similar to the Drosophila Krüppel segmentation gene in the Egr1  genomic  sequence,  the  murine  gene  was  called  Krox24,  for  “Krüppel  box  24”  [20]  or  zif/268  in  reference to three tandem zinc finger sequences [21]. Presentation of recorded bird songs to songbirds  such as canaries and zebra finches induces Egr1 expression in their forebrains. In this case, Egr1 was  referred to by the acronym ZENK [22]. The Egr1/EGR1 gene spans about 3.8 Kb and is located on  chromosome 18 in mice and on chromosome 5 in humans. It is composed of two exons and one 700  bp intron. The first exon includes the first 99 amino acids of the protein and the second exon includes  the three tandem zinc finger motifs [23]. The Egr1/EGR1 gene is highly conserved between mouse,  rat, chicken, zebrafish, chimpanzee, dog, cow, and human. 2.2. Numerous Extracellular Signals Regulate Egr1 Expression via Diverse Intracellular Signalling  Pathways 2.2. Numerous Extracellular Signals Regulate Egr1 Expression via Diverse Intracellular Signalling  Pathways The  analyses  of  5’‐upstream  sequences  of  murine  Egr1  and  human  EGR1  genes  reveal  the  conserved presence of many regulatory  elements, indicating that Egr1 expression is regulated in  response  to  multiple  stimuli. The  seemingly  endless  list  of  extracellular  stimuli  inducing  Egr1  transcription  include  hormones,  growth  factors,  cytokines,  UV,  hypoxia,  stress,  and  mechanical  signals (Figure 2). The MAPK (mitogen‐activated protein kinase) signalling pathways has a central  role downstream of these external stimuli to regulate Egr1 expression. Egr1 expression is activated  by a variety of MAPK‐inducing factors, including TNFs (tumor necrosis factors) in primary human  fibroblasts and rat chondrocytes [24,25], different pro‐inflammatory cytokines IL‐1, TNFα, and IL‐17  in osteoblast‐like cells [26], FGF21 in mouse adipose tissue [27], and the antimicrobial peptide LL‐37  in adipose stem cells [28]. These secreted molecules activate the MAPK pathways, which leads to the  recruitment of the SRF (serum response factor) transcription factor together with the ternary complex  factor (TCF)/Ets family member ELK1 to the serum response elements (SRE) of Egr1 promoter [29]. The contribution of individual SRE in Egr1 transcriptional activation depends on cell types [30,31]. Interestingly, TGFβupregulates Egr1 expression through a Smad‐independent pathway in human  fibroblasts, and activates the MEK1/2/ERK signalling cascade converging on ELK‐1 to induce Egr1  expression  [32]. The  MKK7‐JNK‐cJun  MAPK  pathway  can  also  activate  mouse  Egr1  expression  through  the  formation  of  the  AP1  complex  [33]. AP1  complexes  are  heterodimeric  transcription  factors that bind to Tetracecanoyl Phorbol Acetate Response Elements, also called AP1 elements. AP1  complexes  are  composed  of  combination  of  proteins  belonging  to  the  c‐Fos,  c‐Jun,  activating  transcription factor (ATF), and Jun dimerization protein families reviewed in [34]. cJun directly binds  to the AP1 element of the Egr1 promoter but not to the SREs, meaning that cJun directly activates  Egr1 expression via the AP1 site and indirectly via the SRE sites [33]. Int. J. Mol. Sci. 2020, 21, 1664  4 of 26  Figure 2. Schematic representation of the external stimuli that regulate Egr1 transcription. Int. J. Mol. Sci. 2020, 21, 1664 Figure 2. Schematic representation of the external stimuli that regulate Egr1 transcription. Analysis of deletions in the human EGR1 and mouse Egr1 promoters in fibroblasts revealed that  AP1 and EGR1 binding sites played minor roles in its activation [35]. EGR1 binds to its own promoter  in  mouse  fibroblasts  [36]. 2.2. Numerous Extracellular Signals Regulate Egr1 Expression via Diverse Intracellular Signalling  Pathways This  finding  leads  to  the  observation  that  the  ubiquitously  expressed  transcription factor SP1 (specificity protein 1) binds to a consensus recognition sequence that partially  overlaps with the EGR1 binding site (EBS) [36] and blocks EGR1 recruitment to EBS (Figure 2). Serum  stimulation leads to Egr1 overexpression, which binds competitively with SP1 to EBS. This binding  of EGR1 to its own promoter downregulates Egr1 expression [37], presumably through the MAPK  pathways. In  addition  to  MEK/ERK  pathways,  the  cAMP/PKA/CREB  pathway  mediates  Egr1  activation downstream of hormones or cytokines [38–40]. y Egr1 expression is rapidly but transiently induced by insulin in 3T3‐L1 adipose cell line and in  mouse adipose tissue [41]. Insulin secretion induces a cascade of intracellular events in the adipocyte  cell lineage [42] including the PI3K/Akt pathway responsible for glucose uptake and glycogenesis  and  the  ERK1/2  MAPK  pathway,  which  decreases  insulin  signalling  [43,44]. Glucose  stimulation  induces a rise in cytoplasmic Ca2+ which is necessary for AP1, CRE, and SRE‐mediated activation of  Egr1  expression  [45]. A  high  insulin  and  glucose  concentration  represses  the  phosphorylation  of  AMP‐activated  protein  kinase  (AMPK)  in  rat  tenocytes,  which  prevents  its  positive  role  in  Egr1  expression and alters tendon homeostasis [46]. Several stress signals, such as UV, hypoxia, and mechanical shear stress have been shown to  activate  Egr1/EGR1  transcription. UV  irradiation activates  Egr1  expression  in multiple  cell types,  including mouse and human fibroblasts as well as human fibrosarcoma cells [47]. The UV‐B effect on 5 of 26 Int. J. Mol. Sci. 2020, 21, 1664 Egr1 expression is mediated through the activation and recruitment of the NFB family member p65  to a NFB binding site located on the Egr1 promoter [48]. Under hypoxic conditions, the hypoxia‐ inducible factor‐1α (HIF1α) binds to the EGR1 promoter in adipose stem cells isolated from obese  patients, but not not in hASCs from healthy patients [49]. Hypoxia induces EGR1 upregulation in  hASCs of diabetic patients either through the MAPK/ERK pathway or via the direct recruitment of  HIF1α to the EGR1 promoter [49]. Fluid shear stress activation activates EGR1 transcription in human  endothelial cells and epithelial cells [50]. In summary, Egr1 expression is regulated by a myriad of secreted molecules and stress factors  through numerous regulatory elements located upstream of the Egr1 coding sequence. 2.3. EGR1 Protein: Structure and Transcriptional Activity 2.3. EGR1 Protein: Structure and Transcriptional Activity The EGR1 protein contains 533 and 543 amino acids in mice (Figure 3) and humans, respectively,  with a predicted molecular weight of 58 kDa. However, EGR1 detection by Western‐blot analysis  shows  an  apparent  molecular  weight  running  from  80  to  100  kDa,  presumably  due  to  post‐ translational  modifications  [24,36,51]. EGR1  contains  a  highly  conserved  DNA‐binding  domain  composed of three Cys2‐His2 type zinc fingers [52]. These three zinc fingers recognize a consensus  nine‐base‐pair segment of a G/C rich region of DNA (5’‐GCG(C/G/T)GGGCG‐3’), with each zinc  finger  spanning  three  nucleotides  [36,52,53]. EGR1  also  contains  a  bipartite  nuclear  localization  domain, a strong activation domain, a weak activation domain, and an inhibitory domain [54] on  which NAB1 and NAB2 (NGF1‐A binding proteins) bind to repress transactivation by EGR1 [55,56]. According to EGR1 structure, EGR1 displays activator or repressor transcriptional activities [55–57]. Figure 3. Schematic representation of the post‐translational modifications on EGR1 protein. Figure 3. Schematic representation of the post‐translational modifications on EGR1 protein. The phosphorylation of the different EGR1 domains (Figure 3) is controlled by protein kinases  and phosphatases [58]. Phosphorylation can either enhance or block EGR1 transcriptional activity. In  fibrosarcoma, UV exposure leads to EGR1 phosphorylation by PKC (protein kinase C) and tyrosine  kinases [59], conferring on EGR1 a protective and anti‐apoptotic function [60]. In contrast, EGR1  phosphorylation  by  the  protein  kinase  CKII  has  a  negative  effect  on  EGR1  DNA  binding  and  transcriptional activities [61]. EGR1 can be acetylated (Figure 3) by the CBP/p300 complex [62]. In  summary,  EGR1  activates  or  represses  specific  genetic  programs  according  to  its  “phosphorylation/acetylation pattern”. p p y y p EGR1 can be multi‐ubiquitinated and thus degraded by the ubiquitin‐dependant proteasome  pathway [63]. The coordinated sumoylation (Figure 3) and ubiquitination of EGR1 by SUMO‐1 (Small  Ubiquitin  like  MOdifier  1)  and  UBC9  (ubiquitin  conjugating  enzyme  9)  have  been  shown  to  be  involved in EGF‐induced EGR1 expression and stability in the human endothelial cell line ECV304  [64]. Redox (Reduction‐Oxidation) reactions are involved in various vital cellular functions, such as Int. J. Mol. Sci. 2020, 21, 1664 6 of 26 aerobic cellular respiration, nucleic acid synthesis, and also for the production and elimination of  reactive oxygen species (ROS), which includes superoxide, nitric oxide, hydroxyl radical, hydrogen  peroxide, and hypochlorus acid. At high doses, ROS are toxic for the cell and the damage they cause  was  termed  oxidative  stress. 2.3. EGR1 Protein: Structure and Transcriptional Activity Oxidative  stress  induces  bone  loss  by  stimulating  osteoclastic  bone  resorption and inhibiting osteoblastic differentiation [65–68]. However, hydrogen peroxide at non‐ toxic  doses  increases  the  expression  and  DNA‐binding  activity  of  EGR1  in  mouse  and  human  osteoblastic cells without affecting their differentiation [69,70]. The DNA‐binding properties of EGR1  are modulated by the redox state: EGR1 binding to the DNA depends on the presence of reducing  agents, which are necessary for the correct conformation of the EGR1 zinc‐finger region. Oxidized or  metal‐free EGR1 does not bind to DNA [71]. In the human osteoblastic HOBIT cell line, under non‐ toxic ROS doses, a DNA repair enzyme, the APE1 (apurinic/apyrimidic endonuclease 1) increases  EGR1 binding to DNA with nuclear redox activity. EGR1 also upregulates APE1 gene expression,  showing the existence of a positive‐autoregulatory loop between APE1 and EGR1 proteins [70]. g p g y p p In  summary,  EGR1  displays  diverse  transcriptional  activation  or  repression  functions  depending on its post‐translational modification statues. 2.4. Egr1 Expression Profile In Vivo Egr1  is  expressed  in  numerous  organs  and  cell  types  during  development  and  adult  life. However, Egr1 expression is not ubiquitous. In situ hybridization experiments performed on chicken  and mouse embryos indicate a punctiform location of Egr1 transcripts in various tissues such as  tendon,  cartilage,  bone,  skeletal  muscle,  innervation,  vessels,  and  dermis  [72,73]. In  developing  tendons,  Egr1  transcripts  are  not  observed  in  all  tendon  cells,  but  in  subregions,  such  as  the  myotendinous junction and around long tendons in mouse and chicken embryos [73,74]. In the adult,  the mouse ENCODE transcriptome data set indicates that Egr1 is expressed in many if not all adult  tissues, with high expression in cortex, mammary gland, ovary, and thymus [75]. In situ hybridization  and immunohistochemistry experiments performed on adult mouse tissues show Egr1 expression in  Achilles  tendons  [76],  subcutaneous  adipose  tissue  [77],  hypertrophic  cartilage  [78,79],  and  bone  [80,81]. 3. EGR1 Roles in Connective Tissue Formation, Homeostasis and Healing Consistent  with  the  broad  range  of  Egr1/EGR1  expression  sites,  EGR1  is  involved  in  the  formation and homeostasis of many organs. One powerful tool for addressing gene function is the  use of knock‐out mice. The Egr1 gene was inactivated in mice by homologous recombination with  the insertion of the neomycin resistance cassette upstream of the EGR1 DNA‐binding domain [82]  and with the insertion of the LacZ coding sequence within the Egr1 5’ untranslated region added with  a frameshift mutation upstream of the DNA‐binding domain of Egr1 [83]. Both Egr1 mutant mouse  lines were initially described with no overt phenotype during development or postnatal life [82,83];  with the exception of subtil pituitary and ovarian defects observed in the LacZ insertion mutant mice  [83]. However, connective tissue defects were subsequently described in these Egr1 mutant mice,  affecting tendon, cartilage, bone and adipose tissue formation and homeostasis. The analysis of Egr1  loss‐of‐function in mice has identified numerous EGR1 target genes in connective tissues (reported  in  Table  1). A  striking  point  is  that  the  target  genes  positively  regulated  by  EGR1  are  mainly  components of the extracellular matrix (ECM) or linked to ECM regulation, while those negatively  regulated by EGR1 are cartilage, bone, or adipose tissue differentiation markers (Table 1). 7 of 2 ely re n   omp ssue  omp ssue  nega pose  pose egulated by EGR1 in three connective tissues: tendon, bone, and adipose tissue. 3.1.1. EGR1 Function in Tendon Formation, Homeostasis, and Ageing 3.1.1. EGR1 Function in Tendon Formation, Homeostasis, and Ageing Several studies have addressed the role of EGR1 in tendon biology. In developing limb tendons,  Egr1 is expressed close to the myotendinous junction and delineates the long tendons in mouse and  chicken embryos [73,74]. The Egr1 mutant mice do not display a strong overt tendon phenotype;  however, Egr1−/− mice show a significant decrease in the expression of the key tendon markers, Scx,  Tnmd, and Col1a1 in addition to that of tendon‐associated collagens in developing E18.5 limbs [73]  and adult tail tendons and Achilles tendons [76]. Comparison of adult tail tendons in Egr1−/− versus  Egr1+/+ mice shows a reduced number of collagen fibers in mutant mice. Tail and Achilles tendons  have  collagen  fibrils  with  smaller  diameter  and  impaired  biomechanical  properties  in  Egr1−/−  compared to Egr1+/+ mice [76]. Conversely, Egr1 is sufficient to induce de novo expression of a large  variety of tendon genes (including Scx and tendon‐associated collagen genes) in ectopic contexts in  chicken embryos [73]. The Egr1 gene is sufficient to induce ectopic Scx, Col1a1, Col3a1, Col5a1, and  Col14a1 expression in the neural tube, an unrelated embryonic tissue derived form the ectoderm [73]. Consistently with the in vivo situation, EGR1 is sufficient to induce the expression of a large panel of  tendon  genes  including,  Scx  and  Tnmd,  collagen  associated‐tendon  genes  (Col1a1,  Col1a2  Col3a1,  Col5a1, Col6a1, Col14a1), and tendon matrix‐associated molecules (Tnc, Bgn, Dcn, and Fbn1) in mouse  C3H10T1/2 mesenchymal stem cells [76]. EGR1 also promotes the formation of 3D‐engineered tendon  constructs  made  of  C3H10T1/2  cells  by  increasing  the  expression  of  Scx,  Tnmd,  and  Col1a  genes  [76,85]. Moreover, EGR1 induces tenogenic differentiation in rabbit tendon stem cells [97]. EGR1  mediates the promoting effect of the anti‐miR124 on collagen production in human tendon‐derived  stem cells [98] and the promoting effect of ferulic acid on self‐renewal ability of human tendon‐ derived stem cells [99]. Consistent with the positive regulation of Col1a gene transcription by EGR1  observed both in vivo and in vitro, chromatin immunoprecipitation (ChIP) experiments show the  recruitment of EGR1 to the tendon regulatory regions of the Col1a1 promoter in E18.5 limbs [73] and  to Col1a1 and Col1a2 regulatory regions in adult Achilles tendons [76]. 3.1.1. EGR1 Function in Tendon Formation, Homeostasis, and Ageing Lastly, Egr1 downregulation  has been associated with a loss of the tenogenic differentiation potential in ageing human tendon  progenitor cells, while Egr1 gain‐of‐function has the ability to rescue their tendon differentiation  potential as assessed by the upregulation of Scx, Tnmd, Bgn, Dcn, and Col1a1 gene expression [84]. 3. EGR1 Roles in Connective Tissue Formation, Homeostasis and Healing eferences  Expression in Connective Tissues   o  Physiological /Pathological R in Connective Tissues  (I) Extracellular Proteins:    (1) Extra Cellular Matrix (ECM) Components  [25]  Cartilage  Tendon  ECM component of cartilage negative by EGR1  46,76,84]  Tendon   ECM component of tendon Collagen fibrillogenesis  76,77,84–90]  Tendon   Bone  White adipose tissue   Main ECM structural and functional c tendons  ECM component of adipose ti Fibrosis    76,77,85,91]  Tendon   Bone  White adipose tissue   Main ECM structural and functional c tendons  ECM component of adipose ti Fibrosis  [25,92]  Cartilage  Major ECM component of cartilage  regulated by EGR1  3,76,77,86– 88,90]  Tendon   White adipose tissue  ECM component of tendon and adip Collagen fibrillogenesis   Fibrosis  73,76,77]  Tendon   ECM component of tendon and adip 8 of 2 e White adipose tissue  Collagen fibrillogenesis  7]  Tendon   White adipose tissue  ECM component of tendon and adipose tis Collagen fibrillogenesis  6]  Tendon   White adipose tissue  ECM component of tendon and adipose tis Fibrosis  6,77]  Tendon   White adipose tissue  ECM component of tendon and adipose tis Collagen fibrillogenesis   7,84]  Tendon   White adipose tissue  ECM component of tendon and adipose tis Collagen fibrillogenesis  77]  Tendon   White adipose tissue  ECM component of tendon and adipose tis Collagen fibrillogenesis  7]  Tendon   White adipose tissue  ECM component of tendon and adipose tis Matrix organizer  Collagen fibrillogenesis  5]  Cartilage  Tendon  ECM component of cartilage negatively regu by EGR1  7]  Tendon  Bone  White adipose tissue  ECM component of tendon, bone, and adip tissue  6]  Tendon   White adipose tissue   ECM component of tendon and adipose tis Collagen fibrillogenesis  (2) Secreted Proteins/Hormones White adipose tissue  Collagen fibrillogenesis  7]  Tendon   White adipose tissue  ECM component of tendon and adipose tis Collagen fibrillogenesis  6]  Tendon   White adipose tissue  ECM component of tendon and adipose tis Fibrosis  6,77]  Tendon   White adipose tissue  ECM component of tendon and adipose tis Collagen fibrillogenesis   7,84]  Tendon   White adipose tissue  ECM component of tendon and adipose tis Collagen fibrillogenesis  77]  Tendon   White adipose tissue  ECM component of tendon and adipose tis Collagen fibrillogenesis  7]  Tendon   White adipose tissue  ECM component of tendon and adipose tis Matrix organizer  Collagen fibrillogenesis  5]  Cartilage  Tendon  ECM component of cartilage negatively regu by EGR1  7]  Tendon  Bone  White adipose tissue  ECM component of tendon, bone, and adip tissue  6]  Tendon   White adipose tissue   ECM component of tendon and adipose tis Collagen fibrillogenesis  (2) Secreted Proteins/Hormones ose ti ose ti 9 of 2 [93]  Bone  Bone‐derived hormone involved in bon mineralization  Regulation of glucose homeostasis  [80,81]  Bone  Osteoclast precursors    Osteoclast proliferation and differentiati   [77,93]  Bone  Tendon  Adipose tissue  ECM remodelling enzyme involved in bo formation during skeletal repair  [41,77]  White adipose tissue  Hormone secreted by adipocytes involve energy balance regulation  [73,76,85]  Tendon     Tendon development, homeostasis, and re fibrosis    (II) Transmembrane Proteins  [73,76,84,85]  Tendon  White adipose tissue  Main tendon differentiation marker  Transmembrane glycoprotein involved in te formation, homeostasis, and repair  Increased in obese patients  (III) Cytoplasmic Proteins  [94]  White adipose tissue  Enzyme involved in adipose triglyceride lip to mobilize triglyceride for energy produc   [93]  Bone    Metalloprotein enzyme involved in endoch bone formation and repair Bone‐derived hormone involved in bo mineralization  Regulation of glucose homeostasis  Osteoclast proliferation and differentia [93]  [80,81] 0 of 2 ose  sue White and brown adipose tissues  Thermogenic protein expressed in brown ad tissue  Upregulated in the context of white adipose  browning  Transcription Factors/Nuclear Proteins  White adipose tissue    Adipocyte differentiation  Overexpression induces osteopenia  Tendon   Bone (hypertrophic cartilage)  White adipose tissue  Zinc finger transcription factor involved in t formation, homeostasis, and repair  Egr1 deletion induces bone loss  Egr1 deletion induces white fat brownin Fibrotic factor  Adipose tissue  Increases insulin sensitivity and is down‐egu in type 2 diabetic patients  Tendon  Homeobox protein involved in tendon form and homeostasis  Adipose tissue  Chondrocytes  Positive regulator of adipocyte differentia Negative regulator of osteocyte differentia Anti‐fibrotic factor  85]  Tendon   Main tendon marker   bHLH transcription factor involved in ten development, homeostasis, and repair marked with * are directly regulated by EGR1. 3. EGR1 Roles in Connective Tissue Formation, Homeostasis and Healing Int. J. Mol. Sci. 2020, 21, 1664 11 of 26 11 of 26 3.1. EGR1 is a Potent Inducer of Extracellular Matrix Production in Tendons 3.1. EGR1 is a Potent Inducer of Extracellular Matrix Production in Tendons 3.2. Egr1 is a Mechanosensitive Gene in Tendon Due  to  its  function  to  transmit  load  from  muscle  contraction  to  the  skeleton,  tendon  is  a  mecanosensitive  tissue. Mechanical  signals  are  necessary  parameters  involved  in  tendon  development, homeostasis, and healing, reviewed in [108–110]. Mechanical tendon properties have  been identified in developing embryonic tendons [111]. Muscle contractions are required for tendon  development. In the absence of muscles or muscle contractions, head, axial, and limb tendons do not  form; this muscle‐dependency for tendon formation is observed in zebrafish, chicken, and mouse  embryos, reviewed in [14]. TGFβand FGF signalling pathways prevent tendon gene downregulation  in paralyzed developping limbs in chicken embryos [112]. Since EGR1 has been shown to directly  activate Tgfb2 transcription in mouse developping limbs [73], EGR1 is one possible mechanosensor  protein downstream of mechanical forces and upstream of TGFβ signalling in developing limbs. Consistent  with  this  mechanosensor  function,  Egr1  is  activated  by  mechanical  loading  in  3D‐ engineered tendons made of human tendon cells [113] and equine tenocytes derived from induced‐ pluripotent  stem  cells  [114]. Moreover,  EGR1  regulates  tendon  gene  expression  downstream  of  mechanical signals in 3D‐engineered tendons made of mouse mesenchymal cells [85,113]. Forced‐ EGR1 expression prevents the downregulation of the tendon genes, Scx, Tnmd, Col1a1, Col1a2, and  Tgfb2 in 3D‐engineered tendons after tension release [85]. Mechanical signals are also required for  tendon homeostasis. Loss of mechanical loading induces tendon defects is reviewed in [108,115]. Botox (botuliniumtoxin A) injection into the gastrocnemius muscle of hindlimb induces a decrease of  Egr1 transcription in addition to Scx, Col1a2, and Tgfb2 transcription in mouse Achilles tendons [85]. It is also recognized that the lack of mechanical stimulation is deleterious for tendon healing, while  mechanical stimulation improves tendon healing in rats, mice, and humans [108,115]. Egr1 expression  is increased within 15 min in response to loading during the healing process in injured rat tendons  [100],  indicating  that  Egr1  expression  reflects  a  rapid  transcriptional  response  following  loading  changes  in  healing  tendons. The  increase  of  Egr1  expression  in  injured  Achilles  tendons  after  mechanical loading is followed by an increase of tendon strength, indicating a beneficial role for Egr1  in tendon healing [100]. These observations confirm the mechanosensor role for EGR1 during tendon  healing. Conversely,  Egr1  expression  is  decreased  in  a  mouse  Achilles  tendon  injury  model  in  reduced load conditions [85]. The Egr1 downregulation is concomitent with the downregulation of  tendon genes Scx, Tnmd, Col1a1, Col1a2, but also that of Tgfb2 genes [85]. 3.1.2. EGR1 Function in Tendon Healing EGR1 has been shown to be required for the expression of the key tendon markers and tendon‐ related ECM genes during healing after tendon injury [76]. Tendon injury models where the tension  is maintained (partial rupture) induce a massive increase of tendon gene expression [76,100], while  tendon injury models where the tension is lost (total rupture) induce a loss of tendon gene expression  [101,102]. Different models of tendon injury, where tension is maintained, lead to a massive increase  of Egr1 expression after injury, in mouse or rat Achilles tendons [76,100] and rabbit flexor tendons  [103]. In  addition,  needle‐induced  microlesions  in  healing  rat  Achilles  tendons  increase  Egr1  expression [104]. The transcriptional response i.e., the increase of Scx, Tnmd, Col1a1, Col1a2, Col5a1,  Col6a1, Col14a1, Tnc, and Dcn gene expression in response to longitudinal lesion along the tendon  axis is drastically decreased in Egr1−/− Achilles tendons, showing that Egr1 is required for the injury‐ induced expression of tendon‐related ECM genes and key tendon markers [76]. Moreover, EGR1‐ producing cells promote tendon repair in a rat model of Achilles tendon injury [76] and in a rabbit  model of rotator cuff injury [97]. In  summary,  Egr1  appears  to  be  required  for  the  correct  expression  of  matrix  genes  during  tendon formation, homeostasis, and ageing in vitro and in vivo, but also during tendon healing. A  recent discovery is that tendons are peripheral circadian clock tissues, reviewed in [105], in which  collagen synthesis and homeostasis is under a circadian control [106]. Interestingly, the circadian  clock is disturbed in Egr1−/− mice, associated with impaired locomotor activity and body temperature Int. J. Mol. Sci. 2020, 21, 1664 12 of 26 [107],  suggesting  that  Egr1  could  be  involved  in  the  circadian  synthesis  of  tendon‐associated  collagens. 3.4. EGR1 Regulates Extracellular Matrix Production in Adipose Tissue 3.2. Egr1 is a Mechanosensitive Gene in Tendon EGR1 rescue experiments in  reduced load conditions and after tendon injury increase the expression of tendon‐associated genes,  including Scx, Tnmd, Col1a1, Col1a2, and Tgfb2 [85]. These observations suggest that Egr1 is sufficient  to drive the tendon differentiation program in the absence of mechanical signals during the healing  process in vivo. Mechanical  signals  are  the  driving  force  for  tendon  cell  differentiation  in  different  contexts. Although the scheme is not complete, one attempt to hierarchize mechanical and molecular signals  would be that mechanical signals activate molecular signals such as EGR1 transcription factor, which  then in turn activate the expression of tendon genes such as Scx and Tnmd and Col1a genes (Figure  4). It has to be noticed that Tgfb2 transcription is also induced by EGR1 rescue experiments in unload  conditions in a mouse model of Achilles tendon injury and 3D‐engineered tendons [85]. This data,  combined with the direct binding of EGR1 to Tgfb2 promoter regions in adult tendons [76], leads to  the hypothesis that EGR1 activates Tgfb2 dowstream of mechanical signals, which in turn will activate  tendon matrix genes (Figure 4). This is consistent with the recognized role of TGFβ signalling in the  control  of  tendon  adaptation  to  mechanical  loading,  reviewed  in  [116]. The  link  between  EGR1  transcription factor and the YAP key mechanotransduction pathway [110] remains to be established. 13 of 26 13 of 26 Int. J. Mol. Sci. 2020, 21, 1664 Int. J. Mol. Sci. 2020, 21, 1664 Int. J. Mol. Sci. 2020, 21, 1664  13 of 26  Figure 4. Schematic representation of the position of Egr1/EGR1 in the molecular cascade  downstream of mechanical signals involved in tendon gene expression. Figure 4. Schematic representation of the position of Egr1/EGR1 in the molecular cascade  downstream of mechanical signals involved in tendon gene expression. 3.3. EGR1 and Endochondral Bone Formation and Healing Bone is a supportive connective tissue composed of cells, fibers, and a mineralized solid ground  substance  [117]. Egr1  expression  is  detected  in  several  areas  undergoing  endochondral  bone  formation, such as hypertrophic cartilage [78,79] and periostal regions of the developping long bones  [72,73]. Egr1  is  a  negative  regulator  of  cartilage  markers  [92]. Egr1−/−  mice  display  bone  loss  [80,118,119]. The bone loss upon Egr1 deletion is a consequence of an increased bone resorption, via  the  increased  production  of  the  colony  stimulating  factor‐1  CSF‐1/M‐CSF  known  to  positively  regulate  osteoclast  differentiation  [80]. Bone  development  and  homeostasis  is  controlled  by  the  interplay  between  bone‐forming  osteoblasts  and  bone‐resorbing  osteoclasts  [120],  and  this  equilibrium is lost in Egr1−/− mice. EGR1 is a negative regulator of the osteoclastogenic cytokine CSF‐ 1 production by stromal cells. Phosphorylated‐EGR1 (upon estrogen) blocks Csf1 gene transcription  by preventing the binding of the transcriptional activator SP1 to the Csf1 promoter [80]. The failure  of  estrogen  to  rescue  the  CSF‐1  production  and  consequent  osteoclast  formation  in  Egr1−/−  in  ovariectomized mice positiones Egr1 as a pivotal actor to mediate the anti‐osteoclastogenic effect of  estrogen [80,81]. Transcription  factors  that  play  a  role  in  bone  formation  are  expected  to  participate  in  bone  healing after fracture since endochondral bone formation that occurs in embryos is recapitulated  during bone healing. In a bone fracture mouse model, Egr1 deficiency leads to several bone defects  including  persistant  fibrin  accumulation  in  the  fracture  gap,  abnormal  callus  ossification  with  enlarged areas of cartilaginous tissue, decreased expression levels of Bglap (Osteocalcin), and bone  resorbtion  markers  that  regulate  extracellular  matrix,  including  Acp5  (Tartrate‐resistant  acid  phosphatase)  and  Ctsk  (Cathepsin  K)  genes  [93,118]. This  data  confirms  that  EGR1  controls  the  balance between bone tissue formation and resorption during skeletal repair. Int. J. Mol. Sci. 2020, 21, 1664 14 of 26 Adipose tissue is a loose connective tissue mainly composed of specialized white adipocytes,  held in a framework of collagen fibers, which plays a fundamental role in fat storage, metabolic  control, and thermoregulation [121]. Adipocytes are surrounded by an extracellular matrix, which  serves as mechanical support and is mainly composed of collagens, fibronectin, and elastin [122]. Cells producing ECM in adipose tissue are not clearly indentified; however, it is recognized that  collagens are mostly produced by adipocytes, but also by endothelial cells and adipose stem cells in  normal conditions [123]. 4. EGR1 is a Fibrotic Factor Fibroblast  deregulation  leads  to  fibrosis,  a  process  attributed  to  anarchic  deposition  of  extracellular matrix, in response to injury or in pathological conditions. Fibrosis is observed in almost  any tissue in cases of organ dysfunction, but is also a key process in cancer, inflammation, and ageing. Myofibroblasts  are  the  main  cellular  component  of  fibrosis. Myofibroblasts  are  non‐muscle  contractile cells responsible for the excessive synthesis, deposition, and remodelling of ECM proteins  in  fibrosis;  for  a  recent  review  see  [126]. A  recognized  molecular  driver  of  fibrosis  is  the  TGFβ  signalling  pathway. TGFβ  drives  the  conversion  of  fibroblasts  to  myofibroblasts  to  induce  the  excessive deposition of collagen and inappropriate ECM during fibrosis, recently reviewed in [127]. Generic molecular markers for myofibroblasts are similar to markers for smooth muscle cells, such  as Acta2 (smooth muscle actin), whose expression is regulated by TGFβ signalling [127]. Although  myofibroblasts and TGFβ are recognized to be the respective cellular and molecular hallmarks of  fibrosis,  there  is  no  comprehensive  understanding  of  the  cellular  and  molecular  mechanisms  underlying fibrosis. As EGR1 is a potent regulator of matrix components in different contexts, EGR1  is seen as a fibrotic factor. 3.3. EGR1 and Endochondral Bone Formation and Healing The molecular signature of subcutaneous white adipose tissue with Egr1  deletion identifies a downregulation of ECM genes, including Col1a1, Col1a2, Col3a1, Col5a1, Col5a2,  Col14a1,  Fn1,  Dcn,  and  Post  (Periostin)  [77]. Egr1  deletion  is  also  associated  with  a  spontaneous  browning of subcutaneous white adipose tissue in Egr1 mutant mice compared to wild‐types [77]. The  browning  phenotype  corresponds  to  the  appearence  of  beige  adipocytes  within  the  white  adipose tissue mainly by de novo differentiation of progenitors [124]. In contrast to white adipocytes,  beige adipocytes dissipate excess energy through heat production by a large number of mitochondria,  which exhibit uncoupling activity via the thermogenic protein UCP1 (uncoupling protein‐1) [125]. Consistent  with  the  browning  phenotype,  transcriptomic  analysis  in  Egr1‐deleted  adipose  tissue  shows a concomitant downregulation of the white adipocyte marker, Lep (leptin) and upregulation  of the key beige adipocyte marker Ucp1 [77]. Both positive (Lep) and negative (Ucp1) transcriptional  regulations have been shown to occur via direct EGR1 recruitment to regulatory regions of these  genes [41,77]. The browning process reduces the deleterious consequences of fat accumulation and is  seen  as  possible  mechanism  to  fight  against  obesity  and  to  improve  metabolic  health  [125]. Consistently, Egr1−/− mice are protected from high fat diet‐induced obesity via an increase of energy  expenditure [95]. Conversely to Egr1 loss‐of‐function in mice, Egr1 overexpression in C3H10T1/2 cells  increases  the  transcription  of  matrix  genes  and  prevents  the  beige  adipocyte  differentiation  [77]. Overall, Egr1 deletion leads to a drastic loss of ECM genes associated with a browning phenotype in  subcutaneous white adipose tissue in mice. To summarize, Egr1 loss‐of‐function is associated with reduced ECM production in tendon (a  dense connective tissue), bone (a supportive connective tissue), and adipose tissue (a loose connective  tissue). Although the altered matrix genes upon Egr1 deletion appear to be similar in the different  connective tissues, the reduced ECM production is deleterious for tendon and bone formation and  homeostasis, while being beneficial for white adipose tissue to increase energy expenditure. 4.1. EGR1 and Fibrosis in Metabolic Diseases Linked to Adipose Tissue (Obesity and Diabetes) Fibrosis in adipose tissue is considered a hallmark of metabolically dysfunctional adipose tissue  and is associated with obesity and insulin resistance [86,122,128]. In pathological conditions such as  obesity, the rapid expansion of adipose tissue causes hypoxia since neovascularization cannot keep  up with rapid adipose tissue growth. Hypoxia is followed by the necrosis of adipocytes accompanied 15 of 26 Int. J. Mol. Sci. 2020, 21, 1664 with the infiltration of inflammatory leucocytes and macrophages to remove the dead cells. In these  pathological  conditions,  myofibroblasts  associated  with  the  inflammatory  response,  accumulate  within the adipose tissue and cause ECM thickening, characteristic of fibrosis [121,129]. Adipose  tissue fibrosis results from the imbalance between excess synthesis and impaired degradation of type  I, III, and VI collagens [130]. Consistently, type I, III, and VI collagens are particularly abundant in  adipose tissue of obese patients [131] and collagen content affects tensile strengh of adipose tissue  [132]. Moreover, Col6a1 loss‐of‐function significantly reduces adipose tissue fibrosis in obese mice  [122]. Egr1/EGR1 expression is increased in adipose tissue of obese Lep/Lep mice and patients [95],  and of diabetic db/db mice and type 2 diabetes mellitus (T2DM) patients [133]. T2DM is associated  with insulin resistance and the majority of T2DM patients are overweight or obese [129]. Egr1 gain‐ of‐function in epididymal fat induces insulin resistance [133], while Egr1 loss‐of‐function improves  the whole‐body insulin sensitivity in diabetic mice [133]. One possible mechanism would be that  insulin‐induced EGR1 [41,133] directly inhibits Pnpla2 (ATGL, Adipose TriGlyceride Lipase) expression  in adipocytes that leads to lipolysis inhibition and promote fat accumulation [94]. p y p y p Egr1  overexpression  is  associated  with  metabolically  dysfunctional  adipose  tissues. As  Egr1  regulates matrix production in subcutaneous white adipose tissue and tendon, Egr1 is a credible  regulator  of  inappropriate  ECM  production  in  metabolically  dysfunctional  adipose  tissues. Consistent with this idea, Egr1 transcription is directly activated by insulin and F1α [49] via direct  recruitment to Egr1 promoter. In addition, the insulin and F1α expression is increased in obese and  T2DM mice and HIF1α regulates the expression of ECM genes such as Col1a1, Col3a1, and Col14a1  [86,87]. Egr1 is thus a good therapeutic target to counteract obesity and associated fibrosis since its  loss‐of‐function reduces ECM production and stimulates the white fat browning process. Interestingly, the tendon differentiation gene, Tmnd, involved in ECM regulation in tendons [8],  is also involved in adipose tissue function. 4.1. EGR1 and Fibrosis in Metabolic Diseases Linked to Adipose Tissue (Obesity and Diabetes) TNMD mRNA expression levels are strongly correlated  with the body mass index. TNMD gene expression is significantly higher in obese subjects compared  to lean subjects [134–136], while TNMD gene expression is downregulated in visceral adipose tissue  during diet‐induced weight loss [134]. Although EGR1 and TNMD genes share the same expression  profile in white adipose tissues of obese patients, in contrast to EGR1, TNMD acts as a protective  factor in visceral adipose tissue to alleviate insulin resistance in obesity [136]. 4.2. Tendon Defects in Type 2 Diabetes Mellitus Type II diabetes mellitus (T2DM) is associated with high risk of tendinopathy or tendon tears,  reviewed recently in [137,138]. Various T2DM rodent models, which cannot be dissociated from obese  mouse models, display tendons with decreased collagen content, ECM disorganization, and impaired  mechanical properties, reviewed in [138]. T2DM also impairs the healing process following tendon  injury  and  amplifies  the  fibrotic  process  during  healing,  leading  to  scarred  tendons. There  is  an  increase  of  Col1a1  and  Col3a1  expression  in  FDL  tendons  after  injury  associated  with  decreased  mechanical  properties  in  diet‐induced  obesity  mice  [88]. High  glucose  also  affects  tendon  gene  expression and cell behavior in tendon cell cultures, which induces changes in extracellular matrix  [139]. Interestingly, Egr1 expression was also modified (associated with Mkx, Tgfb1, Col1a2, and Bgn  expression alteration) in rat tendon cells cultured in high glucose for 14 days [46]. 4.4. EGR1 is at the Crossroad of the Molecular Pathways Involved in the Fibrotic Process in Animal Models  for Organ Fibrosis Classical animal models for fibrosis target idiopathic pulmonary fibrosis, renal fibrosis, liver  fibrosis, and heart fibrosis. EGR1 is frequently mentioned as being associated with the progression of  fibrotic process in animal models and in the transcription of fibrotic genes in cellular models. Egr1  deletion is often described as being beneficial to fight fibrosis progression in animal models of organ  fibrosis. Lung fibrosis. EGR1 is involved in lung fibrosis downstream of IGFBP‐5 (insulin‐like growth  factor (IGF) binding protein‐5) to promote fibrotic gene transcription [145] and downstream of TGFβ1  to activate the transcription of the hyaluronan receptor CD44V6 (CD44 containing variable exon 6  (v6)) expression in lung fibroblasts in the context of idiopathic pulmonary fibrosis [146]. Renal fibrosis. In a mouse model of adenine‐enriched diet induced tubulointerstitial nephritis  leading to renal fibrosis, Egr1 is increased in kidney. Egr1−/− mice display reduced TGFbeta activity  and  reduced  renal  fibrotic  zones  and  were  protected  from  renal  failure  [147]. The  miR181  was  identified as an inhibitor of renal fibrosis via Egr1 inhibition, which suppressed the expression levels  of alphaSMA (ACTA2), connective tissue growth factor (CTGF), collagen type I (COL1A1), and type  III collagen (COL3A1) in NRK49F cells [90]. Liver fibrosis. Egr1 has been shown to contribute to liver fibrosis progression downstream of Elk‐ 3 in CCl₄‐induced mouse liver fibrotic tissues and human liver cirrhotic tissues [148]. However, the  beneficial  effect  of  Egr1  deletion  in  the  context  of  liver  fibrosis  is  contradictory. In  an  acute  acetaminophen‐induced  liver  injury  mouse  model,  the  inhibition  on  ERK1/2‐mediated  Egr1  transcriptional  activity  attenuates  hepatotoxicity,  suggesting  that  inhibiting  Egr1  is  beneficial  to  protect against liver fibrosis observed in long‐term application of acetaminophen [149]. By contrast,  another  study  shows  that  livers  of  Egr1−/−  mutant  mice  exhibit  a  more  severe  fibrotic  response  compared  to  those  of  wild‐type  mice  under  acetaminophen  overdose  [150]. The  Egr1  function  remains elusive in liver fibrosis. Heart fibrosis. EGR1 has been shown to be involved in cardiovascular homeostasis and diseases. Notably, Egr1 transcription is activated in hypoxic and ischemic conditions in heart and in calcified  heart valves [151,152]. The miR‐150‐5p retards the progression of myocardial fibrosis by targeting  EGR1 [153]. As EGR1 is systematically mentioned as being involved in fibrosis progession in organ fibrosis  animal  models,  Egr1  is  seen  as  a  putative  target  to  fight  fibrosis. 4.4. EGR1 is at the Crossroad of the Molecular Pathways Involved in the Fibrotic Process in Animal Models  for Organ Fibrosis A  recent  antifibrotic  chemical  component has been identified with the PPARγ agonist (pioglitazone) that inhibits TGF‐β‐driven  fibrosis in animal models for pulmonary, renal, and cardiac fibrosis, reviewed in [154]. Interestingly,  pioglitazone has been shown to repress Egr1 transcription and traduction in kidneys of TGF‐β‐driven  renal fibrosis in mice [155] and in pancreas of a cerulein‐induced acute pancreatitis mouse model  [156]. 4.3. EGR1 Controls Fibrosis in Systemic Sclerosis A classical fibrotic disease  is  systemic sclerosis, also  known as  scleroderma,  which is a rare  disease characterized by excessive collagen deposition resulting in fibrosis in different organs such  as skin and lungs but also the diggestive track (esophagus, stomach, and intestine) and myocard  [140,141]. Systemic sclerosis pathology also displays inflammation and vasculopathy components. Myofibroblasts  are  key  cells  of  the  physiopathology  of  systemic  sclerosis  [140]. TGFβ  and  Wnt  signalling  are  recognized  to  play  a  fundamental  role  in  the  pathogenesis  of  fibrosis  in  systemic  sclerosis, in particular in the differentiation process of activated myofibroblasts [141]. EGR1 transcription factor has been shown to be at the crossroad of the molecular processes  leading to the TGFβ‐dependent fibrosic process in systemic sclerosis, reviewed in [142,143]. EGR1 Int. J. Mol. Sci. 2020, 21, 1664 16 of 26 16 of 26 expression is increased in biopsies of fibrotic skin and lung from patients with systemic sclerosis [32]. In the mouse model of bleomycin‐induced scleroderma, fibrosis is reduced in skin and lung of Egr1−/−  mice,  with  decreased  Col1a1  expression  and  αSMA+  fibroblasts  in  both  tissues  [89]. In  human  fibroblasts, EGR1 upregulates COL1A2 transcription downstream of TGFβ [91]. The high throughput  analysis of EGR1‐responsive genes in human primary fibroblasts identifies over 600 genes involved  in extracellular matrix synthesis, wound healing, and TGFbeta signalling, but also in cell proliferation  and vascular development [144]. This EGR1‐responsive genes signature is enriched in skin biopsies  from  patients  with  systemic  sclerosis  compared  to  healthy  controls  [144]. The  demonstrated  involvement of EGR1 in this fibrotic disease identifies EGR1 as a pertinent target to control fibrosis  in systemic sclerosis [142,143]. 4.4. EGR1 is at the Crossroad of the Molecular Pathways Involved in the Fibrotic Process in Animal Models  for Organ Fibrosis 4.6. EGR1 and Scarred Tendon Abnormal tendon healing is frequent following tendon injury reviewed in [166,167]. Following  accute rupture, tendons undergo a healing process involving the sequential and overlapping phases  of inflamation, cell migration, cell proliferation, ECM production, and remodelling. These successive  phases ultimately result in the production and spatial organization of type I collagen. However, the  healing process is often incomplete in tendons, which leads to scar tendons that do not regain the  mechanical properties of native tendons. The cellular basis of tendon fibrosis is not well understood  and  involves  the  contribution  of  intrinsic  (tendon  sheeths)  and  extrinsic  (circulating  cells)  cell  populations, recently rewiewed by [168]. The molecular basis underlying tendon fibrosis involves the  main fibrotic signalling pathway, TGFβ [102], the transmenbrane protein TNMD [169], and the SCX  transcription factor [170], which are also the main actors involved in tendon development [9,171,172]. Interestingly, SCX directly regulates the transcription of the Acta2 gene (a fibrotic marker) in cardiac  fibrosis [173]. Although Egr1 is required for the correct transcriptional response in healing tendons  [76], EGR1 function in the fibrotic response in tendon has not been established. However, given the  EGR1 involvement in the fibrotic response in organs, EGR1 is very likely to be involved in tendon  scarring. 4.5. EGR1 and Matrix Production in Rheumatoid Arthritis and Osteoarthritis Consistent with Egr1 expression in cartilage and bone, the Egr1 gene is reiteratively cited to be  involved in chronic diseases that lead to articular cartilage degeneration, such as osteoarthritis and Int. J. Mol. Sci. 2020, 21, 1664 17 of 26 rheumatoid arthritis [157]. The molecular cascade underlying the pathogenesis of these two joint  diseases are not well understood. Osteoarthritis leads to cartilage degeneration, reviewed in [158],  while rheumatoid arthritis is an autoimmune and inflamatory disease associated with an increase of  synovial fibroblasts leading to joint degeneration [159]. TNFα levels are increased in the synovial  fluid of patients with osteoarthritis and rheumatoid arthritis [25] and reduce the expression of Col2a1,  Acan (Aggrecan), and Hapln1 through EGR1 recruitment to their promoters [25]. Classical and global  transcriptomic  analysis  identified  high  EGR1  expression  in  articular  cartilage  of  patients  with  osteoarthritis  [96,160,161]  and  in  synovial  tissues  of  rheumatoid  arthritis  patients  [162–164]. Chondrocytes  stimulation  with  interleukin‐1β  (IL‐1)  leads  to  the  recruitment  of  EGR1  to  Pparg  promoter  and  downregulates  its  expression,  preventing  the  protective  effect  of  PPAR  in  osteoarthritis [96]. Ectopic expression of EGR1 in articular cartilage aggravated the degradation of  the cartilage matrix in mice [78]. The excess of EGR1 induced an increase of transcripts and protein  of type I collagen in synovial fibroblasts from rheumatoid arthritis patients [165]. Egr1 represents a  potential target for drug intervention in osteoarthritis or rheumatoid arthritis. References 1. Santos, A.; Lagares, D. Matrix Stiffness: The Conductor of Organ Fibrosis. Curr. Rheumatol. Rep. 2018, 20, 2. 2. Nassari, S.; Duprez, D.; Fournier‐Thibault, C. Non‐myogenic Contribution to Muscle Development and  Homeostasis: The Role of Connective Tissues. Front. Cell Dev. Biol. 2017, 5, 1–17. 2. Nassari, S.; Duprez, D.; Fournier‐Thibault, C. Non‐myogenic Contribution to Muscle Development and  Homeostasis: The Role of Connective Tissues. Front. Cell Dev. Biol. 2017, 5, 1–17. 2. Nassari, S.; Duprez, D.; Fournier‐Thibault, C. Non‐myogenic Contribution to Muscle Development and  Homeostasis: The Role of Connective Tissues. Front. Cell Dev. Biol. 2017, 5, 1–17. p y g Homeostasis: The Role of Connective Tissues. Front. Cell Dev. Biol. 2017, 5, 1–17. 3. Hannan, R.T.; Peirce, S.M.; Barker, T.H. Fibroblasts: Diverse Cells Critical to Biomaterials Integration. ACS  Biomater. 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Rep. 2018, 8, 3155. 11. 5. Concluding Remarks In addition to being involved in matrix production in normal conditions and fibrotic processes,  EGR1 transcription factor has been associated with numerous cancers and has been shown to act as  a tumor suppressor or a tumor promoter depending on cancer types, for reviews see [174,175]. The  reason  for  this  paradoxal/antagonistic  EGR1  function  depending  on  cancer  types  is  not  clear. Interestingly, EGR1 expression is correlated with prostate cancer progression and promotes prostate  cancer metastases [176], which are associated with a massive increase of matrix [177]. Egr1 has been  already targeted to prevent the progression of prostate cancer carcinoma [178]. Interestingly, there is  no associated cancer in tendons. Giant cell tumor of the tendon sheath (GCTTS) very rarely impacts  tendon proper [179]. One attractive hypothesis is that the tendon matrix environement regulated by  EGR1 is protective against cancer. In summary, the EGR1 transcription factor is a key checkpoint in the transcriptional response to  external stimuli. Despite multiple regulatory elements in the Egr1 promoter, Egr1 has been repeatedly  associated with matrix production in connective tissues in homeostatic and pathological conditions. Egr1  deletion  is  a  good  therapeutic  option  for  reducing  fibrosis  in  many  tissues. One  attractive  hypothesis is that EGR1 has a generic function in the transcription of matrix genes. Based on a recent  report of EGR1 function in the brain, in the epigenetic control of the methylome during development  and upon neuronal activity [180], EGR1 could act on the methylome of matrix genes in tendons and  other connective tissues. Int. J. Mol. Sci. 2020, 21, 1664 18 of 26 Funding: This work was funded by Centre National de la Recherche Scientifique and Sorbonne Université. Funding: This work was funded by Centre National de la Recherche Scientifique and Sorbonne Université. 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Evolving Marine Biomimetics for Regenerative Dentistry
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David W. Green 1, Wing-Fu Lai 2 and Han-Sung Jung 1,2,* 1 Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China; E-Mail: dwgreen@hku.hk 1 Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China; E-Mail: dwgreen@hku.hk 2 Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul 120-752, Korea; E-Mail: rori0610@graduate.hku.hk 2 Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul 120-752 Korea; E-Mail: rori0610@graduate.hku.hk * Author to whom all correspondence should be addressed; E-Mail: hsjung@hku.hk; Tel.: +852-2859-0480. * Author to whom all correspondence should be addressed; E-Mail: hsjung@hku.hk; Tel.: +852-2859-0480. * Author to whom all correspondence should be addressed; E-Mail: hsjung@hku.hk; Tel.: +852-2859-0480. Received: 13 March 2014; in revised form: 14 April 2014 / Accepted: 16 April 2014 / Published: 13 May 2014 Received: 13 March 2014; in revised form: 14 April 2014 / Accepted: 16 April 2014 / Published: 13 May 2014 Abstract: New products that help make human tissue and organ regeneration more effective are in high demand and include materials, structures and substrates that drive cell-to-tissue transformations, orchestrate anatomical assembly and tissue integration with biology. Marine organisms are exemplary bioresources that have extensive possibilities in supporting and facilitating development of human tissue substitutes. Such organisms represent a deep and diverse reserve of materials, substrates and structures that can facilitate tissue reconstruction within lab-based cultures. The reason is that they possess sophisticated structures, architectures and biomaterial designs that are still difficult to replicate using synthetic processes, so far. These products offer tantalizing pre-made options that are versatile, adaptable and have many functions for current tissue engineers seeking fresh solutions to the deficiencies in existing dental biomaterials, which lack the intrinsic elements of biofunctioning, structural and mechanical design to regenerate anatomically correct dental tissues both in the culture dish and in vivo. Keywords: regenerative dentistry; biomimetics; marine invertebrates; bone; dentine Keywords: regenerative dentistry; biomimetics; marine invertebrates; bone; dentine Mar. Drugs 2014, 12, 2877-2912; doi:10.3390/md12052877 marine drugs ISSN 1660-3397 www.mdpi.com/journal/marinedrugs Review Evolving Marine Biomimetics for Regenerative Dentistry David W. Green 1, Wing-Fu Lai 2 and Han-Sung Jung 1,2,* 1 Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China; E-Mail: dwgreen@hku.hk 2 Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul 120-752, Korea; E-Mail: rori0610@graduate.hku.hk * Author to whom all correspondence should be addressed; E-Mail: hsjung@hku.hk; Tel.: +852-2859-0480. Received: 13 March 2014; in revised form: 14 April 2014 / Accepted: 16 April 2014 / Published: 13 May 2014 Abstract: New products that help make human tissue and organ regeneration more effective are in high demand and include materials, structures and substrates that drive cell-to-tissue transformations, orchestrate anatomical assembly and tissue integration with biology. Marine organisms are exemplary bioresources that have extensive possibilities in supporting and facilitating development of human tissue substitutes. Such organisms represent a deep and diverse reserve of materials, substrates and structures that can facilitate tissue reconstruction within lab-based cultures. The reason is that they possess sophisticated structures, architectures and biomaterial designs that are still difficult to replicate using synthetic processes, so far. These products offer tantalizing pre-made options that are versatile, adaptable and have many functions for current tissue engineers seeking fresh solutions to the deficiencies in existing dental biomaterials, which lack the OPEN ACCESS Mar. Drugs 2014, 12, 2877-2912; doi:10.3390/md12052877 Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 been designed and fabricated for roles in tissue replacement, biomonitoring and therapeutic delivery of drugs [1]. The more that is understood about the molecular interplay, gene, cellular, biochemical and metabolic regulatory networks in health, disease, inflammation and trauma so the design of biomaterials to match and participate in recovery, regeneration and disease cures has increased in biological complexity. Now there are more sophisticated biomaterials, built from miniatures that are enriched with bioactive elements such as extracellular matrix proteins [2]. There remain startling imperfections in all biomaterials particularly regarding their responsiveness to biology, mechanical design and functional match with normal healthy tissues. Structures made with biomaterials miss important features of design such as, exquisite interfaces, proper integration of different materials and substrates biomimetic surfaces [3–5]. This is really crucial in the dental field, in which individual tissue units are made up of multiple complexes of tissue types. Take for example the periodontium composed as it is of sandwiched cementum, periodontal ligament tissue and bone. Almost every type of biomaterial has been used in clinical or experimental dentistry. Metals and ceramics have been predominant in fixing and replacing the major hard tissues of the tooth. Polymers have also played a major part in dental restorations and replacements [6]. In addition, new roles are being designed for these materials. For example, the versatility that polymers offer has engendered uses in therapeutic delivery such as nucleic acids-SiRNA [7,8] and microRNA’s [9]. New categories of biomaterials are coming to the fore: self-assembled, nanofibrous, nanoparticulate that will eventually provide natural levels of biomimetic sophistication. However, they have had limited impact in dentistry for now due to lower than needed production volumes and technically difficult syntheses. Marine biomimetic structures offer useful advantages before the future onset of truly biomimetic artificial designs. The main advantage is their sophisticated pre-design of structure and architecture. 1. Introduction Biomaterials are widespread in clinical and experimental medicine. Synthetic and biological origins made from polymers or ion complexes, nanoparticles, quantum dots and intricate composites have 2878 1.1. Striving for de Novo Regeneration of Whole Living Tooth Tissues In dentistry, structural biomaterials that enable interactions between epithelial and mesenchymal stem cells have been used to facilitate tooth regeneration [10]. These biomaterials are typically artificial. In reconstructive maxillofacial and periodontal surgery, autologous tissues and autologous blood plasma proteins are introduced into surgery as well to potentiate regeneration and promote rapid healing and sealing [11–13]. For example, calcium phosphate bone substitutes and collagen derivatives are routinely used as structural frameworks that induce tissue rebuilding inside alveolar bone voids and replace missing gum tissue, respectively. These interventions can usually guarantee a clinically acceptable level of anchorage and sealant for metal teeth and implants [14]. The addition of bone morphogenic proteins (BMP’s) and Fibroblast growth factor (FGF), dental matrix proteins, induced Pluripotent Stem (iPS) cells, Periodontal ligament stem cells (PDLSC) and cell sheets into the diseased periodontal ligament have led to accurate regeneration. Recently, the technical feasibility of using scaffolds to facilitate the regeneration of dental structure has been further evidenced by a study that transplanted stem cells with macroporous biphasic calcium phosphate (MBCP) into the dorsal subcutaneous pockets of 5-week-old male immunocompromised mice [15]. The stem cells included cells isolated from the periodontal ligament (PDL) of permanent teeth (pPDLSCs), stem cells from the PDL of deciduous teeth (dPDLSCs), and human bone marrow-derived mesenchymal stem cells (BMMSCs). All three types of stem cells could successfully 2879 Mar. Drugs 2014, 12 produce hard tissues at the periphery of the MBCP eight weeks after the transplantation of the scaffolds [15]. The BMMSC transplants produced bone-like tissues that had a lining of osteoblast-like cells, a lamellar pattern of fiber alignment, and an outer dense fibrous tissue parallel to those osteoblast-like cells. Furthermore, embedded cells were identified to be present in the matrix, which exhibited condensed nuclei-like osteocytes and reduced cytoplasmic space in the lacunae [15]. Additionally, cementum-like tissues were formed in the pPDLSC and dPDLSC transplants [15]. Cementocyte-like cells resembling cellular cementum were also observed in some of the tissues formed. By quantitative RT-PCR and immunohistochemical analyses, higher expression levels of genes related to mineralization (BSP, osteocalcin, and osteopontin) were observed in the BMMSC transplants, whereas in the pPDLSC and dPDLSC transplants, higher expression of genes related to the cementum/PDL complex (CP23 and collagen XII) was observed [15]. These findings act as a proof-of-concept to illustrate that tooth tissue engineering in scaffolds is practically applicable. 1.1. Striving for de Novo Regeneration of Whole Living Tooth Tissues Despite this premise, autologous patient living tissue is ultimately what works best to fasten the biomaterial scaffolds securely for tooth regeneration but is generally in short supply. As a consequence, there must be a viable clinically acceptable alternative. Thus, high-quality dental tissues are being grown in tightly manufactured conditions that can be precisely controlled to match the extent of damage needing replacement. Vital living dental tissue can be grown to a limited extent in petri dishes from donor cells and patient cells extricated from the apical papilla (SCAP), exfoliated deciduous teeth (SHED), periodontal ligament (PDLSC), pulp tissue (DPSC), and dental follicle (DFSC). A more unresolved problem has been in acquiring stem cells for recreating a dental epithelium [16]. Stem cells can be obtained from even non-dental cells and be made to grow in the position of the damaged tooth; odontogenesis in non-dental cells has been induced when exogenous BMP4 was given to intact cultures of second pharyngeal arches [17]. Though no hard tissues (such as enamel and dentine) or identifiable teeth have been obtained, organized cell layers were successfully formed in treated cells derived from the second arch cultures [17]. Further, these cells, which had been treated with BMP4 differentially, expressed Dspp and amelogenin in adjacent layers in a relative orientation similar to that in teeth [17]. These observations confirm the possibility of inducing formation of a complete tooth primordium from non-dental cells by directly manipulating intracellular levels of signaling molecules. Apart from that, tooth primordia have also been successfully procured by tissue recombination. Earlier studies demonstrated that tooth formation can be induced in aboral first-arch mesenchyme and second-arch mesenchyme by applying early oral epithelium (E9-E11) [18,19], which has also been reported to induce tooth formation in the cranial neural crest and trunk neural crest if recombined prior to migration [20]. In a more recent study, the oral epithelium of murine embryos between E9 and E11.5 was also shown to induce tooth formation in non-dental mesenchyme [21]. This accumulated evidence supports the technical possibility of regenerating tooth primordia or bioteeth for subsequent transplantation in an ex vivo environment. Unfortunately, replacing missing dental tissue parts with a cultivated equivalent is preferable to using man-made materials and structures engineered for the same task. It has been impossible to completely emulate the evolutionary perfected design of natural biomaterials synthetically. Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 2880 3 separate types of restoration up to a total of 926 median staying power of the restoration varied from 7.8 years for resins to 14.6 years for crowns [23]. Entire living tissues can be produced in the petri dish with a combination of cellular building blocks, cell and tissue supporting frameworks, and cell activating genes and protein factors in the typical tissue engineering strategy [24,25]. The archetypal petri dish has evolved and metamorphosed into quite sophisticated bioreactors, devices with microcapillaries and micron scale chips [26–28]. A variety of important tissues have been cultivated with a clinically acceptable degree of anatomical accuracy this way such as recently: heart tissue components, optic cup, brain cortical tissue [29–32]. In most other cases though the quantity of tissues generated exclusively in the lab can be too small-as it is restricted to 2 mm thickness for most clinical treatment to be successful [33]. This is because for technical reasons a blood vessel supply is difficult to establish in developing engineered tissues. It has been stated that almost every cell residing in the body is 200 μm from the nearest capillary in order to survive [34]. Laying down a nerve network derived from precursor cells and a conduit, inside petri-dish grown tissues is an equally vital mission to provide full and vital sensory and actuation roles to that tissue. So far synthesis of a nerve and blood vessel network-individually or in combination with the construction of any given tissue has not been achieved. Indeed, there is paucity of clinically acceptable and viable materials and structures in dentistry (to augment and replaced damaged oral and craniofacial structures) that can support and replicate tissue to a sufficient quality of function and offer credible alternatives to conventional techniques and strategies [35]. Dental tissues by nature are stacked together with each other across graded interfaces, so that to regenerate one tissue requires engineering of complete other tissues with which they are set in contact with [35]. One interesting origin for discovering usable items with the utility and versatility necessary to complete tissue generation are the products of evolution from simple animals and plants. Previously, we have demonstrated how this has yielded drug delivery devices, cell sheet templates and frameworks for cartilage regeneration [36–39]. Mar. Drugs 2014, 12 In this article, we look at the possibilities of harnessing structures, substrates and materials from the world of marine biology for use in the tissue engineering of dental tissues, jawbone, dentine and pulp. 1.1. Striving for de Novo Regeneration of Whole Living Tooth Tissues Conventional dental materials are therefore inclined to fail because the structural and biological properties (if they do exist) are not identically matched to their host surroundings of dentine, enamel or periodontal tissue [22]. In one large study treating 426 adults with Mar. Drugs 2014, 12 1.2. Frameworks for Tissue Engineering Marine biomaterials are being renewed as candidates for quite broad biomedical applications. Capacities to extract sufficient marine product yields are increasing too and there is repeated evidence of effects on human cells and biology. Promising examples include collagens from jellyfish [40], polymers from marine Diatoms [41], Chitin from marine sponges [42] and hydroxyapatite and calcium phosphates from fish bone and other organisms [43]. Advantages of marine sourced biomaterials vary from inspiration to engineer materials with new properties and effects, vital biological activities and high bioavailability (Table 1). 2881 Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 Table 1. Comparison between marine biostructures and other biomaterials in regenerative medicine and including dentistry. Marine Biostructures Other Biomaterials and Their Structures Pre-designed and pre-fabricated with numerous possible roles in cell and tissue regeneration. Intrinsic functions can be translated to human biology. Fabricated by designer with many honed functions specific to biomimetic induction, control and regulation of developmental and regeneration processes. Inherent hierarchical structural design. Limited levels of hierarchy can be assembled, so far. High diversity of structure and architecture. Some species highly abundant and sustainably sourced. Structural and architectural specificity is diverse but difficult to generate artificially requiring complicated processing. Intrinsic design features retained for fracture prevention and biorecognition (physical, biochemical, sometimes cellular). Difficulty in generating composites with comprehensive fracture toughening. Short processing times with low cost. Although obtaining marine biostructures from natural habitats can be expensive. Long manufacturing process with many steps at high cost. Some difficulties in sterility and removing all contaminants. High levels of sterility obtained. Safe storage indefinitely usually without special conditions. Variable storage times with possible degradation. Possibility of low-level immunogenicity. Synthetics have no immunogenicity. Some natural biopolymers risk immunogenicity. Low cytotoxicity in properly cleaned biostructures. Low cytotoxicity. Less utility to integrate bioactive elements. Large range of bioactive elements (adhesive ligands, enzyme-sensitive cross-links) can be integrated biomimetically during synthesis. Less accessible in adding new biomimetic properties. Highly accessible design and construction with many biomimetic properties: self actuation, self-adjustment, etc. 2. Biomedical Applications of Marine Products Bioactive compounds from marine organisms have been applied in medicine and steadily in areas of regenerative medicine. Certainly secondary metabolites from marine invertebrates and commensal microbes have been harnessed as potential drug candidates for destroying tumors, killing bacteria stopping inflammation and suppressing pain [44–47]. They are chemical rich and highly diverse although not necessarily highly abundant. However, advances in molecular biology allow prospecting for genes involved in producing bio medically significant chemical compounds, effective synthesis in bacterial vectors or synthesis from the bio molecular structure itself as well as screening methods more accommodating to natural chemical compounds [48]. Finding new niches and habitats for products is ongoing and can be fruitful pursuit for new and abundant natural marine products [44]. Structures, materials and substrates extracted from the plant and animal kingdoms are products that can be implemented in most regions of the tooth in regenerative dental therapies [49–55]. Plant extracts are likely to provide valuable material substitutes made from polysaccharides, phytochemicals 2882 Mar. Drugs 2014, 12 and proteins with superior cell and tissue interactivities than their synthetic counterparts [52]. However, their usage in regeneration therapies has not been fully exploited in practice because of the bias towards well-established restorative materials such as titanium [56], metal alloys [57], zirconia [58], bioactive glasses [59], calcium phosphates [60,61] and glass ionomer resins [62,63]. Many of these are now being gradually being adapted at the Nano metric scale to improve many functions and even superseded by a slowly increasing number of Nano biomaterials and biomimetic and bio inspired materials and structures but these have been limited to fabrication of coatings, new fillers, anti-bacterial factors and for tooth remineralisation strategies [63–67]. We are looking at exploiting those products that have previously evolved in early and simple life-forms exemplified by marine sponges and planktonic organisms. Ultimate biomimicry is the ability to emulate and simulate human biology [37]. In the meantime, there is a strong case for using natural products of all types in regenerative strategies requiring a framework because of their pre-organization into superior and sophisticated structures. The aim is to continue closing the gap between materials and biology and be better able to merge the two into a single product. 2. Biomedical Applications of Marine Products We present data demonstrating the utility for clinical therapy of an assortment of easily obtained marine products for selected roles in regenerative dentistry-incorporating oral tissues and craniofacial tissues in augmenting and replacing missing, damaged and diseased bone, dentine and periodontal ligament tissues (Figure 1). Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 2883 Figure 1. Structural products harnessed directly from marine life. In this show a small selection of marine biostructures with promising properties to rebuild the main individual tooth tissues that are affected by disease: bone, dentine, pulp and periodontal ligament. A further look into marine natural history will offer more structures destined for transplantation into the tooth structure itself. These marine derived structures are required to support dental cells and tissues in their natural position and create structural, crystalline and mineral ion clustered microenvironments for developing mineralized and connective tissues with properties that ensure seamless fusion takes place within existing tooth structures. Tissue boosting molecules such as, growth factor proteins can be stably adsorbed into the marine substitutes and also be entrapped within the mesopores and nanopores. Various routes for appropriating the structures can be employed that increase the number and type of biological roles they have. We can add more roles than the original structure to jump start regeneration. roles they have. We can add more roles than the original structure to jump start regeneration. One other way to obtain organism-built structures and one that minimizes habitat degradation and supply issues is to cultivate the organism of interest in clean and strictly controlled conditions in the laboratory to grow structures for a particular purpose. This promotes the idea of manipulating an organism—Its physiology, genetic coding and perhaps epigenetics—To generate mineralized structures according to the experimenters’ specifications of architecture, shape, size and morphologies by direct modulation in real-time. There are also possibilities to supplement the developing and growing structures with choice additives. These additives can be included in the growth media. For example, the living diatom cell uses silica transporters to shift any excess amount of molecules found in its environment directly into the developing frustule. This system has begun to be manipulated in the lab. In the case of a genetic engineering approach to ―designoid‖ inorganic structures, there have been few concrete examples [76]. Metabolic modulation of growing Diatoms can exert alterations in shell architecture [77]. Gene modifications enacted on the diatom Thalassiosira pseudonana genome have One other way to obtain organism-built structures and one that minimizes habitat degradation and supply issues is to cultivate the organism of interest in clean and strictly controlled conditions in the laboratory to grow structures for a particular purpose. 2.1. Source of Marine Biomaterials and Products In the past, products from the living marine environment have been directly implemented in regenerating bone, cartilage and fat tissue to complete the musculoskeletal system [68]. Pre-made structures synthesized in nature have advantages over artificially made structures in a number of crucial ways. Pre-made structures are hierarchical, exhibit intricate internal structures and are made from superior material constituents. However, there are issues of contaminants (intracrystalline proteins that accompany biogenic crystals) and lengthy biodegradation properties. In addition, there are questions over the ecological impact of stealing marine biostructures away from their natural habitats. It is possible to temper the impact and implications of doing this by implementing the latest updates in farming techniques and growing these marine structures in artificial aquaria [69]. There have been excellent studies aimed towards effectively growing corals and marine sponges in natural habitats in fitting quantities and with the qualities fit for commercial activities and biomedical purposes. In one case, marine sponges have been farmed with the express purpose of producing bioactive metabolites in practical amounts [70] Cloning of the gene coding for the metabolite of interest has been advanced as a solution for this that can be sustained for all needs and applications [71,72]. Similarly, there are options for coral skeletal supplies in guaranteed quantities [73]. Having rapid growth rates of 2.2 mm/month at 18 °C, calcareous algae, Corallina officinalis, may provide more sustained volumes of structures compared with corals, which exhibit an accretion rate of 10 g/m2, to the future hard tissue engineer [74,75]. Cuttlefish bone, sea urchin spines, and seashells have all been tested and evaluated to varying degrees as potential structures to support bone formation [68]. Mar. Drugs 2014, 12 This promotes the idea of manipulating an organism—Its physiology, genetic coding and perhaps epigenetics—To generate mineralized structures according to the experimenters’ specifications of architecture, shape, size and morphologies by direct modulation in real-time. There are also possibilities to supplement the developing and growing structures with choice additives. These additives can be included in the growth media. For example, the living diatom cell uses silica transporters to shift any excess amount of molecules found in its environment directly into the developing frustule. This system has begun to be manipulated in the lab. In the case of a genetic engineering approach to ―designoid‖ inorganic structures, there have been few concrete examples [76]. Metabolic modulation of growing Diatoms can exert alterations in shell architecture [77]. Gene modifications enacted on the diatom Thalassiosira pseudonana genome have 2884 Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 led to adaptations in shell structure in just a few generations [78]. Thus far, the gene modulation process can be used to create gene knockouts and RNA-interfering inserts into the genetic constitution of diatoms (Figure 2). Figure 2. Ideas for re-design of the structure of microshells via genetic and metabolic routes. The architectural features of Coscinodiscus wailesii species of Diatom frustules can be changed via modulation of the chemical and biochemical composition of the growing environment [77]. (A) TEM cross-section through chloroplasts in Diatom cell to show the normal ultrastructure of stacked lamellae. This exact arrangement of organelles molds the areas where silica is deposited and hardened to produce a regular externalized pore structure (B); (C,D) Added Nickel sulfate-in ―sub-lethal quantities‖-in seawater distorts the shape and packing arrangement of lamellae, which in turn affects silica pattern formation at the cell surface. The result of this is to double the pore size and increases the packing density of the pores. This property is something we want to exploit. Gene modulation of Thalassiosira pseudonana has led to the directed evolution of differently structured Diatoms with stark variations in pore size and patterns of pores [78]. This can be a basis for adapting inorganic structures and architectures to desired formats. Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 2885 2.2. Marine Products in Dental Tissue Engineering This approach is designed to lead to the growth and colonization of endogenous tissue throughout the entirety of the construct on the understanding that the local repair and regenerative responses are readied and powerful enough to carry this through. A second option is to use the structures to grow mature tissues in culture that are ready to join and integrate with identical host tissues following transplantation and engraftment [25,86]. There are two valid ways of potentially implementing these structures into the tooth. The first method is to deploy them without cellular contents: to implant a structure infused with regenerative agents into the site of tissue loss and allow the interplay with host biology [84,85]. This approach is designed to lead to the growth and colonization of endogenous tissue throughout the entirety of the construct on the understanding that the local repair and regenerative responses are readied and powerful enough to carry this through. A second option is to use the structures to grow mature tissues in culture that are ready to join and integrate with identical host tissues following transplantation and engraftment [25,86]. The possible products that can be employed in dental tissue engineering include marine sponge skeletons, nacre seashell, Diatom frustules, Foraminifera shells, and coral skeletons [36,38,39,54,87]. A vast number of materials and substrates are being investigated, including marine collagens, alginates from seaweed, chitosans, chitins, and chito-oligosaccharides from crustacean shells. These substrates have been published extensively elsewhere [88–100]. However, there are some less well known and more recent marine-based materials and substrates that have been experimentally implemented in dental hard tissue restoration, cleaning and augmentation roles. This illustrates the wide variety of substances available from life in the oceans. 2.2. Marine Products in Dental Tissue Engineering The marine environment is filled with biodiversity that extends into many levels, chemical and structural. In the past, marine organisms have been examined for their potential to yield new drugs, adhesives and filters. Biomaterials and skeletal structures are relatively recent products that biotechnologists and, to a limited extent, their tissue engineering counterparts have used [36,37,73,79]. Some have been made with strong biomimetic principles (Figure 3A–D). The marine world of structures has also been used as a template for recreating copies of some basic skeletons (inspired mechanistically by the intricate skeletons of Foraminifera, Radiolaria, and Coccolithophores) and for creating new emerging morphological forms [80–83] (Figure 3A–D). Figure 3. Simple inorganic ―designoid‖ objects [76] in nature recreated in the beaker using biomimetic template mediated materials chemistry (A,B) Mesoporous vaterite microsponges produced via mineralization at the interface of a SDS stabilized water microdroplet in oil; Such structures have also been manufactured using plant based surfactants and oils. The stable emulsion is reminiscent of a ―viniagrette― [83,84]; (C) Inorganic surface with multiple patterns that are strongly reminiscent of Diatoms and Radiolaria shells, created by heat treated mesolamellar aluminophosphate vesicles [80]; (D) A single mesoporous sphere of aragonite polymorph of calcium carbonate. The principle behind its formation is analogous to the arrangement of areolar vesicles in diatom mineralization. A thin soapy foam is created from an oil and water mixture. This serves as a template for calcium carbonate mineralization [80]. Using some basic construction principles learnt from nature such as: make vesicles and pack them together like foams and use them as boundaries for mineralization to build on, it is then possible to generate some of the structures and architectural motifs seen amongst Coccolithophores and Foraminifera shells [80,81,83]. These microsponges by virtue of their shape and structure have been successful in drug and gene delivery experiments and in 3-dimensional co-culture with human bone marrow stromal cells. Mar. Drugs 2014, 12 2886 Figure 3. Cont. There are two valid ways of potentially implementing these structures into the tooth. The first method is to deploy them without cellular contents: to implant a structure infused with regenerative agents into the site of tissue loss and allow the interplay with host biology [84,85]. Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 2887 (e.g., Mytilus edulis foot protein-5), which is calcifies onto marine substrates, could adhere to the tooth surface and successfully remineralized etched-in defects [104]. In addition to mussel glue, nacre organic matrix extract has also been used for enamel remineralization. The sophisticated hierarchical structure of human enamel and the construction rules for building it are all well understood, but as yet, materials scientists are unable to match this structure. Artificially created nanocrystals that mimic the size and shape of enamel nanocrystals have been shown to induce enamel reproduction at the tooth surface in the beaker. However, these nanocrystals are not clinically viable because they lack stability inside a defect, are less tough than real enamel and mineralize too slowly and would be washed away in the mouth [62,63,105]. Hydrophobic protein amelogenin is the principal matrix protein that helps shape the crystals of enamel in the early developmental steps [106–108]. Mature enamel is structured by acidic enamelin glycoproteins that coat each individual crystal [83,109]. In one surprising lateral study, a very different type of mineral-building protein from the organic matrix protein of nacre fraction could also shape and pack hydroxyapatite crystals in the exact same parallel bundled orientation as seen in normal enamel and that this translated into a degree of hardness comparable with natural enamel [110]. Perhaps this correspondence in actual enamel and analogous enamel mineralization represents the evolutionary conservation of molecules (proteins) involved continuously throughout the history of biomineralization. Since the water soluble matrix of Pinctada maxima nacre is purported to contain TGF-β class of signaling proteins that can participate in bone remineralization [111,112]. We have seen further accumulating examples of this across evolutionary time and space [69,113–115]. 2.2.1. Tooth Remineralization Methods for in situ tooth remineralization are becoming more diverse and follow biomimetic and bioinspired directions. Inorganic nanoparticles are viewed as fundamental building blocks and seedlings for the emergence of new dentine and enamel crystals with correct shapes, sizes and axes [62,63,101–103]. Overall, these items are an attractive proposition because of their low invasiveness and conceptual simplicity; there is no need for surgery or the inclusion of traditional restoration materials. However, there has been little actual success in formulating substrates that remineralized in place inside the mouth, which is not a constant mineral-enriched environment as all of the in vitro tests imply. One mechanism that possesses the right virtues for stabilization at the tooth surface in the mouth is polydopamine-induced hydroxyapatite formation, which is reported to grow crystals in the image of its surrounding mineralized environment. The main component of mussel glue Mar. Drugs 2014, 12 2.2.2. Biofilm Prevention and Disruption Marine organisms are in a constant arms race to counteract attacks by predators and parasites. Many organisms have sophisticated, mainly chemical, defenses to fend of these attackers. Bacterial biofilms pose a threat, and many organisms have developed defenses to disrupt these biofilms from taking hold [115]. This battle also exists between different bacteria and other micro-organisms. They themselves have been equipped with chemical bioagents that prevent colonization of competitors [116]. Under intense scrutiny right now are polysaccharides from marine bacteria and other organisms such as, marine sponges that perform disruption of biofilms [117,118]. In a special finding during an investigation into shifting films on seaweeds, the bacterial biofilms formed by a ubiquitous Bacillus licheniformis released an enzyme that allowed bacterial dispersal from the embedding medium of the biofilm. The NucB enzyme involved in this process is being developed to disrupt biofilms on the tooth surface after the successful disruption of the biofilms that cause sinusitis [119,120]. However, it remains to be seen whether this enzyme is generic and can effectively dislocate the biofilm structures secreted by other species. The surfaces of marine organisms larger than a single cell are under a constant threat of attack and colonial settlement from microorganisms and parasites. In addition to chemical defenses, organisms possess structural defenses that have evolved to combat bacterial contamination of the outer skin surfaces that can cause physical damage to tissues. One of the best-studied structure-based adaptations is sharkskin, although there are undoubtedly more examples of this phenomenon in other marine organisms [121,122]. Nature has evolved countless interfaces with anti-bacterial defenses using specific nanotopographies. This surface defense is independent of the 2888 Mar. Drugs 2014, 12 effects of chemical secretions. The first application of patterned surfaces aimed at biofilm control resembles diamond-shaped protuberances at the micrometric scale that hinder bacterial contamination. The engineered version is called Skarklet™ and is inspired directly from the micron structure of the scales or dermal denticles of sharkskin [122]. The topography is thought to increase the surface energy at the surface to such a degree that it prevents the expenditure of excessive amounts of energy by bacterial colonies needed to generate a biofilm at the surface. Bacteria die unable to connect with other bacteria cells [122]. The effect of this material has been evaluated in a medicine-relevant study [123]. 2.2.2. Biofilm Prevention and Disruption The synthetically replicated surface hindered the growth of Staphylococcus aureus, Staphylococcus epidermidis, Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Escherichia coli and vancomycin-resistant enterococcus (VRE). 2.2.3. Scaffolds and Biostructures Mar. Drugs 2014, 12 2889 Mar. Drugs 2014, 12 Figure 4. Collagenous Marine sponge comprises of a fibrous framework of bonded fibers and this could be an ideal substitute for a periodontal ligament and bone tissue (A–C). The advantage of selected marine sponge skeletons over many synthetic fiber networks and electrospun constructs is that they are fully fused together. In (A) we see rapid colonisation, proliferation of periodontal ligament stem cells on a collagenous marine sponge within 48 h of dynamic seeding (×200). The PDL stem cells were centrifuged into the marine sponge framework at 1200 rpm for minutes. The fibers provide surfaces for periodontal ligament stem cells to develop a stretched morphology, which is characteristic of their home environment and they span large spaces existing between sponge fibers in all directions. An early form of bone and cartilage tissue is developed in vivo within a collagenous marine sponge skeleton from seeded embryonic stem cells (B,C). The histoarchitecture is early in origin and the organisation is characteristic of osteoid type of bone and cartilage. The bone that was formed showed highly organized matrix when viewed under polarized light, hinting at the possibility of highly organized mineralization in progress (Yellow arrows point to sponge fiber) (4A–C Kindly reprinted from Biomedical Materials [38]); (D) Confocal fluorescence image of osteoblast cell sheets attached and suspended in marine sponge framework at 14 days. The top panel showing cell distribution at high power and the lower panel showing coverage across the whole seeded marine sponge skeleton (red = F-actin staining; green = autofluorescence of sponge fiber; blue = nucleus staining); (E) SEM image of osteoblast cell aggregation on Hippospongia fiber; (Figure D was reproduced with kind permission from Ivyspring International Publisher [54] and Figure E reproduced with kind permission from Wiley-VCH [129]). Figure 4. Collagenous Marine sponge comprises of a fibrous framework of bonded fibers and this could be an ideal substitute for a periodontal ligament and bone tissue (A–C). The advantage of selected marine sponge skeletons over many synthetic fiber networks and electrospun constructs is that they are fully fused together. In (A) we see rapid colonisation, proliferation of periodontal ligament stem cells on a collagenous marine sponge within 48 h of dynamic seeding (×200). The PDL stem cells were centrifuged into the marine sponge framework at 1200 rpm for minutes. 2.2.3.1. Marine Sponge Skeletons The collagenous skeletons of marine sponges have been shown to be effective frameworks supporting a range of cells and tissues and in particular bone tissue [36,54]. The skeletons provide 3-dimensional spaces for cell or tissue colonization and a level of tissue pre-organization. There are sufficient vital structural cues and some other biochemical cues that are able to support and maintain bone and cartilage tissues. The void volume is large, approaching 1000 μm, and is interconnected in all directions. However, the pore gaps are greater than the optimal sizes usually put forward for a prospective tissue colonizing sponge framework. In truth a range of pore sizes are thought necessary to transport fluids, proteins and for cell migration to occur. Typically the optimal pore sizes for cell migration are between 80 and 500 μm [124]. However, the size range is equal to the size of structure for an essential functional unit of tissue [125]. The sponge fibers are fully bonded, and the mixture of collagens blended into the fibers is constitutively made up from analogues (short chain) of basement membrane collagens, such as collagen type IV [126,127]. In one specified marine sponge a type 1 mammalian analogue has been discovered [128]. Initial observations suggested that MSCs hone to fibers, attach and spread even when cultured in serum-free media [36]. We observe the exactly the same response with human Periodontal ligament stem cells (low passage number) cultivated within a Spongia collagenous Marine Sponge (unpublished data). Within an animal model, the subcutaneous responses are dramatic when fetal derived mesenchymal stem cells are embedded in a Spongia collagenous sea sponge for 3 weeks (Figure 4A–C). In a separate in vitro study osteoblasts cultured within Calliospongia framework progressively anchored onto the fibers, expressed alkaline phosphatase and within 3 weeks generated mineral nodules and expressed mid to late osteogenic genes, osteonectin and osteopontin [54] (Figure 4D). In a paper by Zheng et al. marine sponges from the genus, Hippospongia and the family Chalinidae supported mouse osteoblast adhesion, growth and proliferation [129] (Figure 4E). There is also active development of tissue engineering matrices using silica, chitin and collagens from marine sponges [130,131]. The potential of this framework through careful and systematic studies seems promising in bone and the periodontal ligament junction with bone and dentine. 2.2.3.2. Diatom Skeletons These skeletons are showing promise as efficient drug and protein delivery devices [132], an additive for dental Zirconia nanocomposites [133], as a tissue engineering ―space filler‖ and framework and a model for cell encapsulation [134]. Diatoms are microscopic algal cells generate highly intricate shells made of silica glass. The structures are both a small filtration device and an armor plating to protect the organism from predators. These structures possess features and traits that can be exploited for a similar function but for a different set of purposes. Thus, they can be matched to a very different role than that for which they evolved. Diatomaceous earth is a widely used mineral constituent for many industrial applications. In Dentistry it has long been a routine additive for impression materials. Diatom skeletons have been used in technologies spanning sensor devices to paints and now extended vigorously into nanotechnology and biotechnology fields [135–142]. The structures possess an adaptive fitness within hard tissues, dentine, bone and enamel because of their toughness, hardness and mineral compatibility with calcium phosphates [143–146]. Such shells are being adapted for drug delivery [132,147,148] and biosensing roles [149]. We have proposed to use them as agents for cell recruitment, proliferation and migration and as a packing powder for dentine defect filling, and a completed regeneration of mature mineralized tissue. A prime objective would be to reproduce dentine structure in its primary state with high bioceramic fraction and with tubules. Faithful replication of dentine properties can be achieved through chemical self-organization and assembly or via the activities of incorporated dental cells such as, dental pulp stem cells. The primary function of Diatom shells in a tissue engineering context is as a protective container for proteins in cell activation during transplantation and regeneration. The pore sizes range from microscopic to nanoscopic dimensions (200 nm to 1000 nm), which provide the fine capillaries for drawing in soluble proteins and holding them within a tight network (Figure 5A,B). There are two ways that proteins are stably entrapped in the Diatom skeleton. One is via chemical adsorption and the other is by adsorption via weak, low energy van der Waals forces [132]. The electric charge of the protein and its molecular size determine the encapsulation properties, stability and rates of release from the structure. Diatom shells elute proteins at a slower pace compared with comparable hollow and porous particles made from silica. Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 2890 Mar. Drugs 2014, 12 The fibers provide surfaces for periodontal ligament stem cells to develop a stretched morphology, which is characteristic of their home environment and they span large spaces existing between sponge fibers in all directions. An early form of bone and cartilage tissue is developed in vivo within a collagenous marine sponge skeleton from seeded embryonic stem cells (B,C). The histoarchitecture is early in origin and the organisation is characteristic of osteoid type of bone and cartilage. The bone that was formed showed highly organized matrix when viewed under polarized light, hinting at the possibility of highly organized mineralization in progress (Yellow arrows point to sponge fiber) (4A–C Kindly reprinted from Biomedical Materials [38]); (D) Confocal fluorescence image of osteoblast cell sheets attached and suspended in marine sponge framework at 14 days. The top panel showing cell distribution at high power and the lower panel showing coverage across the whole seeded marine sponge skeleton (red = F-actin staining; green = autofluorescence of sponge fiber; blue = nucleus staining); (E) SEM image of osteoblast cell aggregation on Hippospongia fiber; (Figure D was reproduced with kind permission from Ivyspring International Publisher [54] and Figure E reproduced with kind permission from Wiley-VCH [129]). Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 2891 Figure 5. A Diatom shell structure that can deliver incorporated antibiotic. (A) A low power SEM of one species of Diatom shell (cylindrical Aulacoseira sp.) from diatomaceous earth; (B) High power SEM of a Diatom shell to show the nanopores incorporated into the microstructure of discoid Coscinodiscus wailesii; (C) A cumulative release profile for shell incorporated Gentamicin antibiotic lasting up to 14 days before all of the product has been off-loaded. This demonstrates that Diatom shells possess a potential for controlled drug delivery (C kindly reprinted with permission from Future Medicine) [132]. y ( y p p ) [ ] The dentist requires a flowable paste that effectively fills all the spaces, including cracks and fissures within any deep dentine defect. So, we have begun to fabricate varying formulations of Diatom containing pastes that rapidly harden in place in the entire space. Some of the pastes have added to them: calcium phosphate mineral precursors, dental pulp stem cells and phospholipids. In our laboratory we have made mineralized structures with pores similar to normal dentine and we have shown high survival and proliferation rates of Dental pulp stem cells embedded with Diatom shells and an alginate gel. Mechanically, the hardened and crystallized paste containing 75% w/v diatoms behaves as a shock absorber when compressed (at extremely mild loads compared to normal dentine) and yield dramatically more than native dentine. The idea is to provide a shape and void filling mass filled with dental stem cells that supports and cushions an added metal filling above-representing the enamel substitute. Our aim is to keep this as a short-lived transitional structure until cell-originated dentine tissue is reproduced and becomes more densified as calcium phosphate mineral is secreted The dentist requires a flowable paste that effectively fills all the spaces, including cracks and fissures within any deep dentine defect. So, we have begun to fabricate varying formulations of Diatom containing pastes that rapidly harden in place in the entire space. Some of the pastes have added to them: calcium phosphate mineral precursors, dental pulp stem cells and phospholipids. In our laboratory we have made mineralized structures with pores similar to normal dentine and we have shown high survival and proliferation rates of Dental pulp stem cells embedded with Diatom shells and an alginate gel. 2.2.3.2. Diatom Skeletons The release display is time dependent and occurs at a constant rate, thereby representing zero-order kinetics as shown for incorporated Gentamicin sulfate in Figure 5C. Mesoporous silica particles are favored to pack in many different agents for a variety of biological roles such as tumor destruction via the release of anticancer drugs and gene delivery inside the cell [150,151]. Today, these processes can be more tightly controlled with surface molecule decoration [152]. The smallest varieties (80–200 nm) can enter the cell without endangering it. The sizes of the pores and the surface chemistry of the pores can be delimited with accuracy. Being able to specify channel orientation and the size of individual domains will help the control in the precision of encapsulated delivery. Thus, it becomes more realistic to tune the rate, regionality and timing of any particular encapsulated protein. 2.2.3.3. Foraminifera Microskeletons Foraminifera shells are strong contenders as bioactive bone substitutes, drug delivery devices and as an equivalent of bone allograft. Naturally occurring microskeletons are produced by hundreds of thousands of unicellular amoeba like Protists that predominantly reside in all marine habitats throughout the globe. They are in such abundance that one third of the earth surface is coated with dead shells from these primitive organisms. Large benthic, coral reef dwelling Foraminifera shells, without their host creators, can be found deposited in coral beach sands on reef flats and sand cays representing 40% of the total beach sediment. Large benthic Foraminifera have been collected from Bali, Indonesia and exploited in studies for bone healing therapies including osteoporosis [153]. Structurally, these microskeletons are made up of an intricate networking of micro- and nanopores, evolved by natural selection as a particle filtration system as seen under SEM and μCT (Figure 6A,B). These structural properties have been effectively exploited for sustained, targeted drug (bisphosphonate) and antibiotics delivery (Gentamicin) with low toxicity [154]. An additional level of control in delivery is implemented by wrapping up individual spheres with liposome bound coating [154]. The shells are well suited as filler particles in calcified tissue environment because of their mineral content and structure. Hardening the shells by thermal transformation from their original calcium carbonate identity to a calcium phosphate one coupled with an additional nanometric overcoat ensures that the shells can be compacted into bone voids without the loss of the important structure. Antibiotic compounds have been encapsulated into the pores and onto the surface as crystals with 73% loading efficiency. The drug is released gradually with the concomitant dissolution of the mineral. In tests with Staphylococcus aureus, co-cultured macrospheres infused with gentamicin were completely eliminated in 30 min (Figure 6C). Other important bone induction drugs have been offloaded from these efficient structures to increase bone formation and they can reduce the toxic side effects of drugs because of the controlled targeting. Simvastatin-loaded macrospheres exhibited superior effects on bone growth and strength in vivo during prolonged release over 6 weeks compared with control samples in OVX mice [155]. Repeatedly, we see the possibilities of controlled release properties for bone induction agents and the added effect of the release of biogenic ions from the structure upon dissolution. The inductive effects on bone cells of strontium, calcium and magnesium ions released, at physiologically relevant quantities have also been studied [39]. Mar. Drugs 2014, 12 Mechanically, the hardened and crystallized paste containing 75% w/v diatoms behaves as a shock absorber when compressed (at extremely mild loads compared to normal dentine) and yield dramatically more than native dentine. The idea is to provide a shape and void filling mass filled with dental stem cells that supports and cushions an added metal filling above-representing the enamel substitute. Our aim is to keep this as a short-lived transitional structure until cell-originated dentine tissue is reproduced and becomes more densified as calcium phosphate mineral is secreted The dentist requires a flowable paste that effectively fills all the spaces, including cracks and fissures within any deep dentine defect. So, we have begun to fabricate varying formulations of Diatom containing pastes that rapidly harden in place in the entire space. Some of the pastes have added to them: calcium phosphate mineral precursors, dental pulp stem cells and phospholipids. In our laboratory we have made mineralized structures with pores similar to normal dentine and we have shown high survival and proliferation rates of Dental pulp stem cells embedded with Diatom shells and an alginate gel. Mechanically, the hardened and crystallized paste containing 75% w/v diatoms behaves as a shock absorber when compressed (at extremely mild loads compared to normal dentine) and yield dramatically more than native dentine. The idea is to provide a shape and void filling mass filled with dental stem cells that supports and cushions an added metal filling above-representing the enamel substitute. Our aim is to keep this as a short-lived transitional structure until cell-originated dentine tissue is reproduced and becomes more densified as calcium phosphate mineral is secreted Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 2892 from a developing dental cell population, such as DPSC, deposited and is remodeled into a proper, anatomically correct dentine structure. There are several ways to grow microscopic diatom shells on culture dishes in the laboratory for the re-design and partial re-engineering of their structure and architecture. Having control of the conditions in which the shells grow and the composition of their growing media, it becomes possible to alter the pore diameter and architecture such as, the geometrical arrangement of the pores. It has been demonstrated that any new additive in the culture media is gathered into the molecular machinery of skeleton formation. The result is that the inclusion is infused into the amorphous silica framework. Mar. Drugs 2014, 12 Doping of the skeleton is possible because the processing pathways in silica deposition materials chemistry are predisposed to picking up molecules from the local external environment. 2.2.3.3. Foraminifera Microskeletons The ions have been highly effective at promoting activities of a bone-secreting cell while at the same time suppressing the 2893 2.2.4.1. Nacre Seashell Nacre seashell has shown intermittent promise as an osteoinductive bone substitute. The complete story of nacre osteoinduction and its potency has not been revealed and there are still opportunities to further exploit this material for use in oral surgery for example particularly when coupled with bone marrow derived mesenchymal stem cells. The pearly outer layer of seashells in mollusks is nacre. This structural biomaterial has long been subject to study for its superior mechanical strength even when wet (Young’s modulus 60 GPa), which is equivalent to the strength of steel [157]. The fascination for this material is how a brittle ceramic gathered from the marine environment can be made so tough into seashell with a tiny, 0.24% fraction of bioorganic material. Nacre is riddled with fracture-deflecting features that include: advanced ductility by inclusion of water and superior molecular interlinks between the platelets and the matrix into which they are embedded. Nacre is a prime example in which cheap soft materials are exactly structured into a new material that has superior mechanical design and strength. Materials scientists have spent many years striving to emulate the way nacre is built to make stronger and tougher materials using cheap, abundant, soft, unelaborated, mechanically weak starting materials. According to the community of materials technologists, the greatest present and future need is for energy resourcefulness [158]. Nacre, quite by happenstance, may have a significant role to play in mammalian biological tissues because it was discovered that nacre has a bone-forming property [111,112,159]. There is a deep historical perspective to this story as in 1931 Mayan skulls were found to have pure nacre teeth which had fused perfectly with the jawbone [115,160] (Figure 7A). On closer investigation, there is a rational reason for this connection. Molecular processes in biomineralizsation have been conserved throughout evolution, and some key components are found in both invertebrates and human mineralized tissues and have the same or other adaptive functions. The vital constituents of nacre in biological mineralization in bone tissue are the water-soluble matrix proteins. Powders and small chips made from nacre seashell have been used to fill bone voids. This was undertaken based on the knowledge gained from one of the first studies to study nacre led bone mineralization. Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 transformed Foraminifera shells via centrifugation leads to the complete coverage of individual spheres in 7 days via a steady increase in proliferation rate and an increase in secreted matrix proteins [79] (Figure 6D). This result demonstrates how the spheres can be effective as templates for cell accretion in 3-dimensional culture regimes. The expansion of stem cells in the culture dish is one of the important steps towards increasing availability for cell therapies. Thus far, we have merely harnessed these structures in the first steps towards re-building mature bone inside the voids created by disease and traumatic injuries. 2.2.4. Bone Substitutes Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 activity of bone resorbing osteoclast-like cells in the culture dish [79,154]. Thus, the macrospheres possess an intrinsic property for bone homeostasis. Figure 6. Foraminifera spheres are macroscopic shells from marine sediments that grow a highly intricate set of pore structures for the task of passive filtration where food particles are sucked in through channels and by design selected into sizes for the cell inside; (A) The natural structure of interconnected pores and channels has shown a usefulness in tissue engineering for capturing and slowly releasing bone building drugs and supporting proliferation and expansion of stem cells around the outer surface in 3-dimensions (Image generated at the Microstructural Analysis Unit UTS, Sydney, Australia); (B) Close-in cross-sectional view of the macrosphere to show the regular and highly ordered distribution of pores inside the sphere. This image was generated by micro-Tomography (micro-CT at the Australian Centre for Microscopy and Microanalysis); (C) A look at the functional release of Gentamicin antibiotic from macrospheres on the eradication of S. aureus bacteria. At 30 min all bacteria cultured with Gentamicin laden Foraminifera macrospheres were exterminated; (D) An SEM image of a single macrosphere coated with rat adipocyte derived stem cells (ADSC) 7 days after seeding (C Reproduced with kind permission from Future Medicine and amended to improve clarity in reproduction [156] and D Reproduced with kind permission from Anne-Liebert [79]). The idea of using carrier particles to support proliferation of cells has been around for some time. Foraminifera macrospheres have potential to support and proliferate cells in 3-day very rapidly. In one simple test of this, the seeding of Adipocyte derived stem cells (ADSC’s) into calcium phosphate The idea of using carrier particles to support proliferation of cells has been around for some time. Foraminifera macrospheres have potential to support and proliferate cells in 3-day very rapidly. In one simple test of this, the seeding of Adipocyte derived stem cells (ADSC’s) into calcium phosphate 2894 2.2.4.1. Nacre Seashell Nacre chips were found to co-exist with bone chips and eventual weld together with newly formed woven type bone in the presence of human mesenchymal cells, osteoprogenitors and osteoblasts in vitro [161,162] (Figure 7B). Vitalised by this finding, in one of the first experiments undertaken with this biomaterial in a human patient, nacre powder mixed into a slurry using patient’s blood was injected into a jawbone defect [159] (Figure 7C). There was no inflammation and fresh bone 2895 Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 had been generated as the nacre resorbed. Other subsequent experiments have tested nacre’s osteogenic power in few laboratory animals, such as sheep and have reported similar dramatic osteostimulatory effects [163]. In 12 weeks a defect excavated in the lumbar vertebra and filled with nacre powder transformed into spongy bone while the nacre was seen to have dissolved [163]. In comprehending these results we cannot rule out some osteogenic effect from the nacre surface structure itself such as the micro- and nanotopography. In addition, there is proper evidence which shows that the surface chemistry changes at the surface of nacre [164]. Figure 7. (A) Whole nacre incisor replacements found in the lower jaw of an Ancient Mayan individual. Excellent bone fusion is shown by X-ray imaging in some of the nacre implants in panels, b and c; (B) A high power (×600) image of a section at the interface between a nacre piece and metabolically active mesenchymal cells mobilizing into an osteoprogenitor layer. The junction is free of immune cells are present and fibrous tissue [162]; (C) Stained histological sections from human patient biopsy showing the integration between bone and nacre coupled with new bone formation; (D) A high power view of new bone at the nacre surface showing particularly the stained osteoid borders. Also generated are two textures of mature human bone: woven and lamellar; (Plate A, reprinted with kind permission from Bulletin of the History of Dentistry, Copyright (1972) [160]; Plate B reproduced with kind permission of Elsevier [162]. Plates C,D Reproduced with kind permission from Macmillan Publishing Group [115]). Nacre organic matrix proteins, but principally peptides have been demonstrated as the origin of osteoinduction. In the study by Lamghari et al. it was proposed that the matrix peptides stimulated Nacre organic matrix proteins, but principally peptides have been demonstrated as the origin of osteoinduction. In the study by Lamghari et al. it was proposed that the matrix peptides stimulated Nacre organic matrix proteins, but principally peptides have been demonstrated as the origin of osteoinduction. In the study by Lamghari et al. it was proposed that the matrix peptides stimulated 2896 Mar. Drugs 2014, 12 cellular responses that would be expected from treatment with Bone Morphogenic Proteins (BMP’s) and Transforming Growth Factor- beta (TGF-β) [163]. Mar. Drugs 2014, 12 The biochemical responses of bone marrow stromal cells (hBMSC) to nacre for instance are like-for-like following treatment with nanogram quantities of TGF-β. TGF-β is a central protein in dentine building in embryonic development, and it is speculated that TGF-β could play a role in dentine regeneration in adults. The role of BMP in the tooth has been intensively studied. Figure 8. The effectiveness of nacre WSM in bone formation, remodeling potential and osteoporosis treatment; (A,B) Nacre powder and WSM extract induce osteoblast mineralization via consequential molecular signaling pathways e.g., Fos-1, p-ERK, p-JUNK and up-regulate biomineralisation promoting genes BMP-2, cbfa-1 etc. Osteoclast gene activities are suppressed (C). Nacre products also prevent osteoporotic bone in OVX mice (D) (A–D reproduced with kind permission from Elsevier) [165]. A series of efforts have been made to extract, purify and concentrate the key regenerative proteins into a soluble preparation having precisely determined biological effects on different cells acting in bone biomineralizsation. A non-mineralizing route towards extraction of the intrinsic matrix proteins has been asserted as the best way of isolating the osteoinductive constituents rather than a mineralizing mode of extraction [111]. The extracted and concentrated preparation of water-soluble matrix (WSM) has shown considerable potency. The protein extract boosts proliferation and ALP secretion in bone marrow cells in high microgram quantities (135 and 540 μg protein/mL) [112]. It has been shown to induce osteoblast driven mineralization activities much faster than when osteoblasts are placed in a mineralizing medium [166]. Very significant results have come from the research of Kim et al. [165] A series of efforts have been made to extract, purify and concentrate the key regenerative proteins into a soluble preparation having precisely determined biological effects on different cells acting in bone biomineralizsation. A non-mineralizing route towards extraction of the intrinsic matrix proteins has been asserted as the best way of isolating the osteoinductive constituents rather than a mineralizing mode of extraction [111]. The extracted and concentrated preparation of water-soluble matrix (WSM) has shown considerable potency. The protein extract boosts proliferation and ALP secretion in bone marrow cells in high microgram quantities (135 and 540 μg protein/mL) [112]. It has been shown to induce osteoblast driven mineralization activities much faster than when osteoblasts are placed in a mineralizing medium [166]. Very significant results have come from the research of Kim et al. Mar. Drugs 2014, 12 2897 Mar. Drugs 2014, 12 In it nacre WSM extract was demonstrated to effect molecular signaling pathways and regulate gene expression activities differently in osteoblasts and osteoclasts. Osteoblasts were promoted and osteoclasts suppressed in the presence of WSM strongly implicating the importance of nacre in bone remodeling process (Figure 8A–D). In it nacre WSM extract was demonstrated to effect molecular signaling pathways and regulate gene expression activities differently in osteoblasts and osteoclasts. Osteoblasts were promoted and osteoclasts suppressed in the presence of WSM strongly implicating the importance of nacre in bone remodeling process (Figure 8A–D). Thus far, 19 protein fractions have been disclosed via size-exclusion HPLC relating to 110 individual molecules being present. A 60% proportion of the WSM protein is under 1 kDa meaning that they are probably mainly peptides [167]. An earlier study highlighted 4 main fractions with one in particular having the same power of osteoinduction as BMP-2. There is no currently available data featuring nacre-driven mineralization via a cell participation-route in hard dental tissues such as, dentine or enamel, but there is an intriguing study in which tooth slices having the enamel etched away would re-mineralize into almost perfect enamel structure when placed in a solution containing extracted water soluble matrix of nacre (WSM). The newly re-mineralized enamel analogue consisted of hydroxyapatite rods tightly packed together into well-ordered bundles. [110]. Nacre WSM appeared to provide a template for Hydroxyapatite rods and also glued them together into the tight bundles. Mar. Drugs 2014, 12 [165] A series of efforts have been made to extract, purify and concentrate the key regenerative proteins into a soluble preparation having precisely determined biological effects on different cells acting in bone biomineralizsation. A non-mineralizing route towards extraction of the intrinsic matrix proteins has been asserted as the best way of isolating the osteoinductive constituents rather than a mineralizing mode of extraction [111]. The extracted and concentrated preparation of water-soluble matrix (WSM) has shown considerable potency. The protein extract boosts proliferation and ALP secretion in bone marrow cells in high microgram quantities (135 and 540 μg protein/mL) [112]. It has been shown to induce osteoblast driven mineralization activities much faster than when osteoblasts are placed in a mineralizing medium [166]. Very significant results have come from the research of Kim et al. [165] 2.2.4.2. Biotransformed Coral Skeletons Coral skeletons have been used as bone substitutes in surgery for the last 28 years but it was not until the 1990s that it became commercially available as Biocoral and Interpore [168–172]. A pioneering idea for harnessing coralline materials in medicine came from White et al. in 1972 with an innovative process for transforming and generating high fidelity copies of marine skeletons such as coral [51]. Further studies were carried out to assess tissue growth responses to these new implants [49–51]. The first study in which a treated and transformed coral was used in a clinical application happened in 1985 [173]. Now there is a substantial catalogue of published studies attesting to various levels of clinical efficacy to heal widely separated fractures and fill naturally unbridgeable bone voids caused by tumors for example [174]. Bone tissue morphogenesis has been augmented within coral skeletal frameworks by incorporating bone marrow derived stem cells with impressive outcomes (Figure 9A,B). In a large animal defect model mature cortical bone was produced inside coral frameworks housing MSC [175]. Owing to their internal pore and channel structures, crystallinity and composition corals are biomimetic substitutes for different textures of bone but only two principal classes of reef-building coral skeleton are actually used (Porites, Acropora and Goniopora) because they are available in large quantities and possess a highly consistent structure [176]. Their immunogenicity is negligible when thermally treated with only tiny quantities of intracrystalline proteins remaining. Coral skeleton implants are currently limited to non-load bearing regions of the skeletal system as they possess inherent weakness in compression. This use has spurred methods to increase the strength of coral skeletons so that they can support the high compressive forces exerted in load bearing long bones for example. One major way of increasing strength in compression of coral skeleton bone grafts is to chemically transform them from their native calcium carbonate composition into hydroxyapatite. Commercially produced converted coral skeletons for surgery are not completely converted into hydroxyapatite so that some parts will remain as calcium carbonate. Further strengthening is achieved by delivering hydroxyapatite based sol-gel nanocoatings onto the entire surface of struts and 2898 Mar. Drugs 2014, 12 connectors and into the coral micropores and nanopores [177]. The combined appropriation of the calcified material by heat, transformation into calcium phosphate mineral, nanocoating and pore infilling doubles the biaxial strength compared to untreated normal coral [177]. Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 of tooth tissues, it is frequently necessary to build more than one single tissue because there is a gradation of tissue types. For instance, a periodontal ligament substitute will need more than a fibrous web as well as intermediate interfaces for connection with bone and dentine, respectively. Therefore, because the structures are only temporary, we must think of reconfiguring and appropriating such structures to emulate this feature or to build zones of distinct tissues or cellular zones that are fashioned with specific mixes of cell phenotypes and extracellular matrix (ECM) compositions. Figure 10. In building living tooth replacement tissues, addressing issues of mechanical design is paramount. The most potent lesson to learn from nature is the design of smart interfaces. In the right panel is show changes in the strength of compressive force across the whole length of a single tooth. Interfaces between each tissue with different individual structure and composition deflect the force from above without leading to catastrophic failure of the materials involved (Reproduced with kind permission from Elsevier with new labels added) [179]. 3. Limitations and Challenges The tooth organ is made up of an intricate assembly of hard and soft tissues. The softer part is often intercalated within the hardened mineralized tissues. Dentine, enamel, periodontal ligament and bone are structurally dissimilar but are integrated very well with each other. These components are held together with graded interlocking surfaces either with use of embedded fibers or by subtle melding of not-so dissimilar materials inside junctional zones such as the cementum-dentine junction (Figure 10) [179]. At the molecular level, special amphiphilic molecules have been adapted to bond 2.2.4.2. Biotransformed Coral Skeletons It has been shown that an equivalent alkoxide-based hydroxyapatite layer on titanium discs is a direct stimulant for osteoaccelerants, c-fos and Map kinase pathways in attached osteoblasts [178]. Figure 9. Live animal bone repair using coral skeletons (untransformed) laden with mesenchymal stem cells. (A) A comparison of host bone reactions in histological stained section to a segmental defect (control), a segmental defect plugged with coral skeleton (Coral alone), plugged with coral skeleton and FBM and Coral skeleton filled with MSCs. In the group containing coral with MSCs the defect is bridged and repaired in full thickness; (B) X-ray slides comparing the mineralized bone replacement between coral skeleton with FBM and coral skeleton laden with MSCs. Only in the corals with MSCs do we get new mature bone replacement (Reprinted with kind permission from MacMillan Publishing Group) [175]. In our own laboratory we have exploited and appropriated isolated and selected structures from marine bioresources on the basis of biomimetic logic. There are other ways of doing biomimicry but we have described a facile approach with good clinical potentials. Corals exhibit a structure and omposition equivalent to human bone, and the spherical filtration devices have the properties ecessary for drug encapsulation and delivery. These biostructures have been selected purposefully nd simply by observing the structure and knowing something of its function. In partial reconstruction In our own laboratory we have exploited and appropriated isolated and selected structures from marine bioresources on the basis of biomimetic logic. There are other ways of doing biomimicry but we have described a facile approach with good clinical potentials. Corals exhibit a structure and composition equivalent to human bone, and the spherical filtration devices have the properties necessary for drug encapsulation and delivery. These biostructures have been selected purposefully and simply by observing the structure and knowing something of its function. In partial reconstruction 2899 Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 two different materials together, usually through an intermediary organic phase. This organization transforms into vastly increased strengths at the macroscale. This level of sophistication has been hard to match at the level of detail adapted by evolution in living organisms. Such materials will likely have to be grown by cells rather than manufactured in the laboratory. Therefore, it is up to tissue engineering to craft environments and to potentiate the right assemblage of cellular building blocks. Over the years, the pursuit of tooth regeneration and total tooth repair has been one of the main goals of research in regenerative and restorative dentistry. Since the turn of the last century, different biomaterials, including those from marine sources, have emerged as favorable candidates for tissue engineering and regeneration. Although we have discussed how these materials could facilitate tooth repair and regeneration in practical applications, materials and structures built from them do not meet all ideal criteria (including biomechanical, bioresponsiveness, ease of processing and storaging, cell and tissue growing efficiencies) needed for proper biological application (reviewed in Table 1). Marine biostructures are meeting these demands and have other significant advantages over existing biomaterials (Table 1). For example, marine biostructures usually have a hierarchy of built-in. Nevertheless, it is anticipated that with continuous advances in biochemical engineering and the continuous technical advances in materials modification to fine-tune material properties, a window of new opportunities will be opened for tooth regeneration and repair. 3. Limitations and Challenges The tooth organ is made up of an intricate assembly of hard and soft tissues. The softer part is often intercalated within the hardened mineralized tissues. Dentine, enamel, periodontal ligament and bone are structurally dissimilar but are integrated very well with each other. These components are held together with graded interlocking surfaces either with use of embedded fibers or by subtle melding of not-so dissimilar materials inside junctional zones such as the cementum-dentine junction (Figure 10) [179]. At the molecular level, special amphiphilic molecules have been adapted to bond The tooth organ is made up of an intricate assembly of hard and soft tissues. The softer part is often intercalated within the hardened mineralized tissues. Dentine, enamel, periodontal ligament and bone are structurally dissimilar but are integrated very well with each other. These components are held together with graded interlocking surfaces either with use of embedded fibers or by subtle melding of not-so dissimilar materials inside junctional zones such as the cementum-dentine junction (Figure 10) [179]. At the molecular level, special amphiphilic molecules have been adapted to bond 2900 Acknowledgments We would like to thank Associate K. Heimann (James Cook University) for providing us with high quality samples of diatomaceous earth consisting mainly of intact skeletons. Our thanks go to Professor Besim Ben-Nissan for generating microCT images of Foraminifera macrospheres. This study was supported by a grant of the Korea Health Technology R & D Project, Ministry of Health and Welfare, Korea (HI12C02970100). We would like to thank Associate K. Heimann (James Cook University) for providing us with high quality samples of diatomaceous earth consisting mainly of intact skeletons. Our thanks go to Professor Besim Ben-Nissan for generating microCT images of Foraminifera macrospheres. This study was supported by a grant of the Korea Health Technology R & D Project, Ministry of Health and Welfare, Korea (HI12C02970100). Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 with dental stem cells. Owing to the amazing diversity of marine life, there will be more significant discoveries in marine materials and biostructures for future implementation in regenerative dentistry. We envisage this approach to be a bio-based bridge from traditional acellular and non-biological dental biomaterials towards the final objective of a complete biomimetic tissue construct or whole tooth organ. Figure 11. Major application areas for marine biostructures in modern dentistry that cover engineering of entire dental tissues and tissue complexes, disintegration of biofilms, repeat mineralisation of the tooth surface-enamel and shallow dentine defects- and bone substitutes. Acknowledgments We would like to thank Associate K. Heimann (James Cook University) for providing us with high quality samples of diatomaceous earth consisting mainly of intact skeletons. Our thanks go to Professor Besim Ben-Nissan for generating microCT images of Foraminifera macrospheres. This study was supported by a grant of the Korea Health Technology R & D Project, Ministry of Health and Welfare, Korea (HI12C02970100). Acknowledgments 4. Conclusions We have demonstrated how a small selection of structural items from the marine environment can be usefully implemented in regenerative strategies in dentistry to help address common clinical problems, particularly those regarding loss of bone and dentine. These structural products range from small microscopic skeletons to large macroscopic structures. The different applications of marine products in dental tissue engineering have been summarized in Figure 11. Indeed, in addition to their use in dentistry, numerous materials derived from marine sources have been applied in medicine. Thus far, we have appropriated whole structures to create engineered bone, dentine and periodontal ligament tissues. These options are temporary and simple but exhibit potentially good tissue-producing outcomes. The objective is to establish the right environment and conditions for the onset of complete regeneration via a combination of exogenous and endogenous providers. Replicating dentine remains a challenge because the tissues that can be generated produce a reparative style of structure and not the true primary dentine. The periodontal ligament is a more significant challenge because graded joints are necessary on either side for anchorage to the bone and via the cementum layer onto dentine. The entire periodontium complex is an elaborate piece of graded engineering that has not been faithfully emulated artificially, biomimetically or via endogenous routes of action. The point of using natural structures in tissue engineering is to help build competent structural biomaterials that are the same as their natural archetypes or function in a manner that simulates the archetype (jawbone, dentine and periodontium). In these instances, the corals are a faithful mirror image of alveolar spongy bone, whereas the diatom spheres are biomimetic principally in function, striving to provide the right growth factor environment for faithful tissue formation. We have deliberately not looked at the wealth of materials and chemical compounds that could and have served purposes in engineering partial tooth structures. For example, alginates and chitosan have been crafted to engineer pulp tissue replacement 2901 Conflicts of Interest The authors declare no conflict of interest. Mar. 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Radicalization Through the Lens of Situated Affectivity
Frontiers in psychology
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Edited by: Jocelyn J. Bélanger, New York University Abu Dhabi, United Arab Emirates y Jocelyn J. Bélanger, New York University Abu Dhabi, United Arab Emirates Reviewed by: Ana-Maria Bliuc, Western Sydney University, Australia Bjorn Wansink, Utrecht University, Netherlands *Correspondence: Hina Haq hhaq@uni-osnabrueck.de Keywords: radicalization, emotions, situated affectivity, mind invasion, affective scaffolding Radicalization Through the Lens of Situated Affectivity Institute of Cognitive Science, Osnabrück University, Osnabrück, Germany Affective bonding to radical organizations is one of the most prominent features of a recruit’s personality. To better understand how affective bonding is established during the recruitment of youth for radicalization and how it is maintained afterward, it seems promising to adopt new insights and developments from the field of situated cognition and affectivity, particularly the concepts of Affective Scaffolding, Mind Invasion, and Self-Stimulatory Loops of Affectivity (SSLA). The three notions highlight both the intended structuring of the affective bonding by the recruiting organizations and the immersive influence these settings have on the individuals. We will study the affective bonding between an individual and a radical group from two perspectives: first, from an organizational perspective, and second from a personal perspective. The first aims at understanding how extremist organizations “invade the mind” of young people, by providing carefully designed affective scaffolding: (a) during the recruitment process and (b) while being a full member of the organization. The second aims at identifying some of the affective loops which individuals who have joined the radical organization enter. HYPOTHESIS AND THEORY published: 18 February 2020 doi: 10.3389/fpsyg.2020.00205 *Correspondence: Hina Haq Specialty section: This article was submitted to Personality and Social Psychology, a section of the journal Frontiers in Psychology Specialty section: This article was submitted to Personality and Social Psychology, a section of the journal Frontiers in Psychology Received: 09 July 2019 Accepted: 29 January 2020 Published: 18 February 2020 p g There is no simple explanation to why and how a person is radicalized to become a terrorist and suicide bomber. Radicalization is a multi-factor, multi-pathway and complex process (e.g., Taylor and Horgan, 2006; McCauley and Moskalenko, 2008; Sageman, 2008; Schmid, 2013; Hafez and Mullins, 2015). Since 2001, many models and metaphors describing the process of radicalization have been presented by various researchers in (social) psychology, mostly interpreting it as a progression over a period of time and involving different factors and dynamics in individual (lone- actor) and collective settings. Some milestones in the literature on models of radicalization come Keywords: radicalization, emotions, situated affectivity, mind invasion, affective scaffolding INTRODUCTION Reviewed by: Ana-Maria Bliuc, Western Sydney University, Australia Bjorn Wansink, U h U i i N h l d Western Sydney University, Australia Bjorn Wansink, On April 21, 2019, people in Sri Lanka suffered from terror attacks that resulted in the death of 253 people (Safi, 2019). This incident took place after the terrorist group who call themselves the Islamic State of Iraq and Syria (ISIS) lost all its territory in Syria and Iraq, as claimed by the ground forces in March 2019 (Callimachi, 2019). However, ISIS claims responsibility for the Sri Lanka attacks with some Sri Lankan officials initially terming it ISIS’s retaliation of the terrorist attack on the Christchurch Mosques in New Zealand on March 15, 2019 (Miglani and Pal, 2019). Sri Lankan Defense Minister Ruwan Wijewardene stated that most of the terrorists involved in these attacks were well-educated people who stem from middle- or upper-middle-class families, and were financially relatively independent. This statement might speak against what many people commonly believe about suicide bombers, as it seems hard to think of a terrorist as a well-educated and financially stable person with apparently everything to live for, who is deciding to blow themselves up in a suicide attack and kill others (Bergen, 2019). Citation: Such organizations provide their recruits with a well-developed structure and system, which facilitate them to generate positive feelings such as strength, trust, pride, and belongingness within the group, and to generate negative feelings such as hate, anger, and disgust toward an outgroup, to mention the important ones. From the personal perspective, a recruit joining the organization develops an affective bonding with the organization itself. The more that the recruits are exposed to the ideology and objectives of the radical group, and the more they start relating to it, the more their emotions develop in line with the goals of the group. The same holds true for training rituals and other activities within an organization, which gives a further boost to the affective bonding. This bond keeps the recruit in a specific and long-lasting affective state, which leads them toward a deeper involvement in that organization. In sociology, Social Movement Theory (SMT) is an important theoretical framework which is applied to explain radicalization. SMT focuses on broader dynamics and processes of political mobilization leading to radicalization, in which individuals and groups are driven by political goals and rational agents (e.g., Della Porta, 1992; Wiktorowicz, 2005; Dalgaard-Nielsen, 2008; van Stekelenburg and Klandermans, 2010; Beck and Schoon, 2018). Social movement theory, in the context of protest events and campaigns, explain the roles of emotions, which can be generally applied to radicalization and provides an important contribution to understanding emotions in radicalization. Organizations and social movements specifically arouse and use multiple emotions in social networks that play a key role in enabling or inhibiting mobilization for their causes and also sustain a commitment to a cause (e.g., Lofland and Stark, 1965; Khawaja, 1993; Nepstad, 2004; Benski, 2010; Jasper and Owens, 2014; Van Ness and Summers-Effler, 2018). In contrast to already established views that mainly address negative emotions such as humiliation, fear, hate, anger, guilt, contempt, and disgust (e.g., Lindner, 2001; Bar-Tal et al., 2007; Wright-Neville and Smith, 2009; Feddes et al., 2012; Baele et al., 2014; Matsumoto et al., 2015; Baele, 2017; van Stekelenburg, 2017), we would like to also stress the importance of positive emotions, which in combination with negative emotions play a vital role in paving the path to radicalization. Citation: Haq H, Shaheed S and Stephan A (2020) Radicalization Through the Lens of Situated Affectivity. Front. Psychol. 11:205. doi: 10.3389/fpsyg.2020.00205 February 2020 | Volume 11 | Article 205 1 Frontiers in Psychology | www.frontiersin.org Radicalization Through the Lens of Situated Affectivity Haq et al. from Moghaddam (2005), Wiktorowicz (2005), and Silber and Bhatt (2007) who offer different stage models about how an individual becomes radical and eventually violent; Kruglanski (2006) highlights the role of ideology and the “Quest for Significance” (Kruglanski et al., 2009, 2014) and proposes the so-called 3N approach (need, narrative, and network) to understand what drives individuals to radicalization and violent extremism (Webber and Kruglanski, 2017). Furthermore, experts have acknowledged a variety of factors that make people vulnerable toward radicalization as individuals (micro-level), as groups (meso-level) and as societies (macro-level) (e.g., Wiktorowicz, 2005; Sageman, 2008; Kruglanski and Fishman, 2009; Schmid, 2013; Malthaner and Waldmann, 2014). Some of the factors include uncertainty in life (Hogg and Adelman, 2013; Hogg et al., 2013), collective identity problems (Moghaddam, 2012), experiencing alienation (Horgan, 2008; Wilner and Dubouloz, 2010), and grievances stemming from discrimination, stigmatization, rejection, relative deprivation, and humiliation (Moghaddam, 2005; Bindner, 2018). They are usually combined with moral outrage and feelings of revenge (e.g., McCauley and Moskalenko, 2008; Sageman, 2008; Schmid, 2013). Radical groups make use of the factors mentioned above to their advantage in order to influence their followers to build an affective bond with the group and its ideology, which results in mobilizing the recruits to act extremely and violently. above. But we take it as desideratum to investigate particularly the emotional bond between radicalized persons and the groups they belong to, to investigate how such affective bonds between recruits and radical organizations develop and how this supports radicalization, which might prove to be helpful for developing counter radicalization measures.1 Our analysis will help to understand both how radical groups use emotions to influence people and what kind of emotional vulnerability and receptivity makes a person ready to join a radical group and stay committed to it. In that sense, the affective bonds that radical groups develop with their recruits can be accounted for from two main perspectives, that is, an organizational perspective, and a personal perspective. From the organizational perspective, radical organizations structure themselves to provide affective and social platforms for young individuals to connect with the organization and its ideology. Frontiers in Psychology | www.frontiersin.org 2For those already familiar with the framework of situated cognition and affectivity, it might suffice to say that we use the term affective scaffolding instead of “embedded” and “extended affectivity,” the distinction of which we no longer take to be promising in empirical contexts (cf. Stephan, 2018; Stephan and Walter, 2020). Our move is also supported by Kim Sterelny’s claim that the most plausible cases for the extended mind hypothesis are merely “limiting special cases of scaffolded minds,” thus depriving the extended mind paradigm of its heuristic potential, since it “obscures rather than highlights both the continuities and the differences amongst external resources and their contributions to cognitive competence” (Sterelny, 2010, p. 473). Citation: (2018), Delefortrie was raised by an alcoholic and violent mother. He was introduced to Islam by second-generation Moroccans settled in Belgium, having strong family ties. Delefortrie felt it appealing that the religion bans alcohol. He later joined a radical Salafiorganization named “Sharia for Belgium,” where he felt encouraged to undertake complete Salafilifestyle, including that of Jihad. Eventually he traveled to Syria in 2013 to practice Islam. However, he returned to Belgium after spending 5 weeks in Syria to escape battleground and reunite with his wife. After the return, he felt disillusioned with his troubled life in Belgium and expressed his desire to return to Syria in his interview with the authors. Delefortrie idealized the ISIS “Caliphate” and hoped for it to extend to Brussels (Yayla and Speckhard, 2016; Speckhard et al., 2018). His case illustrates how radical groups are successful in instilling hope, pride and ingroup love in their followers in the first place, and creating a strong affective bonding with their recruits by providing affective support which was not available in other societies. The idea of environmental scaffolding,2 which can be traced back to the work of Lev Vygotsky, was reintroduced by Andy Clark to comprise as scaffolds all sorts of environmental structures that can be used for enabling or facilitating certain cognitive tasks (Clark, 1997). In this broad sense, it also applies to various affective processes. We will principally distinguish two different, albeit interconnected, types of affective scaffolding— ways in which our affective life is essentially a matter of more or less intimate dependencies and interdependencies between us and our natural, technological, and social environment. The first type are so-called user-resource interactions; they have been in the center of attention in the early contributions to situated affectivity, the second type is so-called mind invasion (cf. Stephan and Walter, 2020, §§ 3 and 4), which entered philosophy of emotions rather recently, whereas social psychologists studied such phenomena under different terminology before (e.g., Parkinson, 1995). Citation: A radical organization in its ideology provides its followers with various positive emotions such as hope for a better future, pride of belonging to a certain group or religion, feeling of power as being member of a strong and feared group, and love for the radical ideology and those who follow it, fostering a sense of brotherhood among the members of the group. These positive emotions serve as strong pull-factors in recruitment. The manipulation of emotions in different combinations, including both the positive and the negative, by radical groups direct the thoughts and actions of followers and recruits. Therefore, love for the radical group and hate for the outgroup goes side by side (e.g., Brewer, 1999). If we look at the case studies of radicalized individuals, We agree with several of the aforementioned approaches that emotions play an important role in radicalization. In particular, they are key elements in establishing a dynamic affective bonding between a radical group and its followers. It is our goal to better understand how radical groups use a variety of external tools to attract new members, retain them, and direct them to violent actions. To do so we will apply the framework of situated cognition and affectivity (see next section) which is a recent development in cognitive science. In contrast to older (internal) approaches to the human mind it explicitly comprises environmental factors as key components of (cognitive and) affective processes. When we focus on the vital role affectivity plays in the process of radicalization, we do this without downplaying other factors that drive recruitments, such as the psychological, social and political factors mentioned 1This move finds backing also by research in industrial-organizational psychology, where researchers have found that affective commitment, which is the strongest type of bonding, supports an enduring attachment to a company (Allen and Meyer, 1990; Mercurio, 2015). February 2020 | Volume 11 | Article 205 2 Radicalization Through the Lens of Situated Affectivity Haq et al. positive emotions are also at work and may even occur before the rise of negative emotions which lead toward violent actions. Stephan et al., 2014; Colombetti and Krueger, 2015; Colombetti and Roberts, 2015; Wilutzky, 2015; Slaby, 2016). For example, a former fighter for ISIS named Younes (Michael) Delefortrie came back to Belgium from Syria. According to a report on a series of interviews with radicalized individuals by Speckhard et al. Citation: g Among the user-resource interactions, we distinguish cases: (a) with unidirectional material tools for emoting, which are used when we exploit environmental resources in order to regulate our affective life (e.g., visit places that mean something to us, choose music, furnish our apartment)— a strong and relatively permanent example would be the decision to live in a caliphate, Before we start analyzing the recruitment and retention strategies of radical groups, mainly the ISIS and the Taliban, and how members bond to them, we introduce the theoretical framework of situated affectivity, in particular the notions of affective scaffolding, mind invasion, and self-stimulatory loops of affectivity. These three notions help us to understand how environmental structures effect emotions in general, and, in particular, they highlight the affective settings intended by recruiting organizations, and also the immersive influence these settings have on the individuals. (b) with strongly coupled and integrated material tools for emoting, which are used to enact some kind of self- stimulating activity that has been set in place and maintained over time in order to move into a certain emotional process (e.g., a deeply mourning musician whose playing sets up a mutually constraining cycle of affective responding and expression)—many activities taken by members of radical groups afford such kinds of self-stimulatory affective loops (e.g., listening to nasheeds, a form of vocal music that is used to enhance hate toward an outgroup, immersing in social media supporting extreme ideology, engaging in training rituals and learning the use of weapons), Frontiers in Psychology | www.frontiersin.org MIND INVASION IN RADICALIZATION The various types of user-resource interactions, which in the case of radicalization support the affective bonding between a group and its members, present corresponding instantiations of the personal perspective. Often it is the group that provides a variety of possibilities for looping effects that enhance the affective bonding of members to their groups through strong social couplings. In that sense the groups perform various sorts of mind invasion. Slaby (2016) illustrates the concept of “Mind Invasion” with the example of an intern at her new workplace. She has to learn a lot about the activities in her new work environment, how the colleagues interact on different work-related hierarchies, and how they address each other. She learns how showing certain characteristics in the workplace benefits her, and how exhibiting other characteristics does not. She eventually habituates herself to become a professional in her field and progresses to become a full-time employee where she adheres to the workplace culture in a more natural manner. Something which might have felt alien when she first joined the organization now feels natural to her: for example, the long hours at work, continually staying online, answering e-mails even after office hours, feeling anxiety of falling behind on work-related communications even outside the office hours, the ways in which one is expected to show enthusiasm and eagerness for the work, etc. The organization, as Slaby terms it, “hacks the mind” of the new employee, to the extent that she will come to feel as a part of the organization. Her habits and ambitions merge with, and change, according to the organization’s objectives5. SITUATED AFFECTIVITY AND AFFECTIVE SCAFFOLDING In order to understand the influence of environmental structures on individuals and their reciprocal influence, philosophical accounts which are extrapolations of so-called “situated” cognition (Walter, 2014) offer a promising framework. The central thesis of situated cognition is that cognitive processing is not solely an intracranial affair, but that it can be, and often is, supported by extracranial “tools” which ease and simplify cognitive tasks. These tools can be, for example, the morphological and physiological characteristics of our body (embodied cognition), but also our embodied interactions with an appropriately structured natural, technological or social environment (embedded or extended cognition). During recent years, researchers have expanded the debate about situated cognition to the affective domain to investigate to what extent extrabodily factors contribute to our affective life— though not in the trivial sense that most of our emotions are triggered by environmental stimuli in a certain context (e.g., Griffiths and Scarantino, 2009; Colombetti, 2014; Krueger, 2014; (c) (c) with transiently coupled social tools for emoting, which are used when actors choose other people as “tools” for living out or regulating their emotions (e.g., aggressors who provoke others to enter into a fight, confessors who aim at relieving their sorrows through interaction with a priest)— the life of radicals offers several occasions of such a type (e.g., taking conquered women as sex slaves, giving young (c) with transiently coupled social tools for emoting, which are used when actors choose other people as “tools” for living out or regulating their emotions (e.g., aggressors who provoke others to enter into a fight, confessors who aim at relieving their sorrows through interaction with a priest)— the life of radicals offers several occasions of such a type (e.g., taking conquered women as sex slaves, giving young Frontiers in Psychology | www.frontiersin.org February 2020 | Volume 11 | Article 205 3 Radicalization Through the Lens of Situated Affectivity Haq et al. recruits an outlet for aggressive impulses by allowing them to punish prisoners), not even all (h)-cases. As long as the human faculty of critical thinking and distancing from certain positions is not endangered or undermined, we should not treat all kinds of mind invasion as kinds of brain washing. 3Feature (g) affective transformation through social media is not independent from features (f) and (h); it can be part of affective enculturation and it can be used for manipulating people; we introduce it here as a separate feature for analytical reasons. SITUATED AFFECTIVITY AND AFFECTIVE SCAFFOLDING Even if some activities of radical groups can be treated as cases of brain washing, it does not follow that all kinds of mind invasion they provide are instantiations of brain washing; in many interactions with the group we can find volunteering aspects on the side of the members. (d) affectivity instantiated by strongly coupled social systems, when they enact certain types of emotional dynamics over and over again (e.g., old couples enacting cycles of mutual soothing and loving, or cycles of one partner sulking, the other trying to reconcile)—strong social couplings develop both internally among members of radical groups and externally via social media between members of radical groups and followers abroad, We now introduce the notion of mind invasion in more detail by first following Slaby, who coined the term, and then use this perspective to present what extremist groups provide to invade the mind of recruits and members. 4Hereafter, we offer several tools that are used as affective scaffolds by the extremist groups to successfully invade the mind of their targets. (e) with coupled social tools for changing the emotional mind- set, where someone is intentionally looking for another person as a “tool” for modifying her life and mood in order to transform her affective response repertoire in general (e.g., seeking a psychotherapist to work with)—when, for example, someone explicitly volunteers a radical group to get orientation for changing his or her mind and life. 4For those coming from situated affectivity the concept of mind invasion is useful, because it shifts the attention away from the user/resource model and it focuses instead on how the user can be “used” (or “invaded”) by the resource (or scaffold). On the other hand, scholars coming from sociology or psychology (who therefore are familiar with concepts such as enculturation) benefit from mind invasion, because this concept correctly frames the environment as something made of scaffolds and niches. 4For those coming from situated affectivity the concept of mind invasion is useful, because it shifts the attention away from the user/resource model and it focuses instead on how the user can be “used” (or “invaded”) by the resource (or scaffold). On the other hand, scholars coming from sociology or psychology (who therefore are familiar with concepts such as enculturation) benefit from mind invasion, because this concept correctly frames the environment as something made of scaffolds and niches. 5Although what Slaby introduces here as mind invasion, is often referred to as “company socialization” in industrial-organizational psychology (Cooper-Thomas and Anderson, 2006), the term “company socialization” could not easily be extrapolated to cover cases of recruitment and retainment as organized by radical groups. 5Although what Slaby introduces here as mind invasion, is often referred to as “company socialization” in industrial-organizational psychology (Cooper-Thomas and Anderson, 2006), the term “company socialization” could not easily be extrapolated to cover cases of recruitment and retainment as organized by radical groups. MIND INVASION IN RADICALIZATION Through repeated lessons based on the extreme syllabus taught in some of the Madaris, the Taliban intentionally immerse children into an environment that teaches them hatred, anger, and disgust toward the outgroup (those who do not follow their interpretation of religion, and are perceived as threat to the sanctity of the religion). In these environments, a child is introduced to the alphabet letter “hey,” an image of a woman fully covered in black cloth and the word “hijab” along with an illustration of a woman covered completely in black cloths. For the child, the only acceptable way for a woman is when she dressed in a hijab, and any other form of dressing is not acceptable. Along with this kind of radical educational material, the child is also provided with the explanation of how it is sinful for a woman to not properly cover herself, and that she deserves to be punished if she refuses to wear a hijab. Through the Taliban’s interpretation of Islamic law, a woman showing her face to a man who is not related to her is the source of corruption (Gohari, 2000). In a similar manner, the burning image of a guitar in fire is introduced to the child with the word “zunoob” (sin). The child is taught that it is not only bad to listen to or play guitar (or any other kind of music), but rather that the music is a forbidden and punishable sin. This kind of early-age introduction to the radical concepts create a different kind of reality within the minds of children undergoing education in radical organizations. It implies that forcing someone to wear hijab or punishing someone for listening to or playing music is the only acceptable way to live a life. This early enculturation changes the way a child looks at the world. Mind invasion by radical groups is not limited to attract and retain new members. Radical groups spread their ideology and shape vulnerable individuals to adopt the extremist world- view in many ways. For example, in some areas of Pakistan and Afghanistan, the radical group who calls itself Taliban has access to influence people directly via giving religious sermons or running the religious educational institutes, more commonly called Madrassah (pl. Madaris). MIND INVASION IN RADICALIZATION For the letter “hey,” an image of a woman fully covered in black cloth and the word “hijab” is used. For the letter “zey,” the word used is “zunoob” (sin) and the illustration is that of a bonfire made from a pile containing a TV set, a satellite dish, a board game, and a guitar (Paracha, 2011). g For the context of radicalization, it is important to note that environmental structures can be shaped by radical organizations (as tools for mind invasion), to gradually transform the attitudes and the emotional responses of a target group. Accordingly, via social media radical groups attract followers and influence the mindset of individuals [by (g)], to not only join the group but also to be drawn to their more extreme and radical ideas. In that sense, mind invasion facilitates what is described as Identity Fusion (Swann et al., 2009; Paredes et al., 2018), the phenomenon of an individual feeling “fused” or “one” with a group, and it plays a major role in the structuring of recruitment and retention strategies of radical groups. One of such recruitment strategies is called the funnel (Gerwehr and Daly, 2006). As the term indicates, potential recruits are funneled through a carefully structured transformative process (see (e) and (h) above). They emerge from the process as dedicated members of the radical group. This carefully structured and intended process consists of fusing the identity (Swann et al., 2009) of the recruit with the radical group, hazing rituals, commitment to the radical ideology, and support for achieving the desired goals through violence. These processes result in transforming attitudes and radical polarization in those who successfully follow the whole process along the lines as defined by the radical groups. Not all potential recruits follow the whole process. Even if they drop out before they complete the entire process, the minds of the drop-out recruits may still have been invaded to the extent that these recruits will provide benefits to the radical group through, for example, endorsing a positive image of the group among other potential recruits and even navigate others to enter the process of radicalization [according to (f)]. g g Education and curricula based on extreme ideology aim to expose and normalize children to categorical thinking and violence, and results in an us-versus-them mindset. MIND INVASION IN RADICALIZATION Among the cases of mind invasion, we distinguish3: (f) affective enculturation through strongly coupled social systems, which affects all human being in early and later life spans with particular behavior styles, group norms and emotion regimes (e.g., in early childhood, peer groups, companies)—this holds true for everybody who joins a radical group, and particularly for their children, (g) affective transformation through social media by providing possibilities to interact and keep in touch over long distances, but also by spreading hate speeches with all their consequences, or allowing criminal exchanges in the darknet—social media play an important role in the recruitment of new members from abroad, and in directing specific activities including the building of bombs; they also offer lone actors facilities to develop dynamic relationships with other radicals, According to Slaby (2016), organizations structure themselves in a way that influences the affective setup of the individuals working within it; some organizations do this intentionally, while others create this affective environment accidentally. Humans who are inclined to think that they are in pretty good control of their affective life can, therefore, easily underestimate the power of the environment on their emotional repertoire. As it becomes evident from the examples Slaby presents, individuals working (h) tools for manipulating people, which are deliberately launched in order to diachronically modify the attitudes and the emotional set up of the members of a target group (e.g., advertising, political campaigns)—recruitment and retainment of people for extremist groups. In particular, type (h), which corresponds to the organizational perspective, will be relevant for our analysis of the affective scaffolds provided by radical groups. Whereas some very strong instantiations of (h) might result in what is also known as brain washing, not all types of mind invasion are such cases, February 2020 | Volume 11 | Article 205 Frontiers in Psychology | www.frontiersin.org 4 Radicalization Through the Lens of Situated Affectivity Haq et al. within an organizational setup can have their subjectivity hacked through the invasion of their minds by the structuring of the organization. For the letter “tay,” the word used is “takrao” (impact) and an illustration of a plane hitting the Twin Towers in New York is shown. For the letter “jeem,” an image of a white jihadi flag and the word “jihad” is used. For the letter “khay,” an image of a hunting knife (with blood dripping from it) and the word “khanjar” (knife) is used. Frontiers in Psychology | www.frontiersin.org MIND INVASION IN RADICALIZATION One such strategy of ISIS is using different social platforms to create an environment which helps them to spread their ideology [see (g) and (h)]. They invade the minds of young people by showing them a glorified and adventurous face of ISIS and by repeatedly providing its followers glimpses of its ideology through various means, including its mass media propaganda (e.g., Winter, 2015b). The use of social media, nasheeds, and video games, for example, as tools of mind invasion, over time facilitates the transfer of followers from a virtual caliphate to an active recruit either as fighter in ISIS claimed territories or as “lone actor” fighters in other countries. For example, one Birmingham-born ISIS member compared his so-called Jihad in Syria with a famous first-person shooter video game, “Call of Duty”by tweeting; “you can sit at home and play call of duty or you can come here and respond to the real call of duty. The choice is yours” (Kang, 2014). A British foreign fighter of ISIS tweeted that “It’s actually quite fun. It’s really really fun. It’s better than that game Call of Duty. It’s like that but it’s in 3D where everything is happening in front of you” (Klausen, 2015). competition (Vale, 2018). For instance, ISIS regularly publishes a segment under the title of “Hasaad ul Ajnaad (Harvest of the Soldiers)” in its magazines and videos (e.g., Bilger, 2014; Cafarella et al., 2019). Harvest of the Soldiers informs the ISIS followers of the number of successful attacks and operations conducted. These details show the damages and losses they have caused to the outgroup. On the one hand, this propaganda is intended to keep the morale of its followers high by showing how powerful ISIS is. On the other hand, it increases the sense of competition among the recruits in different areas which were controlled by ISIS. By simply publishing the numbers of attacks, ISIS triggers different emotions, like power and pride, which aim at motivating more violent attacks to achieve higher ranks in the within group competition. Another example of reaching out to followers [by (g)] is when ISIS captured a Jordanian pilot on December 24, 2014, and ISIS started a campaign on Twitter to decide how to punish this pilot. MIND INVASION IN RADICALIZATION The pilot was burned alive and run over by a bulldozer in response to an online competition which allowed members, associates, and even passive followers of ISIS to suggest different ways in which the pilot could be executed. Thousands of people used the Arabic Twitter hashtags to suggest that Moaz (the pilot) be cut into pieces by a chainsaw, fed to crocodiles, impaled, set on fire, and even being mutilated using acupuncture needles dipped in acid before being beheaded and having the head sent back to Jordan. A second hashtag carried even more ruthless execution ideas. It was re-tweeted more than 11,000 times (Richards, 2015; The Carter Center, 2015). These tweets with the references of famous video games are reinforced by statements from people who have experienced life under ISIS and describe how adventurous their life seems to be. ISIS uses video games as an external tool to brand itself as being a fun and adventurous organization to join. Through social media postings like these, ISIS members portray the image of the radical group as a powerful and “cool” organization (Vale, 2018). To a young individual looking for orientation, this might look like living the life of a hero participating in a real-life adventure and saving the oppressed from infidels. This also provides a chance to follow an ideology which can transform their prosaic life into a cosmic struggle between good and evil (Kang, 2014). Mind invasion works because it distracts with one thing (for instance, as being a hero) while accomplishing another (that is guiding toward violent radicalization). In addition, ISIS strategically approached young people by providing them a platform to redress the grievances they hold against an outgroup, as well as play their part in building an Islamic community. It provided an opportunity to become a hero and a chance to achieve glory while fighting in a real-life game. With mind-invading strategies like these (e.g., Lakomy, 2019), potential recruits willingly go through the change because of the excitement to be actively part of the action and play the real-time “Call of Duty.” At some point, radicalizable individuals are attracted to radical groups because of the promise of adventure (e.g., Speckhard and Ahkmedova, 2006; Vale, 2018), but the structure of the organization aligns the recruit’s ambitions with the group’s ideology later in the radicalization process. MIND INVASION IN RADICALIZATION This access gives recruiters a platform to directly invade minds during early phases of enculturation (see f) with the extreme ideology and narrative which is based on hatred and anger toward the outgroup. One instance of spreading violence-based ideology was the use of an elementary Urdu language book, which was covertly being used as a part of the syllabus in some local (and low-income) schools in Pakistan. These books were also meant to be sent to Madaris and schools in the north-western region of Pakistan which had some radical ideological presence. The book was published between 2005 and 2010. Unlike the English alphabet books where the letter A is represented by an apple and B by a ball, the content of the elementary Urdu alphabets book (being taught to very young children), however, are all about implanting radical jihadi imagery in the young minds. For example, the word and image used to explain the Urdu alphabet “bey,“ consists of an illustration of a Kalashnikov and the word “bandooq” (gun). The same holds for the concept of Jihad. The radical milieu (Malthaner and Waldmann, 2014) makes it natural for the child to adopt extremist views. When the child grows up, he or she might not join a radical group, but the radical group has already invaded the child’s mind to the extent that he or she accepts and/or supports the extremist ideology. It can also be seen in the children getting education in ISIS territories (e.g., Olidort, 2016). Children who are exposed to this kind of education might not yet be recruits of the radical group, but their lifestyles and ambitions are already developing in alignment with the parameters of the radical group’s ideology (e.g., Al Bayan Center, 2016; Anderson, 2016; NCTV and AIVD, 2017), which raises additional problems for the countries their parents come from, if they return with their mothers or families and join schools in Western countries. Different radical groups use different strategies to shape vulnerable individuals, depending on their organizational structure and aims. Where the Taliban mainly use their easy February 2020 | Volume 11 | Article 205 Frontiers in Psychology | www.frontiersin.org Frontiers in Psychology | www.frontiersin.org 5 Radicalization Through the Lens of Situated Affectivity Haq et al. access to the religious schools and mosques as a tool of mind invasion, ISIS uses more diverse environmental tools to invade the minds of its followers. MIND INVASION IN RADICALIZATION This latter example shows us how ISIS strategically used social media to attract thousands of minds from around the world to participate in a ruthless crime (e.g., Blaker, 2015; Alava et al., 2017). Not all the people who participated in it were radicalized or members of ISIS. However, ISIS appealed to the minds of its followers and recruits by turning a brutal act into an online competition and people participated in dehumanizing another human being without even completely considering what they were doing. The mind invasion performed by the radical group was not limited to their active recruits but also aimed at their sympathizers. Frontiers in Psychology | www.frontiersin.org SPECIFIC TOOLS PROVIDED FOR MIND INVASION The affective scaffolds blocs and groups provide for individuals support the feelings of religious pietism, brotherhood, and belongingness. Radical groups such as ISIS and the Taliban, infuse their group’s ideologies with their interpretation of religion (e.g., Al-Ansari and Hasan, 2018). Such ideologies are easily accepted by their followers and recruits. Any threat toward the radical group is then also considered a threat toward the sanctity of the religion. Therefore, protection of the collective identity against the threat of an outgroup becomes a salient feature of the group and is justified (e.g., Paredes et al., 2018). g g Bloom and Horgan (2019) illustrate instances in which terrorist organizations, specifically those following jihadi narratives, structure themselves to support the culture of martyrdom. Martyrdom is a revered concept among many Muslims in general because it implies the sacrifice of human life for the sake of protection of the religion as a sacred duty and offers honor, opportunities to regain significance, fame and respect (e.g., Bloom, 2005; Güss et al., 2007; Kruglanski et al., 2009; Bélanger et al., 2014; Speckhard and Shajkovci, 2019). In the case of violent jihadi radicals, it explains the motivation of becoming a fighter or volunteering for suicide bombings. The sanctity of martyrdom shows that material incentives do not necessarily matter for all its recruits. For some, the narrative of the highest rank in heaven could be the only spiritual goal. As Bloom and Horgan (2019) report, for younger male suicide bombers, the incentive is of virgins in heaven, along with respect in this world. For younger girls, the incentives based on the jihadi narrative is that they will receive more beauty and will have seventy of their closest relatives join them in heaven, thus allowing the feeling of helping their loved ones and family in the afterlife. On the material level, the recruits are provided with incentives for playing their part for the organization. After the “caliphate” claim, the ISIS leader in June 2014, called out to Muslim professionals such as doctors, engineers, and people with military and administrative expertise to join ISIS (Site Intelligence Group, 2014). Some joined ISIS in response to this call, with the intention of helping Muslims in need there. Others did not join because of the ideology, but rather because of job opportunities (Harissi, 2015). SPECIFIC TOOLS PROVIDED FOR MIND INVASION As indicated in the preceding section, radical organizations use multiple recruitment mechanisms: potential members are sometimes radicalized individually, and sometimes in groups. In this section, we highlight three important ways in which ISIS uses ideological appeals, social appeals, and material appeals as affective scaffolds to invade and radicalize vulnerable minds. While training, new recruits are exposed to literature and videos that paint a vivid picture of Muslim suffering and humiliation by the hands of infidels [see (f)]. The perceived narrative of humiliation by the outgroup (infidels) aggregates the emotions of anger and contempt in recruits, and shames them into taking revenge for their Muslim brothers (Hafez and Mullins, 2015). On an ideological level, radical organizations provide their followers with a strong narrative appealing to the vulnerabilities of the potential recruits (Macnair, 2018; Macnair and Frank, 2018). The radical organization almost always use narratives as affective scaffolds to attract its followers. In places where there is a sense of deprivation, radical organizations almost always present and propagate the narrative of religion in their lands Another strategy which ISIS uses to shape the minds of its current and potential recruits is by evoking the sense of February 2020 | Volume 11 | Article 205 Frontiers in Psychology | www.frontiersin.org 6 Radicalization Through the Lens of Situated Affectivity Haq et al. and among themselves, mixing it with the political narrative of their own. The narrative which radical organizations present is one that is familiar and extremely trusted among the followers because of its sacred value derived from ancient religious roots (Atran and Axelrod, 2008; Ginges et al., 2009). Therefore, many are receptive to this narrative and are also easily manipulated by it (Boutz et al., 2019). Muslims under the sharia law and the protection of Islam against the threat of the outgroup. It should be noted that the calls to join ISIS through its propaganda were not only the narrative of hate and fight against the outgroup, but also the promise of living in a land that will be based on (their interpretation of) the Islamic principles. The organization provides its followers with a trusted and familiar picture of the law set up in their “claimed” lands. The narrative also paints the picture that during the rule of ISIS, individuals would be able to design their Islamic lifestyle, not only on the individual level but also on a collective level (Winter, 2015a). SPECIFIC TOOLS PROVIDED FOR MIND INVASION In the case of ISIS, once it declared itself a “caliphate” in June 2014, it attracted many followers, not only from Iraq and Syria, but also from abroad (e.g., Soufan Group, 2015). ISIS focused on increasing its influence by gaining territory and by calling on Muslims to perform “Hijrah” (migration) to join their newly established caliphate, where sharia law would be practiced (Schmid, 2015). The concept of “Hijrah” is sacred for many Muslims because of its religious significance and history. Therefore, the invitation of calling the Muslims to perform Hijrah held considerable appeal and had aroused the spiritual feelings in not only the followers of ISIS but also those who wished to live under the Islamic law and saw Hijrah to ISIS “caliphate” as a religious obligation (Bin Sudiman, 2017). According to Hogg and Adelman (2013), radical groups provide the vulnerable and potential recruits with a particular identity and give the recruits meaning to their lives. Radical groups lessen the uncertainty of potential followers by telling them how they would feel and behave within the group, and toward the outgroup. The group also instructs on what they should think and how they should retaliate if the ideology becomes threatened (Hogg and Adelman, 2013; Ugarriza and Craig, 2013). We now move on to explain how affective scaffolding is established and used to invade minds on the social level. Radicalization requires a feeling of familiarity, trust, and commitment between the radical organization and its recruits, as it supports clear communication and emotional shaping. The most secure source for recruitment of radical members is from their own families, friends, and like-minded activists within already existing setups (Hafez, 2016), also termed as “Kinship Recruitment.” According to Hafez, kinship recruitment links individuals who share similar beliefs, which helps in creating an immediate collective identity. Kinship recruitment provides the familiar and trusted atmosphere for both the recruiter and the potential-recruit, in which both can attain their desired affective states [see (b) and (d)]. Kinship recruitment also leads to the formation of a bloc within a recruited group, known as bloc recruitment, which refers to the recruitment of multiple individuals from the same family or group. The bloc serves another advantage to the radical organization because it makes it easier for individuals within the bloc to accept radical ideas. We stress again that the affectivity in radicalization is not necessarily based only on violent emotions. Frontiers in Psychology | www.frontiersin.org SPECIFIC TOOLS PROVIDED FOR MIND INVASION According to Anderson (2016), ISIS provides its fighters with salaries and other incentives, such as supplies, gasoline, and women to marry, which helps the organization to retain its recruits and boost their morale. Children are offered gifts and toys as compensation for their loyalty to the group. There were even reports by locals of earning a salary as high as $200 per month for child recruits, which offers the family In the communities where there is chaos because of war or violence, radical groups can provide potential recruits with order and identity. Taking control of the caliphate by ISIS provides its potential recruits with an environment (territory) which facilitates and nurtures the recruits’ religious and spiritual feelings. The new caliphate claim provides its followers with the hope of getting their existential needs answered (e.g., Cottee and Hayward, 2011; McBride, 2011). The caliphate allows a unity of February 2020 | Volume 11 | Article 205 7 Radicalization Through the Lens of Situated Affectivity Haq et al. the opportunity at a better quality of life in the war and violence-torn country. These monetary incentives were usually distributed during events where there were booths displaying and distributing ISIS propaganda (Anderson, 2016). anger, contempt, and disgust toward the outgroup. Within ISIS environment, the recruits (including women and children) are assigned specific roles (e.g., De Leede et al., 2017; Almohammad, 2018; Darden, 2019). The more effectively they perform their duties (e.g., executing prisoners), the more authority and power they can get, and the more empowered they feel. This SSLA pattern becomes reinforced and this loop of power results in deeper involvement of members within the organization. Bloom and Horgan (2019) illustrate other strategies that utilize incentives to radicalize and recruit. One strategy used by ISIS is to make membership for children look desirable and build an atmosphere of competition for member status. Based on the competition, the children were presented with new uniforms and their pictures taken for the propaganda. For adult recruits in ISIS, women were used as a commodity. Women who pledge their allegiance to ISIS are used as potential wives for local and foreign fighters, the purpose of which was to recruit, reward and retain the fighters. SELF-STIMULATORY LOOPS OF AFFECTIVITY IN RADICALIZATION ISIS and its leaders encourage isolation of its members from those who oppose the views of the organization and make an effort to deepening the commitment of their members to the organization. The leaders discourage their recruits to access the mass media of the “outgroup” as well, which, for its members, is the propaganda. The members have access to the materials provided solely by ISIS itself, which keeps certain affective states in a loop, such as anger, hate, and disgust [see (h)]. Web technologies provide a perfect platform for the members of ISIS to design their newsfeeds, not only as affective scaffolds but also as SSLA. The news shared by ISIS and their followers show the suffering of Muslims all over the world, creating further hate, anger, contempt, and disgust toward the outgroup. The more they follow such news, the more negative emotions they feel. ISIS uses their elevated negative emotions to help its recruits to plan more violent attacks against the outgroup. This material also motivates the recruits to become heroes of the religion (Hafez and Mullins, 2015). Self-stimulatory loops of affectivity (SSLA) refer to strong interactions between individuals and their resource [see (b) and (d)], which, when maintained over some time and place, evolve into a form of self-stimulatory activity. The idea of the self- stimulatory loop was coined by Clark (2008) with reference to cognitive tasks. Stronger types of affective individual-resource interactions have been discussed by Colombetti and Roberts (2015). Most examples discussed thus far in the literature relate, however, a user with some technical device as, for example, a musical instrument. It is important to see that strong couplings between users and resources do not only exist toward technical devices, but can also be established with other human beings, or—in the case of mind invasion—with devices, practices, etc., provided by a mind-invading group. To see how self-stimulatory affective loops also contribute to the recruitment of new members to radical organizations, let us look at bloc recruitment. If some individuals of a particular family make a commitment to an ideology, it facilitates the commitment of other members of the same family to follow. The elements that are self-stimulating in the bloc are peer pressure within the bloc, group-thinking, the desire to maintain relationships, and feelings of guilt for staying behind (Hafez and Mullins, 2015). SPECIFIC TOOLS PROVIDED FOR MIND INVASION Women who were captured and did not pledge their allegiance to ISIS are used as sex slaves, and are considered to be “spoils of war.” These incentives serve as scaffolds for the recruits to voluntarily join the radical ideology. Radical organizations also provide the platform which helps to keep the concerned emotions of its recruits within the SSLA. Habitual development of rituals, routines, and training also result in specific affective states for the recruits. According to the propaganda materials by ISIS, the routine of a fighter includes dedicating time to prayers, physical fitness, and activity, learning from religious texts, as well as learning combat and warfare techniques. This routine and training cycle keep the emotions of recruited fighters elevated throughout their attachment with the ISIS. The above examples illustrate how affective bonds are deliberately created by the organization, and what particular scaffolds they use for being successful. Ideological, psychological, and material incentives and motivations created by ISIS, in our view, are directly aimed at building trust and familiarity among its followers, and at the same time providing a sense that the individuals are rewarded by the organization, thus forging a bond with its recruits and followers. In the next section, we analyze how the members of ISIS form and maintain loops of affectivity that support to keep themselves in their desired affective states within the organization, thereby focusing more on the personal perspective. The “bridge-burning” practice, such as recruits burning their government-issued passports to signify loyalty and allegiance to ISIS, is also an instance of SSLA. This diminishes the chance of deserting the group (Hafez and Mullins, 2015). Another instance of bridge-burning, as reported by Hafez and Mullins (2015), is the camera-recorded declaration of the intention to launch an attack. By declaring allegiance to a group and intention of carrying out an act of violence, the recruits bind themselves to such an act and feelings associated with it. If a recruit backs off, he is considered a traitor and has to face humiliation among his comrades. Bridge-burning practices, therefore, keep the recruits in a specific SSLA and prevent recruits from entertaining any thoughts of leaving ISIS. Such practices and rituals also support the emotions that the recruits are all-in and prepared to do anything for the protection of ISIS. Frontiers in Psychology | www.frontiersin.org SELF-STIMULATORY LOOPS OF AFFECTIVITY IN RADICALIZATION propaganda is the use of numbers in the newsletters and videos, specifically the “Hasaad ul Ajnaad (Harvest of the Soldiers)” reports which indicate the attack metrics of ISIS, e.g., they tell which subgroup killed how many infidels (Bilger, 2014; Cafarella et al., 2019). This report not only serves as mind invasion but also as SSLA of competition, designed to motivate its followers, inspire them and keep the fear elevated among the outgroup. propaganda is the use of numbers in the newsletters and videos, specifically the “Hasaad ul Ajnaad (Harvest of the Soldiers)” reports which indicate the attack metrics of ISIS, e.g., they tell which subgroup killed how many infidels (Bilger, 2014; Cafarella et al., 2019). This report not only serves as mind invasion but also as SSLA of competition, designed to motivate its followers, inspire them and keep the fear elevated among the outgroup. For lone actor terrorists, SSLAs can also develop as they intentionally subscribe to virtual environments projecting hate, anger, and disgust toward an outgroup. Virtual environments, facilitated by virtual networking with other extremists in chat rooms, such as that of ISIS’s “virtual caliphate” specifically in the Telegram social network website, can ultimately lead to mobilization of members toward radicalization (Bloom et al., 2019). We find similar patterns in right wing terrorism, where affectively structured virtual environments lead vulnerable minds toward developing loops of negative emotions, which eventually lead them toward violent actions, such as Tarrant’s attack in New Zealand on March 15, 2019. An example of Bangladeshi immigrant Roshonara Choudhry, from the report of Speckhard and Shajkovci (2018), shows how SSLA works in so called lone actors. She was a student at Kings College London. She came across the sermon of Anwar al Awlaki (Yemeni-American preacher and recruiter for Al-Qaeda) on internet [see (g)] and she started relating to Awlaki’s radical ideas of individual Jihad and his anger against United States coalition’s invasion of Iraq. She actively searched for his videos and started withdrawing from the “unbelievers” around her [see (e)]. She developed her own affective loop of hate against infidels and how western countries are discriminating Muslims. She even quit university on the claim that one of her school departments is wrongly supporting Israeli brutality against Palestine. After conducting research on which local parliamentarians voted for the Iraq war, she performed her “jihad” by stabbing Labor MP Stephen Timms in his office, twice. FUNDING The careful manipulation of environmental tools by radical groups to generating customized SSLAs explain how recruits are retained in the group or are inclined toward the group’s ideology, while maintaining the desired emotional state which helps them to form a strong affective bond with the group and its ideology. The article was supported by the DFG Research Training Group Situated Cognition (GRK 2185/1). ETHICS STATEMENT Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. SELF-STIMULATORY LOOPS OF AFFECTIVITY IN RADICALIZATION This example shows how a person developed her own SSLA. She related to the sermons of Awlaki urging revenge, hate and anger against so-called enemies. She on purpose started avoiding people around her to feed her loops of anger and hate in isolation to the extent that it led her to attempt murder. In this particular case, none of the radical organizations was involved, nor was she contacted by any terrorist organization or their members. In many other lone actor cases, however, radical groups are in contact with the actors, and use media as an environmental tool to spread their messages and manipulate vulnerable minds to act according to the group’s ideology. They invade the minds to the extent that the person develops the desired affective loops own their own. AUTHOR CONTRIBUTIONS All authors contributed to outline this contribution and were in charge of subsequent revisions. HH and SS wrote the manuscript, except the section “Situated affectivity and Affective Scaffolding,” which was written by AS. SELF-STIMULATORY LOOPS OF AFFECTIVITY IN RADICALIZATION Another resource provided by ISIS is the propaganda material published and aired from time to time, including its newsletters, videos, and audio recordings in the form of speeches of leaders and nasheeds (Islamic hymns). These evoke the hope of living in a caliphate and a desire to help those who are already living there. The propaganda glorifies martyrdom and is also focused on the dehumanization of the outgroup. An interesting aspect in this When recruits join ISIS, they enter an atmosphere of specific emotions such of love and hope within the group; and hatred, February 2020 | Volume 11 | Article 205 8 Radicalization Through the Lens of Situated Affectivity Haq et al. To understand radicalization, it is important to understand how radical groups or radical organizations manipulate affective aspects of human nature and convince their recruits to willingly commit brutal and violent acts of terrorism. This manipulation of human emotions is not restricted to negative emotions such as anger, disgust, but also includes positive emotions, including power, love, feelings of belongingness. During the process of radicalization, different emotions are addressed in a recruit at the same time; for instance, hate toward an outgroup and love for the ingroup fostering the feelings of belongingness and power within the radical group. Such emotions evolve with time and create a very complex emotional web. In this paper, we have tried to provide the first glimpse of how the framework of situated affectivity (especially the notions of affective scaffolding, mind invasion, and self-stimulatory loops of affectivity) can help us to understand the affective structuring of radicalization. The framework of situated affectivity highlights how radical groups provide affective scaffolding, work on the minds of their recruits, and help recruits to develop certain emotions, which deeply entrench individuals into the ideology of a radical group. The framework of situated affectivity provides a broader range of view on the affective aspects involved in radicalization. It also helps us to understand how radical groups create an environment around their potential recruits that determines who and what these potential recruits will be when they go through continuous, reciprocal, dynamical interactions with the radical groups and its affective environment. Detailing the affective processes give us an opportunity to target those environmental factors and tools which radical organizations are using to lure vulnerable minds toward the violent extremism, and helps in developing counter radicalization measures. Frontiers in Psychology | www.frontiersin.org REFERENCES Cafarella, J., Wallace, B., and Zhou, J. (2019). ISISI’s Second Comeback: Assessing the Next ISIS Insurgency. Washington, DC: Institute for the Study of War (ISW). Al Bayan Center, (2016). The Cubs of the Caliphate: How the Islamic State Attracts, Al Bayan Center, (2016). The Cubs of the Caliphate: How the Islamic State Attracts, Coerces and Indoctrinates Children to its Cause. 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CONCLUSION The authors would like to thank the two reviewers for their very helpful and constructive feedback, and Karsten Müller and members of the Reading Club “Affectivity” of the Institute of Cognitive Science at Osnabrück University for their help. Radicalization is a multi-factor process, and one of the most important of these factors is the affective aspect of radicalization. February 2020 | Volume 11 | Article 205 Frontiers in Psychology | www.frontiersin.org 9 Radicalization Through the Lens of Situated Affectivity Haq et al. REFERENCES From profiles to pathways and roots to routes: perspectives from psychology on radicalization into terrorism. Ann. Am. Acad. Polit. Soc. Sci. 618, 80–94. doi: 10.1177/0002716208317539 Brewer, M. B. (1999). The psychology of prejudice: ingroup love and outgroup hate? J. Soc. 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Psychol. 5, 241–263. doi: 10.1007/s13164-013- 0167-y Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Webber, D., and Kruglanski, A. W. (2017). “Psychological factors in radicalization: a “3 N” approach,” in The Handbook of the Criminology of Terrorism, eds G. LaFree, and J. D. Frontiers in Psychology | www.frontiersin.org REFERENCES Freilich, (Hoboken, NJ: John Wiley & Son), 33–46. doi: 10.1002/9781118923986.ch2 Copyright © 2020 Haq, Shaheed and Stephan. 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. Wiktorowicz, Q. (2005). Radical Islam rising: Muslim extremism in the West. Lanham, MD. Rowman & Littlefield. Wilner, A. S., and Dubouloz, C.-J. (2010). Homegrown terrorism and transformative learning: an interdisciplinary approach to understanding radicalization. Glob. Change Peace Secur. 22, 33–51. doi: 10.1080/ 14781150903487956 February 2020 | Volume 11 | Article 205 Frontiers in Psychology | www.frontiersin.org 12
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Correspondence: Guofeng Zhu (zhugf@nwnu.edu.cn) Correspondence: Guofeng Zhu (zhugf@nwnu.edu.cn) Received: 16 July 2021 – Discussion started: 5 August 2021 Revised: 19 April 2022 – Accepted: 28 June 2022 – Published: 19 July 2022 Received: 16 July 2021 – Discussion started: 5 August 2021 Revised: 19 April 2022 – Accepted: 28 June 2022 – Published: 19 July 2022 Received: 16 July 2021 – Discussion started: 5 August 2021 Revised: 19 April 2022 – Accepted: 28 June 2022 – Published: 19 July 2022 Abstract. The processes of water storage have not been fully understood in different vegetation zones of mountainous ar- eas, which is the main obstacle to further understanding hy- drological processes and improving water resource assess- ments. To further understand the process of soil water move- ment in different vegetation zones (alpine meadow (AM), coniferous forest (CF), mountain grassland (MG) and decid- uous forest (DF)) of mountainous areas, this study monitored the temporal and spatial dynamics of hydrogen- and oxygen- stable isotopes in the precipitation and soil water of the Xiy- ing River basin. The results show that the order of soil wa- ter evaporation intensities in the four vegetation zones was MG (SWLslop: 3.4) > DF (SWLslop: 4.1) > CF (SWLslop: 4.7) > AM (SWLslop: 6.4). The soil water in the AM and CF evaporated from only the topsoil, and the rainfall in- put was fully mixed with each layer of soil. The evapora- tion signals of the MG and DF could penetrate deep into the middle and lower layers of the soil as precipitation quickly flowed into the deep soil through the soil matrix. Each vege- tation zone’s water storage capacity of the 0–40 cm soil layer followed the order of AM (46.9 mm) > DF (33.0 mm) > CF (32.1 mm) > MG (20.3 mm). In addition, the 0–10 cm soil layer has the smallest soil water storage capacity (AM: 43.0 mm; CF: 28.0 mm; MG: 17.5 mm; DF: 29.1 mm). This work will provide a new reference for understanding soil hy- drology in arid headwater areas. Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 https://doi.org/10.5194/hess-26-3771-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Evaporation, infiltration and storage of soil water in different vegetation zones in the Qilian Mountains: a stable isotope perspective Guofeng Zhu1,2, Leilei Yong1,2, Xi Zhao1,2, Yuwei Liu1,2, Zhuanxia Zhang1,2, Yuanxiao Xu1, Liyuan Sang1,2, and Lei Wang1,2 1College of Geography and Environment Science, Northwest Normal University, Lanzhou 730070, Gansu, China 2Shiyang River Ecological Environment Observation Station, Northwest Normal University, Lanzhou 730070, Gansu, China 2 Study area The Xiying River originates from Lenglongling and Kawazhang in the eastern Qilian Mountains (101◦40′47′′– 102◦23′5′′ E, 37◦28′22′′–38◦1′42′′ N) (Fig. 1). As the largest tributary of the Shiyang River, it is formed by the Shuiguan River, Ningchang River, Xiangshui River, and Tatu River converging from southwest to northeast and ultimately flow- ing into the Xiying Reservoir. The average annual runoff of the Xiying River is 388 million m3, which is mainly re- plenished by mountain precipitation and melting water of ice and snow. The runoff is mainly concentrated in summer. The basin elevation is between 2000 and 5000 m, corresponding to a temperate semi-arid climate with strong solar radiation, a long sunshine time, and a large temperature difference be- tween day and night. The average annual temperature of the basin is 6 ◦C, the annual average evaporation is 1133 mm, the annual average precipitation is 400 mm, and the precipitation from June to September accounts for 69 % of the annual pre- cipitation. Precipitation increases with elevation, while tem- perature decreases with elevation in this area (Table 1) (Ma et al., 2018). The zonal differentiation of vegetation in the basin is dominated by DF, MG, cold temperate CF and AM. The soils mainly include lime, chestnut, alpine shrub meadow and desert soil (Fig. 1). In alpine mountains, climate warming has accelerated the melting of glaciers and frozen soil, and the dynamic interac- tion between water bodies stored in different media has be- come the main influencing factor of the water cycle (Penna et al., 2018). Interactions between precipitation and the soil– plant–atmosphere system determine the distribution of water in various storage reservoirs and the subsequent release of water vapor to the atmosphere. These interactions mainly in- clude interception, throughfall, canopy drip, snow accumula- tion and ablation, infiltration, surface and subsurface runoff, soil moisture and the partitioning of evapotranspiration be- tween canopy evaporation, transpiration and soil evapora- tion. As the main links of the hydrological cycle, these pro- cesses have a profound impact on regional water balance and distribution. In the past, studies on the evaporation, infiltration and storage of soil water mostly focused on vegetation types in the same climatic region or different climatic regions. G. Zhu et al.: Evaporation, infiltration and storage 3772 2015; Sprenger et al., 2016, 2017). Evaporation mainly oc- curred in the near-surface part of the soils (0–10 cm), and the light isotope molecules (1H and 16O) evaporated prefer- entially, resulting in the enrichment of heavy isotopes (2H and 18O) on the soil surface (Ferretti et al., 2003). Dansgaard (1964) proposed the concept of deuterium excess, d-excess (d-excess = δ2H-8δ18O), to illustrate the intensity of evap- oration fractionation. Assuming that evaporation occurs in the atmosphere with a humidity of 75 %, it shows that the d-excess value of atmospheric moisture accounts for the d- excess value of 10 ‰ in the atmospheric moisture, which conforms to the worldwide average isotopic labeling of me- teoric waters. Landwehr and Coplen (2006) defined line- conditioned excess (lc-excess) as the difference between the δ2H value of the water sample and the δ18O linear transform value of the same sample, where the linear transformation reflects the relevant referenced meteoric water relationship. Compared with d-excess, lc-excess can explain the evapo- rative fractionation process better. The main reason is that lc-excess of precipitation and soil water changes smoothly and has relatively small seasonal changes (Landwehr et al., 2014). The dynamic changes of isotopes record the signal of soil water evaporation. This enrichment of this dynamic frac- tionation exists in soil water isotopes in different climatic regions. Compared with temperate regions, the evaporation signals in arid and Mediterranean environments penetrate deeper into the soil (Sprenger et al., 2016). After evapora- tion and seepage, some water is stored in the soil. The water storage capacity in humid areas is higher than that in arid ar- eas, that in forest is higher than that in grassland, and that in the surface soil layer is lower than that in deep soil lay- ers with high clay content (Kleine et al., 2020; Milly, 1994; Snelgrove et al., 2021; Sprenger et al., 2019). cle in source areas. In this study, we monitored the stable isotope composition of precipitation and soil water and the spatiotemporal dynamics of soil water storage in four vege- tation zones (alpine meadow (AM), coniferous forest (CF), mountain grassland (MG) and deciduous forest (DF)) at dif- ferent temperatures and humidities in the Xiying River basin. G. Zhu et al.: Evaporation, infiltration and storage To explore the differences in soil water evaporation, infiltra- tion, and storage processes in these four different climates, vegetation types and terrain types, the following research ob- jectives were proposed: (1) to explore the evolution of iso- tope evaporation signals and the “memory effects” of precip- itation input, mixing and rewetting; (2) to understand the soil water storage capacity and influencing factors of four vege- tation areas in mountainous areas. We hope this study can further improve the understanding of the water cycle pro- cess and provide a scientific theoretical reference for water resource utilization and ecological restoration in fragile en- vironments. More importantly, it can provide paradigms for research at different spatial scales (latitude zone, longitude zone, watershed, etc.) based on the knowledge of soil mois- ture evaporation, infiltration, and water storage in mountain vegetation zones. 1 Introduction In arid inland river basins, climate and vegetation changes will affect the hydrological cycle (Sharma et al., 2021; Tet- zlaff et al., 2013). As an essential part of the water cycle, soil water in the unsaturated zone can be converted from precip- itation into the stream or groundwater recharge. Determin- ing soil water evaporation, infiltration and storage properties are critical to understand the regional hydrological cycle and water balance under climate and vegetation changes (Brooks et al., 2010; Dubbert and Werner, 2019; Grant and Dietrich, 2017). As “fingerprints” of water, isotopes have been used to track ecohydrological characteristics, such as evaporation (Barnes and Allison, 1988; Zhu et al., 2021b), groundwa- ter recharge (Koeniger et al., 2016), infiltration paths (Du- vert et al., 2016; Tang and Feng, 2001; Zhu et al., 2021a), evapotranspiration distribution (Gibson et al., 2021; Xiao et al., 2018), and the water absorption by plants (Rothfuss and Javaux, 2017). Water seepage in the unsaturated soil zone and water evap- oration at the air–soil interface are the primary forms of soil water transport. The dynamic water process reflected by the displacement of the isotope signal on the soil pro- file is called the “memory effect”. Understanding the “mem- ory effect” will help us to trace the dynamic changes in cli- mate and soil hydrology (Kleine et al., 2020). The change of stable isotopes in near-surface soil water may reflect the precipitation variation, but these variations decrease with depth unless there is preferential flow (Peralta-Tapia et al., Published by Copernicus Publications on behalf of the European Geosciences Union. Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 3.2 Sample determination The analysis of δ2H and δ18O values of all the above water samples was completed using a liquid-water isotope analyzer (DLT-100, Los Gatos Research, USA) in the stable isotope laboratory of Northwest Normal University. Before analyz- ing the isotope values of soil water, the soil water was ex- tracted from the collected soil samples by a low-temperature vacuum condensation system (LI-2100, LICA United Tech- nology Limited, China). Both the water and isotope standard samples were injected six times during the analysis. To avoid the “memory effect” of isotope analysis, we discarded the first two injection values and used the average value of the last four injections as the final result (Penna et al., 2012; Qu et al., 2020). The analysis results were relative to the Vienna Standard Mean Ocean Water (VSMOW): – Collection of soil samples. Soil samples were collected once a month at depths of 0–10, 10–20, 20–30, 30– 40, 40–50, 50–60, 60–70, 70–80, 80–90 and 90–100 cm from the soil layers in the four vegetation zones. Three duplicate samples were collected for each soil layer. We placed the collected soil sample into a 50 mL glass bot- tle, sealed the bottle mouth with Parafilm and marked the sampling date. We froze the sample for storage until experimental analysis. Each sample was collected sepa- rately in an aluminum box. – Collection of soil samples. Soil samples were collected once a month at depths of 0–10, 10–20, 20–30, 30– 40, 40–50, 50–60, 60–70, 70–80, 80–90 and 90–100 cm from the soil layers in the four vegetation zones. Three duplicate samples were collected for each soil layer. We placed the collected soil sample into a 50 mL glass bot- tle, sealed the bottle mouth with Parafilm and marked the sampling date. We froze the sample for storage until experimental analysis. Each sample was collected sepa- rately in an aluminum box. δ =  Rsample Rstandard −1  × 1000‰, (1) – Collection of precipitation samples. The precipitation samples were collected by a plastic funnel bottle device. After each precipitation event, the collected precipita- tion samples were immediately transferred to an 80 mL high-density polyethylene bottle, and the bottle mouth of the samples was sealed with Parafilm; these samples were also frozen and stored until experimental analysis. 2 Study area Un- derstanding the climatic and hydrological conditions of dif- ferent vertical vegetation zones, and clarifying the regulat- ing role of vegetation in the water cycle, can help to better adapt to climate change’s influences on the hydrological cy- https://doi.org/10.5194/hess-26-3771-2022 Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 3773 G. Zhu et al.: Evaporation, infiltration and storage Figure 1. Study area and location of sampling points. (a) The location of the Xiying River basin in China. (b) The terrain and sampling points of the Xiying River basin. Figure 1. Study area and location of sampling points. (a) The location of the Xiying River basin in China. (b) The terrain and sampling points of the Xiying River basin. 3.1 Sample collection In this study, soil water and precipitation samples were col- lected from four vegetation zones in the Xiying River basin from April to October in 2017 (plant growing season). In 2017, the precipitation in AM, CF, MG and DF were 595.1, 431.9, 363.5 and 262.5 mm, respectively. The average daily temperature in AM, CF, MG and DF were −0.19, 3.34, 6.6 and 7.9◦, respectively (Table 1). 3 Sampling design and analysis – Meteorological data. During the sampling period, the local meteorological data were obtained and recorded by automatic weather stations (Watchdog 2000 series weather stations) set up near the sample plot. – Meteorological data. During the sampling period, the local meteorological data were obtained and recorded by automatic weather stations (Watchdog 2000 series weather stations) set up near the sample plot. 3.3.1 Lc-excess The linear relationship between δ2H and δ18O in precipi- tation and soil water is defined as the local meteoric water line(LMWL) and soil water line (SWL), respectively, which are of great significance for studying the evaporative frac- tionation of stable isotopes during the water cycle. We further calculated the lc-excess for each soil water and precipitation sample. The lc-excess in different water bodies can charac- terize the evaporation index of different water bodies relative to the local precipitation (Landwehr and Coplen, 2006): S = R × W × H × 10, (4) (4) where S is the soil water storage in a certain thickness layer (mm), R is the soil bulk density (g cm−3) and H is the soil thickness (cm). The gravimetric water content, W, is ex- pressed by the following formula: W = M1 −M2 M2 × 100%, (5) (5) lc-excess = δ2H −a × δ2H −b, (2) (2) where M1 is the gravimetric value of wet soil (g) and M2 is the gravimetric value of dry soil (g). where a and b are the slope and intercept of LMWL, respec- tively, and δ2H and δ18O are the isotopic values of hydro- gen and oxygen in the sample. The physical meaning of lc- excess is expressed as the degree of deviation of the isotope value in the sample from LMWL, indicating the nonequilib- rium dynamic fractionation process caused by evaporation. Generally, the change in lc-excess in local precipitation is mainly affected by different water vapor sources, and the an- nual average is 0. Since the stable isotopes in soil water are enriched by evaporation, the average lc-excess is usually neg- ative (Landwehr et al., 2014; Sprenger et al., 2017). 3.3 Analysis method Soil water storage is the thickness of the water layer formed by all the water in a certain soil layer (Milly, 1994) and is expressed by the following formula: 3.2 Sample determination (1) where Rsample is the ratio of 18O / 16O or 2H / 1H in the sample and Rstandard is the ratio of 18O / 16O or 2H / 1H in VSMOW. The test error of the δ2H value does not exceed ±0.6 ‰, and the test error of the δ18O value does not exceed ±0.2 ‰. https://doi.org/10.5194/hess-26-3771-2022 Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 4.1 Hydrological climate The PET and runoff are important indicators that reflect the dry–wet conditions of river basins. During the study period (April–October 2017), in the Xiying River basin, PET was 872.8 mm. The daily evapotranspiration ranged from 7.5 mm (14 July) to 0.9 mm (9 October), showing a fluctuating trend around July, and the PET value in April–July was higher than that in August–October. The input of summer precip- itation and ice/snow meltwater increased runoff, resulting in a trend similar to PET. During the observation period, the total runoff was 3.1 × 109 m, accounting for 89 % of the annual runoff. The variation range of the daily runoff was 286 848 m3 (17 April) to 6 125 760 m3 (13 July). The basin before July was drier than that after July (Fig. 2). G. Zhu et al.: Evaporation, infiltration and storage 3774 Table 1. Basic data of each vegetation zone from April to October 2017 (Longitude (Long), latitude (Lat), altitude (Alt), air temperature (T , daily mean temperature), precipitation (P , total precipitation during the observation period), relative humidity (h, daily mean relative humidity)). Vegetation zone Geographical parameters Meteorological parameters Number of samples Long (◦E) Lat (◦N) Alt (m) T (◦) P (mm) h (%) Precipitation Soil Alpine meadow 101◦51′16′′ 37◦33′28′′ 3637 −0.19 595.1 69.2 72 47 Coniferous forest 101◦53′23′′ 37◦41′50′′ 2721 3.34 431.9 66.6 42 41 Mountain grassland 102◦00′25′′ 37◦50′23′′ 2390 6.6 363.5 60.4 37 54 Deciduous forest 102◦10′56′′ 37◦53′27′′ 2097 7.9 262.5 59.8 40 53 G. Zhu et al.: Evaporation, infiltration and storage Figure 2. Climatic and hydrological conditions of the Xiying River basin. Figure 2. Climatic and hydrological conditions of the Xiying River basin. Figure 2. Climatic and hydrological conditions of the Xiying River basin. to 26.3 ◦C (27 July). The daily average humidity was 54.7 %, and the total precipitation was 250.6 mm, which was con- centrated in the month from late July to late August. The temperature of the studied regions were ordered as follows: AM < CF < MG < DF. The humidities of the studied regions were ordered as follows: AM > CF > MG > DF (Fig. 2). different periods. During the observation period, there were 72 precipitation events in the AM zone, and the total precipi- tation was 534.3 mm, which was relatively evenly distributed each month. In the CF zone, the daily average temperature during the study period was 10.9 ◦C, ranging from −5.4 ◦C (5 April) to 22.0 ◦C (27 July). The daily average humid- ity was 62.5 %, and the precipitation was 400.6 mm, mainly concentrated from early August to late September. Close to the foothills is the MG zone, with a daily average tempera- ture of 14.9 ◦C, ranging from −0.7 ◦C (5 April) to 25.3 ◦C (27 July). The average daily humidity was 51.1 %, and the precipitation of the vegetation zone during the observation period was 327.2 mm, mainly from late July to mid-August. During the observation period, the daily average temperature in the DF zone was 15.8 ◦C, ranging from −1.2 ◦C (6 April) 3.3.2 Potential evapotranspiration The potential evapotranspiration (PET) was calculated based on the Penman–Monteith equation (Allen, 1998): PET = 0.4081(Rn −G) + γ 900 T +273 u2 (es −ea) 1 + γ 1 + 0.34 u2 , (3) (3) where PET is the daily potential evapotranspiration (mm d−1), Rn is the net radiation (MJ m2 d−1), G is the soil heat flux density (MJ m2 d−1), γ is the psychrometric con- stant (kPa◦−1), u2 is the wind speed at 2 m height (m s−1), T is the mean daily air temperature at 2 m height (◦), 1 is the slope of the vapor pressure curve (kPa◦−1), ea is the actual vapor pressure (kPa) and es is the saturated vapor pressure (kPa). These data come from nearby weather stations. To explore the differences in the natural environment in different vegetation zones, air temperature, atmospheric hu- midity and precipitation were used to indicate each research site’s temperature and moisture conditions. The hilltop is a typical AM zone, with a daily average temperature of 6.1 ◦C, ranging from −9.7 ◦C (5 April) to 16.8 ◦C (27 July). The daily average humidity was 68.2 %, with little difference in https://doi.org/10.5194/hess-26-3771-2022 Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 G. Zhu et al.: Evaporation, infiltration and storage 3775 G. Zhu et al.: Evaporation, infiltration and storage 4.3 Spatial variation in water stable isotopes in different vegetation zones Isotope data of precipitation and soil water obtained from dif- ferent vegetation zones are shown in the dual-isotope space in Fig. 5. At the AM observation station, the slope (8.4) and intercept (23) of the LMWL were higher than those of the global meteoric water line (GMWL). The slope of LMWL in the other three vegetation zones was lower than that of GMWL and gradually decreased with decreasing altitude. With the decrease in altitude, the slope of SWL decreased in all vegetation zones except for DF (AM: 6.4; CF: 4.7; MG: 3.4; DF: 4.1), indicating that the evaporation of soil moisture increased. On the one hand, the vegetation coverage of the DF site was higher. On the other hand, the Xiying Reservoir enhanced the regional air humidity and decreased the local water vapor circulation driving force. The monthly variation in soil water isotopes records the signal of precipitation input and evaporation. The low- temperature environment and abundant precipitation events in AM make the monthly average δ2H and δ18O of soil wa- ter more depleted than in other vegetation zones (−69.4 ‰ to −51.6 ‰, δ2H; −7.5 ‰ to −10.3 ‰, δ18O). Despite this, the SWlc-excess of most samples at this station was still negative, and there were different degrees of evaporation in the process of precipitation penetrating the soil and mix- ing with original pore water, among which evaporation frac- tionation was stronger in July (−11.9 ‰ lc-excess) and Oc- tober (−14.5 ‰ lc-excess). The soil water isotopes of CF gradually changed seasonally. From April to July, precipi- tation was scarce, the temperature rose, and the isotopes of soil water were gradually enriched on the surface (−52.7 ‰ to −29.5 ‰, δ2H; −7.0 ‰ to −2.1 ‰, δ18O), reaching the peak value of the observation period in July (−29.5 ‰, δ2H; −2.1 ‰, δ18O), and continuous rainfall input from late July to mid-August resulted in soil water isotope depletion (−57.0 ‰, δ2H; −8.1 ‰, δ18O). The SWlc-excess was an obvious fractionation signal in contrast to the trend of the isotope change, reaching the lowest value (−26.3 ‰) dur- ing the sampling period in July, and the change in air tem- perature and precipitation controlled the evaporation inten- sity. From April to July, the isotopic value of surface soil water in MG was higher (δ18O was greater than zero), and SWlc-excess was lower than −30 ‰. G. Zhu et al.: Evaporation, infiltration and storage The high temperature caused the precipitation to undergo strong below-cloud evaporation during the fall, leading to the en- richment of isotopes. In addition, the atmospheric precipita- tion isotopes of the four vegetation zones had similar tempo- ral variations: from April to August, the fluctuations in δ2H and δ18O increased, reached the maximum in mid-August, and then gradually decreased (Fig. 3). δ18O), reached a peak in June when there was no rainfall event (−18.2 ‰, δ2H; 0.2 ‰, δ18O), and then became de- pleted (−53.2 ‰, δ2H; −5.2 ‰, δ18O). In addition, due to the influence of the Xiying Reservoir and vegetation cover- age, the isotopic enrichment degree of soil water in this veg- etation zone was lower than that in MG. As the most intuitive form of water change, the gravimetric water content was al- ways at a low value in July (AM: 21.0 %; CF: 14.8 %; MG: 11.9 %; DF: 14.9 %), when the evaporation was the strongest, and it was most obvious in shallow soil (Table 3) (Fig. 4). δ18O), reached a peak in June when there was no rainfall event (−18.2 ‰, δ2H; 0.2 ‰, δ18O), and then became de- pleted (−53.2 ‰, δ2H; −5.2 ‰, δ18O). In addition, due to the influence of the Xiying Reservoir and vegetation cover- age, the isotopic enrichment degree of soil water in this veg- etation zone was lower than that in MG. As the most intuitive form of water change, the gravimetric water content was al- ways at a low value in July (AM: 21.0 %; CF: 14.8 %; MG: 11.9 %; DF: 14.9 %), when the evaporation was the strongest, and it was most obvious in shallow soil (Table 3) (Fig. 4). 4.2 Temporal variation in water stable isotopes in different vegetation zones Influenced by different water sources and complex weather conditions in the precipitation process, the isotopic compo- sitions of precipitation in the four vegetation zones were dif- ferent during the study period. The mean values of δ2H and δ18O in the AM zone (number of samples: 72) were −73.1± 36.3‰ (−163.9 ‰–13.7 ‰) and −10.0 ± 4.3 ‰ (−23.1 ‰ https://doi.org/10.5194/hess-26-3771-2022 Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 G. Zhu et al.: Evaporation, infiltration and storage G. Zhu et al.: Evaporation, infiltration and storage 3776 to −1.3 ‰), respectively. The average δ2H and δ18O val- ues of the CF zone (number of samples: 42) were −42.0 ± 37.2 ‰ (−117.8 ‰–13.0 ‰) and −7.1 ± 4.7‰ (−17.4 ‰ to −0.1 ‰), respectively. The average δ2H and δ18O val- ues of the MG zone (number of samples: 37) were −37.4 ± 30.5 ‰ (−103.1 ‰–4.2 ‰) and −5.9 ± 3.9‰ (−15.1 ‰ to −0.9‰), respectively. The average δ2H and δ18O values of the DF zone (number of samples: 40) were −31.8 ± 42.8 ‰ (−110.2 ‰–23.2 ‰) and −5.8 ± 5.5‰ (−15.2 ‰–3.2 ‰), respectively (Table 2). The maximum isotopic values of the four vegetation zones appeared on 4 August (AM: 13.7 ‰, δ2H; −1.3‰, δ18O), 10 August (CF: 13.0 ‰, δ2H; −0.1 ‰, δ18O), 7 August (MG: 4.2 ‰, δ2H; −0.9 ‰, δ18O) and 13 August (DF: 23.2 ‰, δ2H; 3.2 ‰, δ18O). The highest tem- perature in each vegetation zone appeared on 27 July. The high temperature caused the precipitation to undergo strong below-cloud evaporation during the fall, leading to the en- richment of isotopes. In addition, the atmospheric precipita- tion isotopes of the four vegetation zones had similar tempo- ral variations: from April to August, the fluctuations in δ2H and δ18O increased, reached the maximum in mid-August, and then gradually decreased (Fig. 3). to −1.3 ‰), respectively. The average δ2H and δ18O val- ues of the CF zone (number of samples: 42) were −42.0 ± 37.2 ‰ (−117.8 ‰–13.0 ‰) and −7.1 ± 4.7‰ (−17.4 ‰ to −0.1 ‰), respectively. The average δ2H and δ18O val- ues of the MG zone (number of samples: 37) were −37.4 ± 30.5 ‰ (−103.1 ‰–4.2 ‰) and −5.9 ± 3.9‰ (−15.1 ‰ to −0.9‰), respectively. The average δ2H and δ18O values of the DF zone (number of samples: 40) were −31.8 ± 42.8 ‰ (−110.2 ‰–23.2 ‰) and −5.8 ± 5.5‰ (−15.2 ‰–3.2 ‰), respectively (Table 2). The maximum isotopic values of the four vegetation zones appeared on 4 August (AM: 13.7 ‰, δ2H; −1.3‰, δ18O), 10 August (CF: 13.0 ‰, δ2H; −0.1 ‰, δ18O), 7 August (MG: 4.2 ‰, δ2H; −0.9 ‰, δ18O) and 13 August (DF: 23.2 ‰, δ2H; 3.2 ‰, δ18O). The highest tem- perature in each vegetation zone appeared on 27 July. G. Zhu et al.: Evaporation, infiltration and storage Table 2. General characteristics of precipitation δ2H and δ18O in different vegetation areas from April to October 2017. 2. General characteristics of precipitation δ2H and δ18O in different vegetation areas from April to October 2017. Vegetation δ2H/‰ δ18O/‰ Zone Max Min Mean SD Max Min Mean SD AM 13.7 −163.9 −73.1 36.3 −1.3 −23.1 −10.0 4.3 CF 13.0 −117.8 −42.0 37.2 −0.1 −17.4 −7.1 4.7 MG 4.2 −103.1 −37.4 30.5 −0.9 −15.1 −5.9 3.9 DF 23.2 −110.2 −31.8 42.8 3.2 −15.2 −5.8 5.5 e 3. Time series of rainfall and isotope characteristics in different vegetation zones of the Xiying River basin, with dotted te of soil water sampling. Figure 3. Time series of rainfall and isotope characteristics in different vegetation zones of the Xiying River basin, with dotted lines indicating the date of soil water sampling. 4.3 Spatial variation in water stable isotopes in different vegetation zones During this period, the evaporation and fractionation of shallow soil water were in- tense. As in CF, the input of heavy precipitation from late July to mid-August led to the depletion of soil water isotopes in MG. There was only sporadic rainfall in DF from April to July, and the soil water isotopes were gradually enriched on the surface (−46.1 ‰ to −18.2 ‰, δ2H; −4.7 ‰–0.2 ‰, During the study period, compared with that in other veg- etation belts, the surface isotopic value of the soil water in MG was relatively enriched (−24.3 ‰, δ2H; −0.8 ‰, δ18O), the lc-excess was smaller and deeper into the middle and lower soil layers (−25.8 ‰), and the gravimetric water con- tent was relatively low (8.4 %). Due to the difference in veg- etation types and the influence of reservoirs, this change did not have an obvious elevation effect. Although the el- evation was low, the soil water of DF had more depleted isotopic characteristics and higher soil moisture than those of MG in most samples. Soil profiles obtained from differ- ent vegetation zones can reflect the evaporation signals of water. The low-temperature natural environment made AM soil less affected by evaporation (lc-excess > −20 ‰), and the gravimetric water content was high (gravimetric water content > 20 %) during the whole study period. The surface soil water of CF was easily affected by climate and had a higher isotopic composition (−29.5 ‰, δ2H; −2.1 ‰, δ18O) and lower lc-excess (−26.3 ‰). Due to evaporation, soil wa- ter isotopes in the MG and DF areas were enriched in the surface soil layer. In particular, in MG, the average values of δ2H and δ18O in the 0–10 cm soil layer were as high as −24.4 ‰ and −1.2 ‰, respectively, and SWlc-excess was lower than −25 ‰, even close to −40 ‰ in some samples. In this case, the evaporation signals can easily penetrate the deep soil, making the gravimetric water content values at all the sampling sites lower than 20 % (Figs. 4, 6). https://doi.org/10.5194/hess-26-3771-2022 Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 3777 4.4 Variations in the water storage capacity of the 0–40 cm soil layer in different vegetation areas monthly average water storage capacity was lowest at 0– 10 cm (43.0 mm) and highest at 30–40 cm (51.7 mm). The water storage capacity of CF gradually decreased from April to July (150.1–101.2 mm), and increased after July (101.2– 160.0 mm). The monthly average water storage capacity was lowest at 0–10 cm (28.0 mm) and highest at 30–40 cm (40.0 mm). The water storage capacity of MG gradually de- creased from April to July (80.3–64.0 mm), and increased af- This study used soil water to calculate the water storage of the 0–40 cm soil layer in the four vegetation zones during the observation period (Fig. 7). The water storage capac- ity of AM gradually decreased from April to July (209.7– 167.2 mm), and increased after July (167.2–201.8 mm). The Table 3. General characteristics of soil water δ2H, δ18O, lc-excess and gravimetric water content (GWC) in different vegetation areas from April to October 2017. of soil water δ2H, δ18O, lc-excess and gravimetric water content (GWC) in different vegetation areas from Table 3. General characteristics of soil water δ2H, δ18O, lc-excess and gravimetric water content (GWC) in different vegetation areas from April to October 2017. pril to October 2017. Table 3. General characteristics of soil water δ2H, δ18O, lc-excess and gravimetric water content (GWC) in different vegetation areas from April to October 2017. Month Vegetation δ2H/‰ δ18O/‰ lc-excess/‰ GWC/% zone Max Min Mean Max Min Mean Max Min Mean Max Min Mean 4 AM −55.2 −70.7 −65.6 −8.5 −10.8 −10.1 −2.7 −7.1 −4.7 25.9 23.0 24.7 CF −52.7 −72.2 −63.9 −7.0 −9.9 −8.9 −4.0 −12.0 −8.4 27.6 14.9 20.0 MG −7.32 −50.6 −41.0 2.8 −5.8 −3.9 −8.8 −36.8 −19.4 21.7 6.5 14.7 DF −46.1 −69.4 −62.1 −4.7 −9.9 −8.5 −2.5 −23.2 −9.7 27.7 19.4 21.8 5 AM −46.1 −76.5 −66.4 −7.4 −12.2 −10.1 −2.6 −7.7 −4.9 32.6 23.2 28.9 CF −45.8 −61.9 −53.5 −5.3 −8.4 −7.0 −9.3 −17.7 −13.0 22.6 9.0 16.1 MG −6.7 −47.3 −39.2 2.9 −6.5 −4.3 −4.5 −36.2 −14.4 15.7 7.6 11.2 DF −30.8 −63.5 −53.8 −1.9 −9.4 −6.9 −3.2 −30.1 −13.6 26.0 11.7 17.7 6 AM −62.5 −83.9 −69.4 −8.9 −12.6 −10.3 −1.5 −8.4 −5.8 33.3 21.9 26.0 CF −45.8 −78.4 −58.7 −5.1 −12.0 −7.8 5.5 −26.6 −8.5 32.1 10.0 21.3 MG −19.7 −74.9 −46.9 0.8 −11.8 −5.8 13.0 −33.7 −11.0 19.3 7.5 14.2 DF −18.2 −64.9 −51.7 0.2 −9.0 −5.9 −4.6 −38.2 −19.4 13.5 8.4 11.1 7 AM −47.3 −60.1 −51.6 −6.9 −8.4 −7.5 −8.8 −14.8 −11.9 25.4 19.0 21.0 CF −29.5 −51.4 −41.6 −2.1 −7.9 −5.6 −2.6 −26.3 −11.2 24.3 7.2 14.8 MG −10.6 −48.4 −39.2 2.3 −6.4 −4.1 −5.8 −35.8 −16.1 18.7 6.3 11.9 DF −35.1 −69.0 −54.1 −1.7 −8.7 −5.5 −14.8 −35.3 −24.5 18.2 11.8 14.4 8 AM −58.5 −80.3 −66.6 −8.4 −11.6 −9.6 −6.1 −15.4 −9.7 28.1 19.5 25.1 CF −57.0 −75.5 −66.4 −8.1 −9.8 −9.2 −2.5 −13.1 −8.3 21.4 8.7 16.3 MG −34.2 −53.8 −44.0 −3.2 −5.5 −4.4 −14.7 −22.6 −18.7 11.3 9.5 10.4 DF −53.2 −84.3 −67.6 −5.2 −13.5 −9.2 6.8 −26.1 −9.6 23.6 14.7 20.6 9 AM −48.0 −79.2 −61.0 −7.8 −11.1 −9.2 −4.3 −10.4 −7.2 29.9 20.3 25.3 CF −52.5 −67.7 −60.7 −7.8 −10.1 −8.8 −0.1 −11.3 −6.0 31.3 9.3 20.5 MG −32.3 −45.3 −38.8 −3.5 −4.4 −4.0 −9.1 −23.8 −16.5 15.3 9.1 13.0 DF −30.5 −77.0 −59.8 −3.1 −11.4 −8.2 −1.8 −19.3 −9.3 25.8 14.7 19.1 10 AM −42.4 −73.5 −58.9 −6.1 −9.8 −7.9 −12.2 −18.2 −14.5 36.2 25.4 29.5 CF −59.1 −66.3 −61.7 −8.8 −10.5 −9.5 5.1 −5.3 −1.5 30.0 16.8 23.1 MG −50.3 −66.7 −58.3 −5.6 −8.3 −7.1 −5.5 −18.4 −11.9 18.3 11.4 15.8 DF −38.0 −61.8 −48.3 −2.7 −8.2 −4.9 −11.9 −34.8 −23.9 25.5 8.9 17.2 5 Discussion ter July (64.0–104.6 mm). Table 3. General characteristics of soil water δ2H, δ18O, lc-excess and gravimetric water content (GWC) in different vegetation areas from April to October 2017. The monthly average water stor- age capacity was lowest at 0–10 cm (17.5 mm) and highest at 20–30 cm (22.0 mm). The water storage capacity of DF gradually decreased from April to June (159.3–104.0 mm), increased from June to August (104.0–154.0 mm), and there was a decrease from August to October (154.0–111.8 mm). The monthly average water storage capacity was lowest at 0–10 cm (29.1 mm) and highest at 20–30 cm (35.0 mm). In general, the soil water storage capacity of the 0–10 cm soil layer was less than that of the other soil layers. The order of the water storage capacity of the 0–40 cm soil layer in the four vegetation zones is AM (46.9 mm) > DF (33.0 mm) > CF (32.1 mm) > MG (20.3 mm). https://doi.org/10.5194/hess-26-3771-2022 https://doi.org/10.5194/hess-26-3771-2022 Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 https://doi.org/10.5194/hess-26-3771-2022 G. Zhu et al.: Evaporation, infiltration and storage 3778 G. Zhu et al.: Evaporation, infiltration and storage G. Zhu et al.: Evaporation, infiltration and storage Figure 4. Heat map of the soil depth profile of δ2H, δ18O, lc-excess and GWC in different vegetation zones, and the layer lacking measure- ment is indicated by the gray color. Figure 4. Heat map of the soil depth profile of δ2H, δ18O, lc-excess and GWC in different vegetation zones, and the layer lacking measure- ment is indicated by the gray color. Figure 5. Dual-isotope space of precipitation (a) and soil water (b) in isotope data of four vegetation zones. In the box plots, the box represents the 25th–75th percentile, the line in the box represents the median (50th percentile), the required line indicates 90th and 10th percentile, and the point indicates the 95th and 5th percentile. Figure 5. Dual-isotope space of precipitation (a) and soil water (b) in isotope data of four vegetation zones. In the box plots, the box represents the 25th–75th percentile, the line in the box represents the median (50th percentile), the required line indicates 90th and 10th percentile, and the point indicates the 95th and 5th percentile. Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 5.1 Evaporation of soil moisture in different vegetation zones In the arid river source area, the replenishment of soil mois- ture mainly comes from precipitation. The slope of the re- gional atmospheric precipitation line can reflect the strength of local evaporation. Due to a low atmospheric temperature, low cloud base height, and low air-saturated water vapor loss, the AM zone was weakly affected by secondary evapora- tion during precipitation. There, the slope of LMWL (8.4) was even higher than that of the GMWL (Hughes and Craw- ford, 2012). As the altitude decreased, the secondary evap- oration under clouds strengthened, and the LMWL slope of each vegetation zone decreased (Pang et al., 2011). The slope of SWL can indicate the strength of soil moisture evapora- tion in each vegetation zone. The evaporation intensity re- sults of the four vegetation zones followed the order of MG Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 https://doi.org/10.5194/hess-26-3771-2022 3779 G. Zhu et al.: Evaporation, infiltration and storage https://doi.org/10.5194/hess-26-3771-2022 Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 3780 G. Zhu et al.: Evaporation, infiltration and storage G. Zhu et al.: Evaporation, infiltration and storage Figure 6. The variation of δ2H, δ18O, lc-excess and GWC in different vegetation zones in each sampling. Figure 6. The variation of δ2H, δ18O, lc-excess and GWC in different vegetation zones in each sampling. (SWLslop: 3.4) > DF (SWLslop: 4.1) > CF (SWLslop: 4.7) > AM (SWLslop: 6.4) (Fig. 5). The dynamic changes in lc- excess of soil profiles in different vegetation areas reflect the process of soil water evaporation caused by drought during the study period. The monthly average value of SWlc-excess in the AM zone was less than 0, and the minimum value was −11.9 ‰ (July). Although the vegetation belt was subject to different degrees of evaporation each month, it was less affected by drought, and it was difficult for evaporation to penetrate into the middle and lower soil layers. The SWlc- excess of the CF belt was greater than that of AM from April to June. The evaporation was the strongest in July (−11.2 ‰ lc-excess). As in AM, the evaporation mainly occurred in the topsoil of the CF belt. The vegetation coverage of the MG zone was low, and the arid environment made the isotopes of the surface soil produce strong evaporation signals (lc-excess was close to −40 ‰). In most samples, the SWlc-excess of the 60–80 cm soil layer was negative. The evaporation sig- nal shifted to the lower layer of the soil (Barnes and Allison, 1988; Zimmermann et al., 1966). Similar evaporation signals have been found in the Mediterranean and arid climate re- gions (McCutcheon et al., 2017; Sprenger et al., 2016). Evap- oration signals exist in only the surface soil of humid areas, and there is no difference between lc-excess and 0 in the soil layer below 20 cm (Sprenger et al., 2017). The monthly sur- face soil evaporation of DF was less than that of MG from April to June, and it was greater than that of MG after July, mainly due to the influence of the vegetation and reservoirs. There were commonalities in the soil moisture changes in different vegetation zones characterized by more enriched isotopes, stronger evaporation signals, and lower moisture content in the shallow soil. https://doi.org/10.5194/hess-26-3771-2022 With increasing soil depth, the isotope gradually became depleted, and the evaporation sig- nal was gradually weakened until it disappeared. The evo- lution of the investigated isotopes, lc-excess, and gravimet- ric water content in the unsaturated soil showed differences among different vegetation zones. From a high altitude to a low altitude, the isotopic value of the surface gradually in- creased, and the evaporation signal increased (Figs. 4, 6). G. Zhu et al.: Evaporation, infiltration and storage The soil moisture profile showed a trend of water increasing from top to bottom, indicating the influence of the previous precipita- tion. The soil was humid, so the replenishment of soil water by precipitation had the characteristics of top-down piston re- plenishment. Preferential infiltration showed high variability in isotopic signals (Brodersen et al., 2000), and the rainwa- ter in MG and DF flowed into the deep soil rapidly through the soil matrix via exposed soil fissures and roots. This re- sulted in the sudden depletion of soil isotopes at a depth of 60–100 cm. This may be due to the more recent depleted pre- cipitation that quickly reached this depth and the preferen- tial infiltration into the soil. Water movement and mixing in the unsaturated zone can be observed in the spatiotemporal variation in isotopes within 1 m of the soil profile, and the AM and CF zones underwent considerable rainfall. After a short period of weak evaporation, the soil was rewetted by the next rainfall. In AM, the soil moisture remained above 20 % each month. The MG and DF zones had only sporadic precipitation from mid-May to late July, and the soil mois- ture evaporated rapidly. With the decrease in air temperature and the occurrence of continuous precipitation after July, the soil was rewetted after 2 months of drought, and both vegeta- 5.2 Memory effects of precipitation input, mixing and rewetting The changes in soil water isotopes and soil moisture can eval- uate the input, mixing and rewetting precipitation process in different vegetation areas. The main methods of precipitation input are plug flow and preferential flow. Plug flow is the complete mixing of water through the soil matrix with shal- low free water. Under the action of plug flow, precipitation infiltrates along the hydraulic gradient, pushing the original soil water downward. Preferential flow means that precipita- tion uses soil macropores to quickly penetrate shallow soil to form deep leakage (Tang and Feng, 2001). After precipita- tion, the variability of isotope signals at a certain soil depth can identify the seepage method of water (Peralta-Tapia et al., 2015). During the study period, the soils of the AM and CF areas were seasonally frozen and thawed year-round, and Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 https://doi.org/10.5194/hess-26-3771-2022 3781 G. Zhu et al.: Evaporation, infiltration and storage G. Zhu et al.: Evaporation, infiltration and storage G. Zhu et al.: Evaporation, infiltration and storage Figure 7. Monthly variation of soil water storage in 0–40 cm soil layer of different vegetation zones. Figure 7. Monthly variation of soil water storage in 0–40 cm soil layer of different vegetation zones. the difference in the soil isotope profile was small. The soil moisture profile showed a trend of water increasing from top to bottom, indicating the influence of the previous precipita- tion. The soil was humid, so the replenishment of soil water by precipitation had the characteristics of top-down piston re- plenishment. Preferential infiltration showed high variability in isotopic signals (Brodersen et al., 2000), and the rainwa- ter in MG and DF flowed into the deep soil rapidly through the soil matrix via exposed soil fissures and roots. This re- sulted in the sudden depletion of soil isotopes at a depth of 60–100 cm. This may be due to the more recent depleted pre- cipitation that quickly reached this depth and the preferen- tial infiltration into the soil. Water movement and mixing in the unsaturated zone can be observed in the spatiotemporal variation in isotopes within 1 m of the soil profile, and the AM and CF zones underwent considerable rainfall. After a short period of weak evaporation, the soil was rewetted by the next rainfall. In AM, the soil moisture remained above 20 % each month. The MG and DF zones had only sporadic precipitation from mid-May to late July, and the soil mois- ture evaporated rapidly. With the decrease in air temperature and the occurrence of continuous precipitation after July, the soil was rewetted after 2 months of drought, and both vegeta- tion zones showed the replacement and mixing of soil water isotopes and precipitation. The results showed that the soil water storage capacity of the mountain grassland (MG) was seriously insufficient, reflecting the incomplete rewetting of the soil by precipitation at the end of the study. In addition, low soil water storage capacity will enrich the remaining soil water isotopes (Barnes and Allison, 1988; Zimmermann et al., 1966). We observed the “memory effect” of soil rewet- ting caused by precipitation input and the mixing of different vegetation areas during the entire study period. The changes in soil moisture in each vegetation area reflect different cli- matic and hydrological characteristics (Figs. 4, 6). the difference in the soil isotope profile was small. 6 Conclusions This work provides further insights into the movement and mixing of soil water in different vegetation zones in arid source regions. During the study period, the dynamic changes in lc-excess in the soil profiles of different veg- etation zones reflected the evaporation signals caused by drought. Soil water evaporation in spring and summer, and insufficient precipitation during the drought period, were the main driving forces of isotopic enrichment in the surface soil. The soil water evaporation intensity of the four vege- tation zones followed the order of MG (SWLslop: 3.4) > DF (SWLslop: 4.1) > CF (SWLslop: 4.7) > AM (SWLslop: 6.4). In the MG and DF zones, drought caused the evaporation signal to penetrate deep into the middle and lower soil lay- ers. The SWlc-excess below 70 cm of the ground surface re- mained negative. Soil water isotopes and gravimetric water content record the process of soil rewetting caused by precip- itation input and mixing. The AM and CF zones have many precipitation events. After a short period of weak evapora- tion, the soil was rewetted by the next precipitation event. There was only sporadic precipitation in the MG and DF belts from mid-May to late July. After July, the temperature dropped, and continuous precipitation wet the soil again af- ter 2 months of drought. The MG and DF zones had only sporadic precipitation from mid-May to late July. With the decrease in air temperature and continuous precipitation af- ter July, the soil was rewetted after 2 months of drought. Moisture and temperature conditions were the key factors that restricted the soil water storage capacity in the different vegetation zones. The water storage capacity of the 0–40 cm soil layer results followed the order of AM (46.9 mm) > DF (33.0 mm) > CF (32.1 mm) > MG (20.3 mm). The water storage capacity of the surface soil in each vegetation zone was weak, and more water was stored in the middle and lower soil layers with higher clay contents. The research results can be applied to arid and semi-arid alpine regions and can be valuable for latitude and longitude differentiation. This study mainly emphasized the spatiotemporal heterogeneity of soil water evaporation, infiltration, and water storage in different vegetation zones. These results are important for understand- ing regional hydrological processes and ecological restora- tion services in environmentally fragile areas. Code availability. G. Zhu et al.: Evaporation, infiltration and storage The forest acted as a reservoir due to its strong water storage and soil conservation capacity (Sprenger et al., 2019). The water storage capacity of the 0–40 cm soil layer in DF was higher than 100 mm at each sampling time. In addition, the water content of the 0–40 cm soil layer in each vegetation zone increased with the deepening of the soil layer, and the water storage capacity of the surface soil was weak. The difference in soil properties also led to more water storage in the middle and lower soil layers with higher clay contents (Milly, 1994) (Fig. 7). Climate warming and the spa- tiotemporal imbalance of water resources have disturbed the ecological water balance of different vegetation zones in in- land river source areas (Liu et al., 2015). Plant growth mainly depends on the water stored in shallow soil layers (Amin et al., 2020). Drought reduces soil water storage and inhibits plant growth (Li et al., 2020). To effectively improve and i id l i G. Zhu et al.: Evaporation, infiltration and storage vegetation zones is necessary. This will provide a reference for the formulation of ecological policies. vegetation zone replacement, the precipitation decreased, the temperature rose, the groundwater level dropped, and the soil water storage capacity was weak (Coussement et al., 2018; Kleine et al., 2020). The soil water storage capacity of the AM zone with low temperature and rainy weather was higher than that of other vegetation zones (results of the 0– 40 cm soil layers from April to October: AM: 187.8 mm; CF: 128.4 mm; MG: 81.2 mm; DF: 132.1 mm). During the study period, the soil water storage capacity (0–40 cm) ex- ceeded 165 mm each month. With the decrease in altitude, the monthly difference in dry–wet conditions in each veg- etation zone gradually became obvious. With the increase in temperature during summer, the environment became dry, and the soil water storage capacity weakened (Sprenger et al., 2017). The soil water storage capacity of the CF zone be- gan to decrease in April, and the water storage capacity of the 0–40 cm layer reached the minimum value (101.2 mm) in July. The variation in temperature and precipitation was the main reason for the monthly difference (Dubber and Werner, 2019). Although there was a certain water storage capacity in CF with some transpiration loss, the soil water storage capac- ity in this vegetation zone was not strong. The water storage capacity of MG soil was lower than that of the other vegeta- tion zones. The continuous dry and warm weather in spring and summer led to the water storage capacity of 0–40 cm soil being lower than that of 100 mm every month. In particular, drought stress leads to insufficient soil moisture, making it difficult to maintain plant demand, resulting in sparse veg- etation and large-scale exposed surface soil, which further accelerates surface water loss. The continuous precipitation from the end of July prevented further drought development, and the water input gradually restored the soil water stor- age capacity (Kleine et al., 2020). The DF had hydrothermal conditions similar to those of MG, but the soil porosity of the forest zone was obviously larger than that of the barren land, and its permeability was higher than that of the barren land. Precipitation infiltrated the ground through roots and turned into groundwater. 5.3 Influencing factors of soil water storage capacity in arid headwater areas As the temperature decreased rapidly with increasing height, precipitation and humidity increased to a certain extent, and the vegetation showed a strip-like alternation approximately parallel to the contour line, forming zonal vegetation with obvious differentiation (Yin et al., 2020). The dry–wet con- ditions of different vegetation zones restricted the soil water storage capacity in the basin. In the process of low-altitude https://doi.org/10.5194/hess-26-3771-2022 Hydrol. Earth Syst. Sci., 26, 3771–3784, 2022 3782 G. Zhu et al.: Evaporation, infiltration and storage oxygen, J. Hydrol., 100, 143–176, https://doi.org/10.1016/0022- 1694(88)90184-9, 1988. Data availability. The data sets that have been analyzed in this pa- per are available from the authors upon request. oxygen, J. Hydrol., 100, 143–176, https://doi.org/10.1016/0022- 1694(88)90184-9, 1988. Brodersen, C., Pohl, S., Lindenlaub, M., Leibundgut, C., and Wilpert, K. V.: Influence of vegetation structure on isotope con- tent of throughfall and soil water, Hydrol. Process., 14, 1439– 1448, 2000. Author contributions. GZ and LY conceived the idea of the study; YX and QW analyzed the data; ZS and LY were responsible for field sampling; ZZ participated in the experiment; LW participated in the drawing; LY wrote the paper; LS, XZ and YL checked and edited language. 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This research was financially supported by the National Natural Science Foundation of China (41661005, 41867030, 41971036). G. Zhu et al.: Evaporation, infiltration and storage The authors much thank the colleagues in the Northwest Normal University for their help in fieldwork, labo- ratory analysis and data processing. Gibson, J. J., Holmes, T., Stadnyk, T. A., Birks, S. J., Eby, P., and Pietroniro, A.: Isotopic constraints on water balance and evapo- transpiration partitioning in gauged watersheds across Canada, J. Hydrol., 37, 100878, https://doi.org/10.1016/j.ejrh.2021.100878, 2021. Financial support. This research has been supported by the Na- tional Natural Science Foundation of China (grant nos. 41661005, 41867030, 41971036). Grant, G. E. and Dietrich, W. E.: The frontier be- neath our feet, Water Resour. Res., 53, 2605–2609, https://doi.org/10.1002/2017WR020835, 2017. Review statement. This paper was edited by Jorge Isidoro and re- viewed by three anonymous referees. 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Analisis Teori Belajar Pembelajaran Seni Tari Di Sanggar Seni Kinanti Sekar
Jurnal pendidik Indonesia/Jurnal Pendidik Indonesia
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Analisis Teori Belajar Pembelajaran Seni Tari Di Sanggar Seni Kinanti Sekar Analisis Teori Belajar Pembelajaran Seni Tari Di Sanggar Seni Kinanti Sekar Anggraeni Wahyu Arini PGSD, Universitas Ahmad Dahlan, Yogyakarta, Indonesia; anggraeni2100005188@webmail.uad.ac.id Febriana Putri Lestari PGSD, Universitas Ahmad Dahlan, Yogyakarta, Indonesia; febriana2100005195@webmail.uad.ac.id, Argandaru Dimas Priambodo PGSD, Universitas Ahmad Dahlan, Yogyakarta, Indonesia; argandaru2100005211@webmail.uad.ac.id Anggraeni Wahyu Arini PGSD, Universitas Ahmad Dahlan, Yogyakarta, Indonesia; anggraeni2100005188@webmail.uad.ac.id Febriana Putri Lestari PGSD, Universitas Ahmad Dahlan, Yogyakarta, Indonesia; febriana2100005195@webmail.uad.ac.id, Argandaru Dimas Priambodo PGSD, Universitas Ahmad Dahlan, Yogyakarta, Indonesia; argandaru2100005211@webmail.uad.ac.id Anggraeni Wahyu Arini PGSD, Universitas Ahmad Dahlan, Yogyakarta, Indonesia; anggraeni2100005188@webmail.uad.ac.id Febriana Putri Lestari PGSD, Universitas Ahmad Dahlan, Yogyakarta, Indonesia; febriana2100005195@webmail.uad.ac.id, Argandaru Dimas Priambodo PGSD, Universitas Ahmad Dahlan, Yogyakarta, Indonesia; argandaru2100005211@webmail.uad.ac.id *Coresponding Author Info Artikel: Dikirim: 20-02-2023 ; Direvisi: 20-03-2023-; Diterima: 20-04-2023 Cara sitasi: Arini Wahyu Anggraeni. Lestari Putri Febriana & Priambodo Dimas Argandari. (2023).Analisis Teori Belajar Pembelajaran Seni Tari Di Sanggar Senni Kinanti Sekar. JPIn: Jurnal Pendidik Indonesia, vol(06), 01 *Coresponding Author Info Artikel: Dikirim: 20-02-2023 ; Direvisi: 20-03-2023-; Diterima: 20-04-2023 Cara sitasi: Arini Wahyu Anggraeni. Lestari Putri Febriana & Priambodo Dimas Argandari. (2023).Analisis Teori Belajar Pembelajaran Seni Tari Di Sanggar Senni Kinanti Sekar. JPIn: Jurnal Pendidik Indonesia, vol(06), 01 Abstrak. Penelitaian ini mengkaji tentang proses pembelajaran seni tari di Sanggar Seni Kinanti Sekar. Penelitian ini berlandaskan teori belajar behavioristik, Kontruktivisme dan Sosiokultural. Jenis penelitian ini adalah deskriftif kualitatif yang mendeskripsikan proses pembelajaran tari. Teknik mengumpulkan data yaitu observasi, wawancara, dokumentasi kemudian dilakukan analisis data dengan melakukan reduksi data, penyajian data, dan menarik kesimpulan. Proses pembelajaran seni tari di Sanggar diikutu oleh anak anak dan orang dewasa dimana dalam setiap pertemuan 1 jam hingga 1.5 jam. Instrument penilaian tes praktik yaitu hafalan gerak,hafalan pola lantai dan menyesuaikan iringan musik. Proses pembelajaran ini peserta didik menirukan terlebih dahulu ragam gerak yang diajarkan oleh pelatih, kemudian peserta didik diberi kesempatan berlatih mengulang ragam gerak yang sudah dipelajari dan diharapkan mampu menghafal ragam gerak yang telah diajarkan hingga tergolong dalam kategori baik. Selain itu peserta didik juga dapat belajar dari teman sebayanya, mereka tidak dipaksa untuk melakukan pergerakan melainkan eksplorasi gerak datang dari dirinya sendiri. Kata Kunci: Teori Belajar. Seni Tari, Sanggar Seni Kinanti Sekar Abstract. This research examines the process of learning dance art at the Kinanti Sekar Art Studio. This research is based on behavioristic, constructivism and sociocultural learning theory. This type of research is descriptive qualitative which describes the dance learning process. JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 Analisis Teori Belajar Pembelajaran Seni Tari Di Sanggar Seni Kinanti Sekar Techniques for collecting data are observation, interviews, documentation and then data analysis is carried out by reducing data, presenting data, and drawing conclusions. The dance learning process in the Studio is attended by children and adults where in each meeting it takes 1 hour to 1.5 hours. The practical test assessment instruments are rote memorization, floor pattern memorization and adjusting musical accompaniment. In this learning process students first imitate the range of motion taught by the coach, then students are given the opportunity to practice repeating the range of motion that has been learned and are expected to be able to memorize the range of motion that has been taught so that it is classified in the good category. In addition, students can also learn from their peers, they are not forced to make movements, but exploration of motion comes from themselves. Keywords: Learning Theory. Dance Arts, Kinanti Sekar Art Studio Pendahuluan (12pt, bolt) Pendahuluan (12pt, bolt) Hal ini berarti bahwa berhasil atau tidaknya pencapaian tujuan pendidikan itu amat bergantung pada proses belajar yang dijalani siswa baik pada saat dia berada disekolah atau berada JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 di lingkungan rumah atau di lingkungan keluarganya sendiri. Untuk itu pemahaman yang benar tentang konsep dasar belajar dengan segala aspek serta bentuk dan manifestasinya sangat mutlak dibutuhkan oleh para pengajar. Adanya kekeliruan atau ketidak lengkapan persepsi mereka akan proses belajar dan hal-hal yang berkaitan dengannya mungkin dapat mengakibatkan kurang bermutunya hasil belajar yang dicapai murid. JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 di lingkungan rumah atau di lingkungan keluarganya sendiri. Untuk itu pemahaman yang benar tentang konsep dasar belajar dengan segala aspek serta bentuk dan manifestasinya sangat mutlak dibutuhkan oleh para pengajar. Adanya kekeliruan atau ketidak lengkapan persepsi mereka akan proses belajar dan hal-hal yang berkaitan dengannya mungkin dapat mengakibatkan kurang bermutunya hasil belajar yang dicapai murid. di lingkungan rumah atau di lingkungan keluarganya sendiri. Untuk itu pemahaman yang benar tentang konsep dasar belajar dengan segala aspek serta bentuk dan manifestasinya sangat mutlak dibutuhkan oleh para pengajar. Adanya kekeliruan atau ketidak lengkapan persepsi mereka akan proses belajar dan hal-hal yang berkaitan dengannya mungkin dapat mengakibatkan kurang bermutunya hasil belajar yang dicapai murid. Berhasil atau tidaknya pencapaian tujuan pendidikan itu amat bergantung pada proses belajar yang dijalani siswa baik pada saat dia berada di sekolah atau berada di lingkungan tempat tinggal atau dalam keluarganya. Untuk itu pemahaman konsep belajar dari segala aspek serta bentuk manifestasinya sangat dibutuhkan oleh pengajar, dengan begitu pemahaman tentang konsep belajar sangat penting untuk mencegah kekeliruan dari persepsi akan proses belajar dan hal-hal yang ada kaitannya dengan konsep belajar hingga dapat mengakibatkan kurang bermutunya hasil belajar yang akan dicapai. Pemahaman konsep dalam pembelajaran saling terkait dengan penggunaan teori belajar. Karena teori belajar sangat penting untuk dikuasai oleh seorang tenaga pendidik, dengan adanya teori belajar dan pembelajaran dapat membuat tenaga pendidik menjadi guru yang profesional. Misalnya dengan merumuskan tujuan pembelajaran yang sesuai, memilih strategi yang tepat, memberikan bimbingan atau pengarahan kepada peserta didik, manfasilitasi serta memotivasi terkait belajar kepada peserta didik, menciptakan suasana belajar yang kondusif, berinteraksi dengan peserta didik, memberikan evaluasi juga penilaian secara adil terhadap hasil belajar dan pembelajaran yang dilakukan. Metode Penelitian ini merupakan penelitian deskriptif dengan menggunakan pendekatan kualitatif. Penggunaan penelitian deskriptif kuantitatif dalam penelitian ini dimaksudkan untuk memperoleh informasi yang dapat mendukung peneliti untuk mendeskripsikan penerapan konsep serta teori- teori belajar dan pembelajaran di Sanggar Seni Kinanti Sekar. Waktu dan Tempat Penelitian Penelitian ini dilaksanakan di Sanggar Seni Kinanti Sekar yang beralamatkan di Prawirodirjan, Kec. Gondomanan, Kota Yogyakarta, DIY. Adapun waktu observasi dan wawancara dilaksanakan pada hari Selasa, 20 Desember 2022 hingga Jum’at 23 Desember 2022. Pendahuluan (12pt, bolt) Mungkin tidak setiap persoalan dapat diselesaikan oleh teori, tetapi tanpa ada teori belajar , pembelajaran tidak akan tahu kemana arah yang dituju serta bagaimana harus memulainya. Pendahuluan (12pt, bolt) p Pendidikan adalah suatu proses pembelajaran yang dilakukan secara terencana dan dijalankan secara sadar untuk mengembangkan potensi akademik ataupun bakat yang dimiliki oleh peserta didik. Sebagaimana telah dijelaskan dalam undang-undang Nomor 20 Tahun 2003 pasal 1 tentang Sistem Pendidikan Nasional, dijelaskan bahwa pendidikan merupakan usaha sadar dan terencana untuk mewujudkan suasana belajar dan proses pembelajaran agar peserta didik secara aktif mengembangkan potensi dirinya untuk memiliki kekuatan spiritual, keagamaan, pengendalian diri, kepribadian, kecerdasan, akhlak mulia, serta keterampilan yang kelak akan diperlukan oleh dirinya, masyarakat, bangsa serta negaranya. Menurut Feida Noorfaila dalam bukunya yang berjudul teori-teori belajar dalam pendidikan Belajar adalah kunci yang paling utama dari setiap usaha yang dilakukan dalam lingkungan pendidikan, dengan demikian tanpa belajar tidak akan pernah ada yang namanya pendidikan. Belajar sebagai suatu proses dan belajar selalu mendapat tempat yang luas dalam berbagai disiplin ilmu yang ada kaitannya dengan upaya pendidikan. Belajar bukan sekedar pengalaman, belajar berlangsung secara aktif dan intergrative dengan berbagai bentuk perbuatan untuk mencapai suatu tujuan. Sebagai mana menurut Gagne, belajar merupakan suatu proses dimana suatu organisasme berubah perilakunya sebagai akibat pengalaman. Perubahan yang terjadi di sini dimaksudkan perubahan perilaku dalam proses belajarnya. Menurut Gagne (Dahar, 2011:2) belajar merupakan suatu proses dimana suatu organisme berubah perilakunya sebagai akibat pengalaman. Ada penekanan bahwa belajar itu menyangkut perubahan dalam suatu organisme. Perubahan yang terjadi di sini adalah perubahan perilaku dalam proses belajar. Kemudian menurut S. Nasution M.A., belajar sebagai perubahan kelakuan, pengalaman, dan latihan. Jadi belajar membawa suatu perubahan pada diri individu yang belajar. Perubahan itu tidak hanya mengenai sejumlah pengalaman, pengetahuan, melainkan juga membentuk kecakapan, kebiasaan, sikap, pengertian, minat, peyesuaian diri. Dalam hal ini meliputi segala aspek organisasi atau pribadi individu yang belajar. Sardiman A.M. berpendapat bahwa belajar itu merupakan perubahan tingkah laku atau penampilan dengan serangkaian kegiatan misalnya dengan membaca, mengamati, mendengarkan, meniru, dan sebagainya. Ngalim Purwanto juga berpendapat bahwa belajar berhubungan dengan perubahan tingkah laku seorang terhadap sesuatu situasi tertentu yang disebabkan oleh pengalamanya yang berulang-ulang dalam situasi itu, dimana perubahan tingkah laku itu tidak dapat dijelaskan atau dasar kecerendungan respon pembawaan, kematangan atau keadaan-keadaan sesaat seseorang. Konsep dasar belajar merupakan kegiatan yang berproses dalam memakai unsur yang sangat fundamental dalam penyelenggaraan setiap jenis dan jenjang pendidikan. Subjek Penelitian Subjek penelitian ini dipilih berdasarkan pada pertimbangan bahwa informan dapat memberikan informasi yang lengkap dan terlibat langsung dalam penerapan konsep teori belajar, seperti tujuan, strategi, pengarahan yang diberikan, motivasi hingga evaluasi di sanggar Seni Kinanti Sekar. Subjek penelitian ini adalah Pendiri Sanggar Seni, Tenaga pendidik dan pelatih sanggar serta peserta didik Sanggar Seni Kinanti Sekar. p gg p gg Nama : Nur Alfiyah Jabatan : Manajer di Sanggar Seni Kinanti Sekar Hasil Penelitian Berdasarkan data yang diperoleh maka dapat diuraikan hasil penelitian sebagai berikut : 1. Deskripsi Tempat Penelitian Sanggar Seni Kinanti Sekar dapat dikatakan sebagai Sekolah Non Formal, sanggar ini mendapat pembinaan langsung dari Dinas Kebudayaan Yogyakarta. Berdiri pada bulan Juli tahun 2015 di Prawirodirjan, Kec. Gondomanan, Kota Yogyakarta, DIY. Didirikan oleh perempuan yang bernama Kinanti Sekar Rahina dan di pimpin oleh Bagas Arga Santosa. Seiring berjalannya waktu, Sanggar Seni Kinanti Sekar ini tidak hanya terbatas pada pengajaran tari tradisional dan daerah melainkan juga pembelajaran budaya Jawa lainnya seperti aksara Jawa dan tetembangan. Selain kelas tari ada beberapa program yang memang aktif diajarkan disana seperti ; Klub tembang jawa, klub kerawitan, dan klub baca tulis aksara jawa. Namun untuk ketiganya belum mulai aktif kembali dikarenakan adanya pandemi Covid-19 yang menyerang di Indonesia tahun 2019 silam. Sekolah non formal Sanggar Seni Kinanti Sekar memiliki orientasi yang baik dibuktikan dengan pendidik atau para pengajar dan sistem mengajarnya. Peserta didik disana dipersiapkan dengan matang untuk dapat dan mampu berkolaborasi dalam dunia kesenian. Selain itu tujuan utamanya adalah melestarikan budaya lokal jawa dan diharapkan bermanfaat untuk melatih kepercayaan diri serta memiliki rasa sosial yang tinggi agar lebih peka terhadap lingkungan sekitarnya. Sekolah non formal Sanggar Seni Kinanti Sekar memiliki orientasi yang baik dibuktikan dengan pendidik atau para pengajar dan sistem mengajarnya. Peserta didik disana dipersiapkan dengan matang untuk dapat dan mampu berkolaborasi dalam dunia kesenian. Selain itu tujuan utamanya adalah melestarikan budaya lokal jawa dan diharapkan bermanfaat untuk melatih kepercayaan diri serta memiliki rasa sosial yang tinggi agar lebih peka terhadap lingkungan sekitarnya. jenis program dan kelas yang ditawarkan dan bisa diikuti oleh umum dimulai dari anak usia 8 tahun hingga tidak terbatas, dengan artian semua dapat merasakan program kesenian yang mereka inginkan. Selain kelas pelatihan yang mencakup kelas tari anak- anak, kelas tari dewasa (kreasi dan klasik), kelas aksara Jawa, tetembangan, dan kelas pantomime. Ada banyak program yang bisa diikuti oleh masyarakat di berbagai kalangan. Dimulai dari Workshop, Podcast Cerita Kinanthi, Jumat Wagen, Kelas Alam, Berkunjung ke Rumah Nenek, Klithih Kwaruh, hingga sowan sanggar yang ditawarkan disana. Tentunya dalam setiap kelas dan program yang disajikan memiliki daya tarik tersendiri bagi para pendidik dan peserta didiknya. jenis program dan kelas yang ditawarkan dan bisa diikuti oleh umum dimulai dari anak usia 8 tahun hingga tidak terbatas, dengan artian semua dapat merasakan program kesenian yang mereka inginkan. Hasil Penelitian Selain kelas pelatihan yang mencakup kelas tari anak- anak, kelas tari dewasa (kreasi dan klasik), kelas aksara Jawa, tetembangan, dan kelas pantomime. Ada banyak program yang bisa diikuti oleh masyarakat di berbagai kalangan. Dimulai dari Workshop, Podcast Cerita Kinanthi, Jumat Wagen, Kelas Alam, Berkunjung ke Rumah Nenek, Klithih Kwaruh, hingga sowan sanggar yang ditawarkan disana. Tentunya dalam setiap kelas dan program yang disajikan memiliki daya tarik tersendiri bagi para pendidik dan peserta didiknya. g p p p y 2. Deskripsi Data Penelitian Berdasarkan perolehan data hasil penelitian dari observasi, wawancara dan dokumentasi yang dilakukan secara langsung di Sanggar Seni Kinanti Sekar, maka dapat diuraikan dan disajikan dalam pembahasan hasil penelitian sebagai berikut : a. Sistem pembelajaran dan Proses belajar tari maupun aksara jawa di Sanggar Seni Kinanti Sekar a. Sistem pembelajaran dan Proses belajar tari maupun aksara jawa di Sanggar Seni Kinanti Sekar JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 Berdasarkan hasil penelitian di Sanggar Seni Kinanti Sekar Sistem pembelajaran yang digunakan sama halnya seperti di sekolah pada umumnya karena terdapat sistem caturwulan (4 bulan sekali), dimana mereka melaksanakan ujian. Sistem ini berlaku di kelas tari. Kelas tari sendiri terbagi menjadi dua yaitu kelas tari anak dan kelas tari dewasa. Proses belajar aksara Jawa seperti kelompok belajar dimana setiap bulannya memiliki siswa yang berbeda- beda. Dalam satu bulannya merekrut maksimal 10 orang untuk mengikuti KBM selama empat kali pertemuan, dihari Selasa akan mendapatkan beberapa materi tentang aksara Jawa. Untuk bulan berikutnya memberikan kesempatan pada siswa lain. adi memang memberikan kesempatan kepada semua orang untuk dapat terlibat dalam proses belajar aksara Jawa. Tapi kalau kelas tari ada sistem caturwulan jadi 4 bulan sekali diadakan ujian dan KBM dilaksanakan pada hari Senin sampai Sabtu. JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 Berdasarkan hasil penelitian di Sanggar Seni Kinanti Sekar Sistem pembelajaran yang digunakan sama halnya seperti di sekolah pada umumnya karena terdapat sistem caturwulan (4 bulan sekali), dimana mereka melaksanakan ujian. Sistem ini berlaku di kelas tari. Kelas tari sendiri terbagi menjadi dua yaitu kelas tari anak dan kelas tari dewasa. Proses belajar aksara Jawa seperti kelompok belajar dimana setiap bulannya memiliki siswa yang berbeda- beda. Hasil Penelitian Dalam satu bulannya merekrut maksimal 10 orang untuk mengikuti KBM selama empat kali pertemuan, dihari Selasa akan mendapatkan beberapa materi tentang aksara Jawa. Untuk bulan berikutnya memberikan kesempatan pada siswa lain. adi memang memberikan kesempatan kepada semua orang untuk dapat terlibat dalam proses belajar aksara Jawa. Tapi kalau kelas tari ada sistem caturwulan jadi 4 bulan sekali diadakan ujian dan KBM dilaksanakan pada hari Senin sampai Sabtu. j p p b. Arahan dan Bimbingan yang diberikan Pelatih atau Pengajar dalam pembelajaran Budaya Jawa berupa Tarian Tradisional di Sanggar Seni Kinanti Sekar b. Arahan dan Bimbingan yang diberikan Pelatih atau Pengajar dalam pembelajaran Budaya Jawa berupa Tarian Tradisional di Sanggar Seni Kinanti Sekar Berdasarkan hasil penelitian di Sanggar Seni Kinanti Sekar Seluruh pelatih atau pengajar di Sanggar Seni Kinanti Sekar harus paham betul mengenai budaya jawa dan tarian tradisional jawa yang akan diajarkan. Seperti contohnya tari klasik, pengajar atau pelatih disana harus tau bagaimana cara untuk memakai Jarik, memakai sampur, stagen dan properti-properti yang digunakan dalam Kegiatan Belajar Mengajar. Dengan demikian tenaga pendidik disana dapat memberikan arahan dan bimbingan kepada peserta didik kaitannya dengan gerakan tari yang benar serta pemakaian properti yang benar. Point plus dalam belajar bukan hanya dari pemahaman tentang gerak tari, tapi juga memahami seluruh pendukung atau properti dalam tarian tersebut. c. Cara Pelatih atau Pengajar dapat mengidentifikasi karakteristik setiap siswanya, kaitannya dengan cepat tidaknya siswa dalam menangkap pembelajaran c. Cara Pelatih atau Pengajar dapat mengidentifikasi karakteristik setiap siswanya, kaitannya dengan cepat tidaknya siswa dalam menangkap pembelajaran c. Cara Pelatih atau Pengajar dapat mengidentifikasi karakteristik setiap siswanya, kaitannya dengan cepat tidaknya siswa dalam menangkap pembelajaran Berdasarkan hasil penelitian di Sanggar Seni Kinanti Sekar Hal tersebut biasanya terjadi di kelas anak yang usianya rentan 4 hingga 7 tahun. Di usia tersebut mereka memiliki karakter yang beragam sekali. Ada yang pendiam, ada yang hiperaktif hingga ada yang sampai harus ditemani orangtuanya namun pelatih atau pengajar disana akan mengarahkan mereka. Pelatih atau pengajar disana lebih kalem dan titen untuk memperhatikan karakteristik para siswanya, untuk siswa yang pendiam pelatih atau pengajar disana tidak terlalu memaksa keinginan anak untuk bergerak sesuai dengan teman lainnya. Karena hal tersebut harus timbul dari dalam diri anak yang bersangkutan. Namun yang paling mengesankan jika didalam satu kelas terdapat lima anak dan diantara mereka terdapat anak yang pendiam tapi terdapat satu anak yang hiperaktif hal tersebut akan sangat membantu, karena anak yang hiperaktif biasanya diarahkan untuk mengajak teman-temannya. Anak tersebut dapat memberikan energi yang baik untuk teman teman lainnya. Sehingga mereka akan berani dalam mengekspresikan dirinya. Selain dukungan dari pelatih atau pengajar dan teman temannya hal tersebut juga tidak luput dari dukungan orang tua. Jadi dapat disimpulkan untuk mengidentifikasi karakter akan sangat terlihat di kelas anak yang berusia dini berkisar 4 hingga 7 tahun. d. Pelatihan ragam gerak bagi siswa yang baru berkecimpung dalam dunia tari Berdasarkan hasil penelitian di Sanggar Seni Kinanti Sekar Palatih atau pengajar disana akan lebih fokus pada anak yang baru pertama kali menari, mereka akan tetap sabar dan tekun dalam membimbing anak tersebut. Biasanya anak yang telah menguasai tarian (Senior) akan diberikan pengertian untuk membantu anak baru, karena hal tersebut dapat memberikan energi baik pada anak anak lain agar mereka tergerak untuk bisa. Kelas didalam sanggar dimulai dari tingkatan yang paling mudah, sedang kemudian susah. Pada tingkatan mudah bagi siswa yang baru mulai berkecimpung dalam dunia tari butuh extra perhatian dalam memperhatikan setiap detail gerakannya namun itu tidak menjadi masalah bagi tenaga pendidik disana. Semua siswa memiliki solidaritas yang tinggi, satu energi, satu rasa dengan begitu semua akan mudah dilalui, tidak ada yang memiliki ego besar dan merasa harus menunggu anak yang baru belajar. e. Alokasi Waktu Pembelajaran Seni Tari dalam Sanggar Seni Kinanti Sekar e. Alokasi Waktu Pembelajaran Seni Tari dalam Sanggar Seni Kinanti Sekar Berdasarkan hasil penelitian di Sanggar Seni Kinanti Sekar Kelas anak di Sanggar Kinanti Sekar memiliki alokasi waktu 1 jam dalam setiap pertemuannya, hal tersebut untuk menghindari rasa jenuh setiap siswanya karena anak anak mudah terpecah konsentrasi dan cepat merasa bosan. c. Cara Pelatih atau Pengajar dapat mengidentifikasi karakteristik setiap siswanya, kaitannya dengan cepat tidaknya siswa dalam menangkap pembelajaran Jika kelas dilaksanakan terlalu lama akan mempengaruhi mood mereka. Sedangkan kelas dewasa memiliki alokasi waktu 1,5 jam untuk seriap sesi kelasnya, kelas ini dimulai pada malam hari. Seluruh alokasi waktu tersebut dapat JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 dikatakan efektif karena sudah disesuaikan dengan keadaan siswanya, karena bagi mereka menari adalah sebagian relise dari semua keperakan. JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 JPIn: Jurnal Pendidik Indonesia dikatakan efektif karena sudah disesuaikan dengan keadaan siswanya, karena bagi mereka menari adalah sebagian relise dari semua keperakan. dikatakan efektif karena sudah disesuaikan dengan keadaan siswanya, karena bagi mereka menari adalah sebagian relise dari semua keperakan. f. Kendala dalam Pembelajaran Seni Tari dalam Sanggar Seni Kinanti Sekar h. Langkah- langkah dalam proses Pembelajaran di Sanggar Seni Kinanti Sekar Berdasarkan hasil penelitian di Sanggar Seni Kinanti Sekar langkah langkah untuk proses pembelajaran yang pertama adalah saling mengenal satu sama lain, langkah kedua pelatihh atau pengajar disana harus paham mengenai karakter siswa begitu juga sebaliknya, kemudian setelahnya berdoa sesuai kepercayaan masing masing dan membahas tentang pokok materi mengenai gerak tari. Terdapat kesempatan bagi siswa untuk menggunakan salah satu properti, dimana mereka harus menggunakan jarik, hal ini bertujuan untuk memberikan pemahaman kepada siswa tentang budaya, kemudian dilanjutkan tentang pembelajaran tari, belajar tentang pola lantai. Selain tari Sanggar juga melaksanakan kegiatan pendukung lainnya untuk mendukung mereka seperti Jumat wagen khusus untuk pembelajaran budaya jawa dan juga workshop penunjang seperti tentang cara memakai sanggul, memakai pakaian jawa yang baik dan benar, ada juga kelas alam yang menjadi salah satu sumber inspirasi bagi siapapun untuk mencipta, lalu mengenalkan siswa untuk kembali lagi ke alam, menguasai wiraga, wirama, wirasa, dengan begitu akan tercipta moment bahagia ketika pelatih atau pengajar dekat dengan para siswanya. f. Kendala dalam Pembelajaran Seni Tari dalam Sanggar Seni Kinanti Sekar Berdasarkan hasil penelitian di Sanggar Seni Kinanti Sekar Kendala sebenarnya ada pada waktu, karena memang sebagian besar dari kelas dewasa merupakan mereka yang sudah bekerja dan kuliah, kadang dalam setiap kelasnya ada anak anak yang harus masuk kelas tari tapi mendapat sebuah pekerjaan, jadi anak tersebut harus mendahulukan pekerjaannya. Akhirnya materi yang didapatkan kurang maksimal. Kemudian kendala dari segi Bahasa, sebagian anak yang mengikuti kelas tari bukan penduduk lokal jogja melainkan dari berbagai daerah, jadi penggunaan bahasa jawa akan kurang maksimal dalam pemahaman materinya. Kemudian juga anak sekolah yang dimana anak tersebut mengikuti kegiatan disekolahnya hingga mengakibatkan sanggar harus menyesuaikan kegiatan mereka disekolah. Tenaga pendidik disana juga memperhatikan bagaimana membuat suasana yang baru untuk anak anak ketika mereka sampai sanggar, agar mereka lebih fresh, happy, santai, karena itu sebelum kelas dimulai tenaga pendidik disana akan berdoa kemudian dilanjutkan dengan yal-yel yang identik dengan rasa semangat bertujuan untuk memulai lagi suatu pembelajaran dengan hal yang membahagiakan. g. Cara menciptakan suasana belajar di Sanggar Seni Kinanti Sekar g. Cara menciptakan suasana belajar di Sanggar Seni Kinanti Sekar Berdasarkan hasil penelitian di Sanggar Seni Kinanti Sekar Hal pertama yang dapat dilakukan yaitu saling mengenal siswa, tahu detail- detail tentang siswa tersebut seperti asal sekolah, kesibukan yang sedang dilakukan, tempat tinggal serta hal lainya, hal tersebut dimaksud agar siswa ataupun guru dapat saling mengenal satu sama lain. Biasanya sebelum kelas dimulai seluruh tim wajib mengenal anggotanya, lucunya setelah selesai melakukan pembelajaran anak didik akan mengajarkan pelatih atau pengajar untuk bermain tiktok atau melakukan hal lainya seperti sharing cerita. Dengan begitu mereka bukan seperti menemukan sosok guru yang menakutkan dan formal namun justru malah teman barunya yang memang harus dihormati. Ketika hal tersebut telah terjadi, kedepanya proses belajar dan pembelajaran akan lebih nyaman, tidak ada rasa sungkan. Ketika mereka ingin bercerita tentang apapun pelatih atau pengajar disana bisa menjadi pendengar yang baik, kemudian ketika mereka merasa sedang dalam keadaan atau kesulitan apapun termasuk kesulitan gerak dalam tarian mereka akan lebih berani untuk mengungkapkanya dan sebisa mungkin akan dicari solusinya. Dimulai dari saling mengenal lalu intens untuk cerita hal apapun random (terbuka) dapat menciptakan suasana belajar yang sangat menarik untuk siswanya. Bermain tiktok bukan masalah yang besar walaupun sanggar basicnya adalah budaya tradisi, tapi hal ini juga dapat di manfaatkan sebagai media promosi dan juga media untuk mengenalkan tentang budaya jawa. i. Metode pembelajaran yang diterapkan di Sanggar Seni Kinanti Berdasarkan hasil penelitian di Sanggar Seni Kinanti Sekar menggunakan metode inqury, metode tersebut merupakan metode pembelajaran dimana tenaga pendidik mengajak siswa untuk berpatisipasi dalam proses belajar. Di sanggar tersebut bukan hanya pelatih atau pengajar yang memberikan pemahaman materi namun siswa disana ikut berperan didalamnya, sehingga terjadi timbal balik komunikasi dalam pembelajaran seni antara guru dan siswa. Dengan adanya komunikasi yang baik maka pembelajaranya pun akan terasa menarik atau asik bagi para peserta didik, lebih menarik juga dengan metode konstektual, dengan metode ini dapat mendekatkan peserta didik kepada lingkungan sekitar dan peserta didik pun akan lebih peka dengan peristiwa yang terjadi di lingkungan sekitar dan dapat menciptakan tarian yang disesuaikan dengan konteks JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 masyarakatnya. Dengan begitu peserta didik akan mengetaui tentang lingkungan mereka seperti apa maka rasa kepekaan itu akan muncul oleh pesertanya sendiri kemudian akan ada rasa untuk mengembangkan dan melestarikan budaya. Di Sanggar Tari Kinanti Sekar juga terdapat metode dimana siswa itu akan mencoba lebih ekspresif, jadi setiap pengajar akan menggali potensi yang dimiliki oleh setiap peserta didiknya, dan juga mencoba untuk lebih percaya diri dengan ruang-ruang yang diciptakan. JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 masyarakatnya. Dengan begitu peserta didik akan mengetaui tentang lingkungan mereka seperti apa maka rasa kepekaan itu akan muncul oleh pesertanya sendiri kemudian akan ada rasa untuk mengembangkan dan melestarikan budaya. Di Sanggar Tari Kinanti Sekar juga terdapat metode dimana siswa itu akan mencoba lebih ekspresif, jadi setiap pengajar akan menggali potensi yang dimiliki oleh setiap peserta didiknya, dan juga mencoba untuk lebih percaya diri dengan ruang-ruang yang diciptakan. J.Jenis tari dalam pembelajaran di Sanggar Seni Kinanti Sekar Berdasarkan hasil penelitian di Sanggar Seni Kinanti Sekar jenis tarian yang diajarkan di sanggar yang pertama adalah Tari klasik yang terdiri dari tari Nawung Sekar, tari Sari Kusumo, dan tari Golek Ayun-ayun. Selanjutnya untuk tari Kreasi sebagian besar adalah tari tarian yang diciptakan oleh founder Sanggar Seni Kinanti Sekar yaitu Kinanti Sekar Rahina terdiri dari tari Kenes Gandes, tari Gelegar Nusantara, tari Jampi Gugat, tari Nyawiji, tari Padhang Bulan, dan tari Jaranan. i. Metode pembelajaran yang diterapkan di Sanggar Seni Kinanti Dalam pembelajaran seni tari disana teruntuk anak anak di usia 4-7 tahun biasanya menggunakan tari kreasi, karena pada usia tersebut gerak dan lagu akan lebih ekspresif untuk suasana bahagia dan lebih fun untuk mereka ikuti. Teruntuk usia 7-12 tahun menggunakan tari kreasi yang memang di ajarkan dan di ciptakan oleh founder dari Sanggar yaitu Kinanti Sekar Rahina. Sanggar Kinanti Sekar menggabungkan kedua tarian tersebut dari tari klasik gerakannya yang gerakannya memadukan antara karakter dengan tarianya karena tentunya karakter seorang itu berbeda beda. Tetapi tari kreasi itu awal gerakannya dari tari klasik akan tetapi tari kreasi dari irama dan gerakannya lebih cepat sedikit dibandingkan dengan tari klasik. PEMBAHASAN Penelitian dalam bentuk Observasi dan Wawancara dilakukan di Sanggar Seni Kinanti Sekar di daerah Prawirodirjan, Kecamatan Gondomanan, Kota Yogyakarta, DIY. Peserta didik perlu dibimbing yang dibantu dengan penerapan metode pembelajaran seni tari, banyak sekali jenis metode yang dapat digunakan, seperti metode ceramah, metode diskusi, metode tanya jawab, metode demonstrasi, metode eksperimen, metode resitasi dan lain sebagainya. Kaitannya dengan kegiatan pelatihan seni tari yang meliputi Tari Klasik (Nawung Sekar, Sari kusumo, Golek Ayun ayun) dan Kreasi (Sebaguan besar yang merupakan tari tarian yang diciptakan oleh founder Kinanti Sekar Rahina. Tari Kenes Gandes, tari Gelegar Nusantara, tari Jampi Gugat, tari Nyawiji, tari Padhang Bulan, dan tari Jaranan). Adapun temuan pada proses pelatihan ragam gerak tari Klasik ataupun Kreasi di Sanggar Kinanti Sekar adalah dengan sebuah metode mengajar yang menekankan pada pelatihan, metode ini memiliki kelebihan yang dapat dirasakan peserta didik pada proses pembelajaran tari klasik ataupun kreasi karena membantu peserta didik memahami dengan jelas jalannya suatu proses perpindahan setiap ragam gerakan yang ada dalam suatu tarian. Dalam metodologi pengajaran dapat diukur dari baik buruknya suatu metode pengajaran yang terletak pada tujuan pembelajaran yang hendak dicapai. Tentunya jenis metode pembelajaran sangat banyak dan dalam proses mengajar atau membimbing tenaga pendidik diperbolehkan untuk memilih salah satu atau beberapa metode gabungan yang menurutnya dapat direalisasikan dan dipakai secara efektif kaitannya dalam mencapai tujuan pendidikan yang telah ditentukan di satuan pendidikan. Pelatih atau pengajar hingga teman-teman terutama (senior) yang membawa suasana positif dapat berperan penting dalam proses pelatihan yang sedang berlangsung di sanggar, karena dalam awal pelatihan tentunya peserta didik belum menghasilkan keterampilan gerak yang sempurna. Untuk itu diperlukan proses latihan untuk mengetahui karakteristik dan kesukaran yang dimiliki peserta didik, sehingga dapat memilih atau menentukan evaluasi gerakan yang perlu diperbaiki. Hal tersebut sejalan dengan pendapat para ahli bahwa metode latihan adalah cara mengajar tenaga pendidik yang dimana peserta didik melaksanakan kegiatan- kegiatan latihan, agar peserta didik memiliki ketangkasan dan keterampilan yang lebih tinggi dari apa yang dipelajari (Sagala,213:217). Dengan begitu metode latihan sangat sesuai dan tepat diterapkan dalam pendidikan nonformal terutama pada sanggar Seni Kinanti Sekar. Terdapat juga pembelajaran menggunakan metode “ Mencontoh” dimaksudkan peserta didik mencontoh gerakan yang dicontohkan oleh pelatih atau pengajar di Sanggar Seni Kinanti Sekar. Metode mencontoh dalam pengajaran adalah cara pelatih atau pengajar mengajarkan atau membimbing dalam melatih seni, dimana tujuan yang hendak dicapai adalah hasil akhir yang mirip hingga sama dengan gerakan yang dicontohkan. PEMBAHASAN Mencontoh merupakan cara belajar atau melatih seni tari yang didasarkan atas pertimbangan pikiran, mencontoh merupakan hal yang JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 mudah karena hanya mengamati obyek di depannya namun metode ini juga memerlukan ketajaman pengamatan. Dengan mengamati gerakan yang dilakukan pelatih atau pengajar tari secara berulang-ulang akan menjadikan anak terampil dalam gerak menari, maka dari itu metode mencontoh dapat juga dikatakan efektif. mudah karena hanya mengamati obyek di depannya namun metode ini juga memerlukan ketajaman pengamatan. Dengan mengamati gerakan yang dilakukan pelatih atau pengajar tari secara berulang-ulang akan menjadikan anak terampil dalam gerak menari, maka dari itu metode mencontoh dapat juga dikatakan efektif. mudah karena hanya mengamati obyek di depannya namun metode ini juga memerlukan ketajaman pengamatan. Dengan mengamati gerakan yang dilakukan pelatih atau pengajar tari secara berulang-ulang akan menjadikan anak terampil dalam gerak menari, maka dari itu metode mencontoh dapat juga dikatakan efektif. Metode berikutnya adalah dengan metode Inqury, metode ini mengajak siswa untuk berpartisipasi dalam proses belajarnya. Bukan hanya pengajar atau pelatih yang dapat memberikan pemahaman atau materi tentang seni tari namun siswa juga harus dapat memahami materi tersebut sehingga ada timbal balik komunikasi antara tenaga pendidik dan siswanya. Dengan adanya komunikasi maka pembelajaran akan terasa lebih menarik. Kemudian juga terdapat metode kontekstual, metode ini mendekatkan siswa kepada lingkungan sekitarnya, yang dapat membuat peserta didik lebih peka dengan lingkungan sekitar. Rasa kepekaan itu akan muncul dalam diri peserta didik sehingga akan ada dorongan untuk mengembangkan dan melestarikan budayanya. Di sanggar Kinanti Sekar juga terdapat metode dimana siswa disana mencoba lebih ekspresif, setiap pengajar atau pelatih disana akan menggali potensi yang dimiliki oleh setiap siswanya, dan juga mencoba untuk lebih percaya diri dengan ruang-ruang yang diciptakannya. Berdasarkan pengamatan dilapangan dengan menggunakan metode demonstrasi pada kegiatan pembelajaran yang dilakukan oleh peneliti selama tiga kali pertemuan ini, maka penulis menyimpulkan bahwa proses pembelajaran tari klasik dan kreasi melalui langkah-langkah penerapan dengan menggunakan metode demonstrasi pemberian materi, mencontohkan gerakan, memberikan waktu untuk latihan, mengoreksi gerakan yang dilakukan oleh peserta didik, meminta peserta untuk mengulang kembali dan memberikan evaluasi pada akhir pembelajaran dan pengambilan nilai kelompok. Pembelajaran ini dilakukan secara bersama- sama proses pembelajaran demonstrasi ini dilakukan di setiap pertemuannya. Proses pembelajaran tari klasik dan kreasi ini telah melakukan tahap-tahap pembelajaran demonstrasi, yaitu dari proses pembelajaran dimana pelatih memakai teori behavioristik. PEMBAHASAN Dilakukan dengan memberikan penjelasan mengenai teori yang akan dipelajari, mencontohkan, memberikan waktu latihan, dilakukan dengan memberikan waktu latihan kepada siswi untuk berlatih bersama teman- temannya secara perlahan. Alokasi waktu yang digunakan dalam proses pelatihan juga relatif sesuai berkisar 1 jam hingga 1,5 jam, mengingat pelatihan tari dalam penyampaian materi tidak harus terburu-buru. Hal ini sesuai dengan teori abdulhak (2012;21) bahwa waktu penyelenggara relative singkat dan pada umumnya berkesinambungan. Selain teori belajar Behavioristik terdapat juga teori Teori belajar konstruktivisme menjelaskan bahwa siswa akan membangun pengetahuannya sendiri berdasarkan pengetahuan yang berasal dari pengalaman sebelumnya, pengetahuan tentang objek di dunia, dan biasanya akan dimulai dari karakteristik manusia masa depan yang diharapkan, konstruksi pengetahuan, serta proses belajar menurut teori konstruktivisme. Piaget menyatakan bahwa anak-anak yang ingin mengetahui dan mengkonstruksi pengetahuan tentang objek di dunia, mereka mengalami dan melakukan tindakan tentang objek yang diketahuinya dan mengkonstruksi objek itu berdasarkan pemahaman mereka. Karena pengertian mereka terhadap objek itu dapat mengatur realitas dan tindakan mereka.(Hendrowati& Yuni,Tri, 2015:4). Teori belajar kontruksivisme ini tentunya melibatkan kemampuan kreativitas untuk mengkontruksi pengetahuan. Kreativitas merupakan proses kemampuan seseorang untuk menciptakan produk baru dengan kombinasi, untuk mencipta sesuatu yang baru. Hal ini sejalan dengan penjelasan Hendrick bahwa kreativitas adalah proses mengungkapkan pengalaman masa lalu dan menempatkan pengalaman bersama dalam pola baru, ide, atau produk baru (Maharani, 2017:3). Ditemukan juga teori belajar Sosiokultural yang dimana menghendaki agar dalam proses pembelajaran individu dilibatkan secara aktif dalam suatu setting sosial dan interaksi sosial. Dengan demikian, proses pembelajaran harus memberikan tempat bagi nilai-nilai budaya. Koentjoroningrat menyatakan bahwa budaya adalah "daya dari budi" yang berupa cipta, karsa, dan rasa. Sedangkan kebudayaan adalah hasil dari cipta, karsa, dan rasa tersebut (Sujarwa, 2010: 28). Pembelajaran berbasis budaya merupakan strategi penciptaan lingkungan belajar dan perancangan pengalaman belajar yang mengintegrasikan budaya sebagai bagian dari proses pembelajaran. Pembelajaran berbasis budaya merupakan perluasan konsepsi teori belajar kultural karena telah menempatkan budaya dalam proses pembelajaran untuk mencapai tujuan pembelajaran yang diharapkan. Disamping telah memenuhi penanaman nilai-nilai universal sebagai bagian yang tidak terpisahkan dalam proses pendidikan. Pendidikan merupakan proses untuk merubah tingkah laku ke arah yang positif, sehingga pendidikan yang terbaik adalah pendidikan yang tidak hanya berfokus pada penguasaan kognisi, tetapi juga afeksi dan psikomotor siswa. Sesuai dengan metode yang digunakan dalam sanggar yaitu metode Inqury dan Kontektual, dimana keduanya sama-sama menekankan pada pembelajaran di lingkungan sekitar dan dapat menimbulkan rasa kepekaan dalam diri peserta didik untuk mengembangkan dan melestarikan budaya. PEMBAHASAN Metode pembelajaran inquiry merupakan bentuk dari pendekatan pembelajaran yang berorientasi kepada siswa (student centered approach). Inquiry menyediakan siswa beraneka ragam pengalaman konkrit dan pembelajaran aktif yang mendorong dan memberikan ruang dan peluang kepada siswa untuk mengambil inisiatif dalam mengembangkan keterampilan pemecahan masalah, pengambilan keputusan, dan penelitian sehingga memungkinkan mereka menjadi pebelajar sepanjang hayat. Inqury melibat komunikasi yang berarti tersedia suatu ruang, peluang, dan tenaga bagi siswa untuk mengajukan pertanyaan dan pandangan yang logis, obyektif, dan bermakna, dan untuk melaporkan hipotesis mereka. Tugas guru adalah menyediakan ruang untuk memberikan kesempatan kepada siswa mengembangkan hipotesis dan secara terbuka membuktikan kebenaran hipotesis yang diajukannya (Trianto 2007:140). Maka dari itu komunikasi yang baik antara tenaga pendidik dan peserta didik sangatlah penting karena JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 dengan begitu terdpat timbal balik dalam pembelajaran, siswa di sanggar paham akan materi dari dan lingkungan atau suasana belajar menjadi lebih menarik. JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume 06, No. 01, 2023 dengan begitu terdpat timbal balik dalam pembelajaran, siswa di sanggar paham akan materi dari dan lingkungan atau suasana belajar menjadi lebih menarik. dengan begitu terdpat timbal balik dalam pembelajaran, siswa di sanggar paham akan materi dari dan lingkungan atau suasana belajar menjadi lebih menarik. Ucapan Terima Kasih (12 pt, bolt) p p , Ditujukan kepada berbagai pihak yang membantu penelitian, misalnya sponsor penelitian dan narasumber. Ucapan terima kasih bersifat optional, dapat dituliskan ataupun tidak. Daftar Pustaka (12 pt, bolt) Dahar, Ratna Wilis. 1998. Teori-teori Belajar. Jakarta: P2LPTK Simpulan (12 pt, bolt) Berdasarkan hasil penelitian Deskriptif kualitatif yang berjudul Analisis Teori Belajar Pembelajaran Seni Tari di Sanggar Seni Kinanti Sekar di Prawirodirjan, Kec. Gondomanan, Kota Yogyakarta diperoleh dan dapat menjawab rumusan disetiap proses pembelajaran. Terdapat beberapa metode yang digunakan seperti metode latihan, metode mencontoh, metode inqury, metode kontektual serta metode yang membuat peserta didik disana lebih ekspresif Ucapan Terima Kasih (12 pt, bolt) Daftar Pustaka (12 pt, bolt) JURNAL TEORI DAN PRAKTIK, VOL. 5, NO. 2 TAHUN 2020. Nugraha, Erika Sulistya. (2018). Inquiry Berwawasan Sosiokultural Untuk Meingkatkan Keterampilan Proses Sains Dan Motivasi Belajar Siswa Pada Pembelajaran IPA. Semarang Putra, I. M. D. A. (2021). Eksplorasi Gamelan Angklung dan Selonding Sebagai Media Ungkap Dalam Penciptaan Karya Musik Segara Widya: Jurnal Hasil Penelitian dan Pengabdian Masyarakat, Vol. 9, No. 1 Tahun 2021. Rachman Poppy,dkk. (2022). Manajemen Pembelejaran di Sanggar Belajar Sejati Desa Jatisurip. Jurnal Pendidikan dan Konseling, Vol.04, No. 4. Tahun 2022. https://journal.universitaspahlawan.ac.id/index.php/jpdk/article/downl oad/5577/4 014 Sagala. (2013). Konsep dan makna pembelajaran. Bandung: Alfabeta. Daftar Pustaka (12 pt, bolt) Damayanti Susi, dkk. (2021). Sosialisasi Peran Apresiasi Untuk Meningkatkan Kesiapan Belajar Anak di Sanggar Genius Yayasan Yatim Mandiri Cabang Kediri. Jurnal Pengabdian Masyarakat Pendidikan Dasar, Vol.1, No. 1 Tahun 2021. https://ojs.unpkediri.ac.id/index.php/dedikasi/article/download/16065/2102 Hanum Mutia Laila. (2020). Penerapan Konsep Belajar Merdeka di Taman Anak Sanggar Anak alam Nitiprayan Kasihan Bantul Yogyakarta. Jurnal Pendidikan anak usia dini, Vol. 9, No.5 Tahun 2020. https://journal.student.uny.ac.id/index.php/pgpaud/article/download/16970/16388 Hanum Mutia Laila. (2020). Penerapan Konsep Belajar Merdeka di Taman Anak Sanggar Anak alam Nitiprayan Kasihan Bantul Yogyakarta. Jurnal Pendidikan anak usia dini, Vol. 9, No.5 Tahun 2020. JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume xx, No. xx, Edisi Tahun asimilasi dan akomodasi teori kontruktivisme Piaget. Jurnal e- DuMah, Vol. 1, No.1 Tahun 2015. Isti`adah, Noofalia Feida. (2020). Teori-Teori Belajar Dalam Pendidikan. Jawa Barat: EDU Publisher. II. MAHARANI, A. (2020). MENINGKATKAN PEMAHAMAN BAHASA INDONESIA LISAN, KREATIVITAS DAN KETERAMPILAN SOSIAL ANAK MELALUI CERITA BERGAMBAR. JURNAL TEORI DAN PRAKTIK, VOL. 5, NO. 2 TAHUN 2020. Nugraha, Erika Sulistya. (2018). Inquiry Berwawasan Sosiokultural Untuk Meingkatkan Keterampilan Proses Sains Dan Motivasi Belajar Siswa Pada Pembelajaran IPA. Semarang Putra, I. M. D. A. (2021). Eksplorasi Gamelan Angklung dan Selonding Sebagai Media Ungkap Dalam Penciptaan Karya Musik Segara Widya: Jurnal Hasil Penelitian dan Pengabdian Masyarakat, Vol. 9, No. 1 Tahun 2021. Rachman Poppy,dkk. (2022). Manajemen Pembelejaran di Sanggar Belajar Sejati Desa Jatisurip. Jurnal Pendidikan dan Konseling, Vol.04, No. 4. Tahun 2022. https://journal.universitaspahlawan.ac.id/index.php/jpdk/article/downl oad/5577/4 014 Sagala. (2013). Konsep dan makna pembelajaran. Bandung: Alfabeta. Setinawati Rahmida, dkk. (2022). Meningkatkan Kemampuan Eksplorasi Gerak Tari dengan Metode Karya Wisata. Jurnal Pendidikan Tari, Vol.3, No. 1 Tahun 2022. http://journal.unj.ac.id/unj/index.php/jpt/article/download/29370/1 3606 Sujarwa. (2010). Ilmu Sosial Budaya Dasar ( Manusia dan Fenomena Sosial Budaya). III. YOGYAKARTA : PUSTAKA PELAJAR. Suciyanti,dkk. 2012. Teori Belajar Kultural Dan Perluasan Konsepsinya. Yogyakarta Trianto. (2009). Model Pembelajaran Terpadu Dalam Teori dan Praktek. Jakarta : Prestasi IV. BELAJAR PUBLISHER JPIn: Jurnal Pendidik Indonesia p-ISSN 2722-8134, e-ISSN 2620-8466 Volume xx, No. xx, Edisi Tahun asimilasi dan akomodasi teori kontruktivisme Piaget. Jurnal e- DuMah, Vol. 1, No.1 Tahun 2015. asimilasi dan akomodasi teori kontruktivisme Piaget. Jurnal e- DuMah, Vol. 1, No.1 Tahun 2015. Isti`adah, Noofalia Feida. (2020). Teori-Teori Belajar Dalam Pendidikan. Jawa Barat: EDU Publisher. II. MAHARANI, A. (2020). MENINGKATKAN PEMAHAMAN BAHASA INDONESIA LISAN, KREATIVITAS DAN KETERAMPILAN SOSIAL ANAK MELALUI CERITA BERGAMBAR. JURNAL TEORI DAN PRAKTIK, VOL. 5, NO. 2 TAHUN 2020. II. MAHARANI, A. (2020). MENINGKATKAN PEMAHAMAN BAHASA INDONESIA LISAN, KREATIVITAS DAN KETERAMPILAN SOSIAL ANAK MELALUI CERITA BERGAMBAR. Sagala. (2013). Konsep dan makna pembelajaran. Bandung: Alfabeta. Setinawati Rahmida, dkk. (2022). Meningkatkan Kemampuan Eksplorasi Gerak Tari dengan Metode Karya Wisata. Jurnal Pendidikan Tari, Vol.3, No. 1 Tahun 2022. Setinawati Rahmida, dkk. (2022). Meningkatkan Kemampuan Eksplorasi Gerak Tari dengan Metode Karya Wisata. Jurnal Pendidikan Tari, Vol.3, No. 1 Tahun 2022. http://journal.unj.ac.id/unj/index.php/jpt/article/download/29370/1 3606 Sujarwa. (2010). Ilmu Sosial Budaya Dasar ( Manusia dan Fenomena Sosial Budaya). III. YOGYAKARTA : PUSTAKA PELAJAR. Suciyanti,dkk. 2012. Teori Belajar Kultural Dan Perluasan Konsepsinya. Yogyakarta Trianto. (2009). Model Pembelajaran Terpadu Dalam Teori dan Praktek. Jakarta : Prestasi IV. BELAJAR PUBLISHER Sujarwa. (2010). Ilmu Sosial Budaya Dasar ( Manusia dan Fenomena Sosial Budaya). 1
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Role of occupation on hypertension among women in Bangladesh
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Role of occupation on hypertension among women in Bangladesh Md. Kawsarul Islam  (  kawsar@ms.butex.edu.bd ) Bangladesh University of Textiles Ahbab Mohammad Fazle Rabbi  Bangladesh University of Textiles M. Ershadul Haque  University of Dhaka Zillur Rahman Shabuz  University of Dhaka Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/16 Abstract Background: Hypertension is a major non-communicable disease all over the world. This study determines the role of occupation along with other potential covariates on hypertension for Bangladeshi women. Methods: The analysis had been used the data from Bangladesh demographic and health survey 2017- 18. Univariate and bivariate analyses were performed to check patterns and correlates of hypertension. To explore the influence of demographic, socioeconomic, and biological factors affecting hypertension, binary logistic regression models were used. Results: The prevalence of hypertension among non-working women was 30.31%. The role of occupation on hypertension was significant in both the unadjusted and adjusted models. Women who were not working during the survey were 1.176 times more likely to have hypertension than physically working women while controlling for other variables. In Bangladesh, after controlling the effect of occupation other significant risk factors for hypertension were age, BMI, marital status, education, and region. Conclusion: Occupation is an essential subject for our life and livelihood. Since physically active women were less likely to be hypertensive than non-working women, occupation could be utilized as a significant tool to prevent hypertension. Background High blood pressure, commonly known as hypertension, is a significant public health issue worldwide. According to WHO, hypertension is responsible for heart disease, stroke, kidney failure, disability, and several other non-communicable diseases (NCDs) [1]. Hypertension is a pivotal reason for different cardiovascular diseases (CVDs) [2]. Hypertension-related cardiovascular mortality rates are rising in tandem with the increasing global prevalence of chronic kidney disease, diabetes mellitus and obesity [3]. Almost 26.4% of the adult population in the world had hypertension in 2000 and it was forecasted to 29.2% by 2025 [4]. In 2010 nearly one-third of people aged 20 years or older had hypertension globally [5]. From 1990 to 2019, the number of hypertensive people aged 30–79 become doubled in the world [6]. The prevalence of hypertension is higher in low- and middle-income countries (31.5%) than in high- income countries (28.5%) and it was estimated that three-quarters of the world’s hypertensive population reside in low- and middle-income countries [5]. In low- and middle-income countries the prevalence of hypertension raises due to rapid urbanization, increased life expectancy, unhealthy diet, and lifestyle changes in recent years [7]. The increase in hypertension is remarkable specifically among South Asian countries [8] and other low- and middle-income countries. Between the year 2000 and 2010 the prevalence of hypertension in high-income countries decreased by 2.6% whereas in low-and middle- income countries the prevalence of hypertension increased by 7.7%. A study in 12 high-income countries revealed that Canada, South Korea, Australia, and the UK had the lowest prevalence of hypertension, and Finland had the highest [9]. Page 2/16 Page 2/16 A study in thirteen European countries revealed no relationship between unemployment and hypertension [10]. Findings from Japanese working population proof a little bit that job strain was significantly related to hypertension [11]. There was a positive association between hypertension and occupational stress comes from a combination of high work demands and low job decision latitude [12]. In India, working women were found to have less hypertension compared to their counterparts. It may be fair to presume that a higher risk of hypertension among nonworking women may be due to “inactivity” or “sedentary life” [13] and its concomitant social and economic pressures. In Bangladesh, the prevalence of hypertension was recorded for adults first time in 1976 and it was 1.10% [14]. Background For adults, this scenario has increased over the year ranging from 1.10% in 1976 to more than 25% in 2018. But for the people aged 35 or older, the prevalence of hypertension increased significantly from 32–45% among women and from 20–34% among men during 2011 to 2018 [15]. Several researches have been conducted on pattern and trend of women’s hypertension across the globe [16–22]. In the South Asian region; particularly in India and Nepal; men were more likely to be hypertensive than women but in Bangladesh the scenario was completely opposite [23]. The evidence from other studies depicted that the prevalence of hypertension was higher among women compare to men in Bangladesh [24–26]. Using BDHS 2017-18 data, Khan et al. identified that age, sex, educational level, working status, body mass index (BMI), and diabetes were the main predictors of hypertension [27]. The risk factors associated with hypertension among adults in rural Sylhet district of Bangladesh were age, education, wealth status, body mass index (BMI), waist circumference, smoking status, fruits and vegetables intake, and physical activity [28]. Islam et al. found that area of residence, sex, age, education, occupation, wealth index, body mass index (BMI), blood glucose levels, and waist circumference as the predictors for hypertension [29]. The data of national NCDs Risk Factor Survey of Bangladesh conducted in 2010 revealed that sex, age, education, occupation, wealth index, BMI, alcohol use, diabetes, and tobacco use were the potential risk factors [30]. Hypertension was significantly associated with older age, sex, education, place of residence, working status, wealth index, BMI, and diabetes [31]. A recent study analyzed prevalence, awareness, treatment, and control of hypertension and the association of potential predictors with it [25]. Job-related stress had significant role on hypertension [32, 33]. The status of hypertension of an individual may be influenced by the activities that would undertake a person while doing jobs/income generating process. There is a research gap in explaining how the income generating activities influence the hypertension of women. In this study, emphasize was given to explore the role of employment on pattern of hypothesis among women in Bangladesh. The paper is organized in 5 sections. Section 2 represents the materials and methods including data source and study design, and statistical analysis. The results are presented in Section 3 and the findings are discussed in Section 4. Concluding remarks are placed in Section 5. Variables The response variable of interest was the status of hypertension that has two categories – presence and absence of hypertension. According to NIPORT (2020), individuals were classified as having hypertension if, (i) they had an average systolic blood pressure (SBP) level of 140 mmHg or above, and (ii) they had an average diastolic blood pressure (DBP) level of 90 mmHg or above, or (iii) they were taking antihypertensive medication at the time of the survey [15]. The main covariate of our study was types of occupation and we classified all the mentioned professions into four categories. Respondents were classified as “non-worker” if they were not in a job during survey time; “physical worker” if they worked at home, factory and outside; “professional” if they were in a specific skilled profession and “businessmen” were those people who make their livelihood from different types of small or large business. The wealth index factor scores, obtained from the principal component analysis of the number and kinds of consumer goods the household have, were categorized into poor, middle and rich. The eight administrative divisions were grouped into three regions; (i) Chattogram and Sylhet as Eastern region, (ii) Barishal, Dhaka, and Mymensingh as Central region and (iii) Khulna, Rajshahi, and Rangpur as Western region. Other covariates used in this paper were age at birth (15–34, 35–54, ≥ 55), BMI (categorized as thin, normal, overweight, obese), marital status (never married, married, widowed/divorced), education level (no education, primary, secondary, higher), and place of residence (urban, rural). The response variable of interest was the status of hypertension that has two categories – presence and absence of hypertension. According to NIPORT (2020), individuals were classified as having hypertension if, (i) they had an average systolic blood pressure (SBP) level of 140 mmHg or above, and (ii) they had an average diastolic blood pressure (DBP) level of 90 mmHg or above, or (iii) they were taking Data And Methods Data Page 3/16 A secondary dataset from the BDHS 2017-18 was used for this study, which is available in https://dhsprogram.com/data/available-datasets.cfm. The BDHS 2017-18 is a nationally representative survey in which the whole country was divided into eight administrative divisions: Barishal, Chattogram, Dhaka, Khulna, Mymensingh, Rajshahi, Rangpur, and Sylhet. Each division was divided into zilas, and each district into upazilas. The survey was based on a two-stage stratified sample of households. In the first stage, 675 enumeration areas (EAs) were selected with probability proportional to EA size 250 from the urban areas and 425 from the rural areas. As EA consisting of an average of 120 households. In the second stage a systematic sampling procedure was applied to select an average of 30 households from each EA. A total of 20,250 households were selected from the 675 EAs and 5,063 households (one-fourth of the total households) were selected for collecting biomarker measurements (blood pressure and blood glucose) on all adult age 18 and older. Blood pressure was measured three times in a seated position by trained technicians using digital oscillometric blood pressure measuring device. There were at least five minutes intervals between the measurements. The average of the last two measurements was used to report the respondent’s blood pressure values [15]. In this study, we have considered all women who participated in the biomarker measurements. The selected dataset was filtered for missing values of the covariates considered in this study. Finally, a total of 7295 women were used for statistical analysis. bl Statistical Analysis Univariate analysis (frequency distribution) were performed to understand the basic characteristics of the explanatory variables in the sample. Chi-square tests were carried to examine the association of selected explanatory variables and hypertension status. Binary logistic regression model was employed to examine both the unadjusted and adjusted effects of covariates on hypertension. Covariates found to Page 4/16 Page 4/16 have significant association with the hypertension in the bivariate analysis were considered in the regression analysis. Data analysis were performed using STATA 14 and svyset command was used in both bivariate and regression analysis to address the stratified two-stage cluster sampling design. have significant association with the hypertension in the bivariate analysis were considered in the regression analysis. Data analysis were performed using STATA 14 and svyset command was used in both bivariate and regression analysis to address the stratified two-stage cluster sampling design. Results Approximately 59% of women were not working at the time of the survey whereas only around 3% were professional and businessman. Almost half of the women were aged between 15 to 34 years while only 17.64% of them had age 55 or higher. More than half of the respondents (54.86%) had normal BMI whereas a few (6.15%) of women were obese. Most of the women (79.41%) were married while only 5.47% were never married. The percentage of women who had completed primary education (29.44%) was very close to secondary education (30.57%) whereas about half (14.08%) of them had higher level of education. It was revealed that an equal proportion of women (33.34%) belong to poor, middle class and rich families. The maximum number of women resided in the western region (38.16%) whereas the minimum being in the eastern region (26.24%). Approximately two-third (64.11) of the women lived in rural areas and the remaining lived in urban areas. Variable Frequency n (%) Hypertension (%) p value No Yes Wealth Poor Middle Rich 2432 (33.34) 2432 (33.34) 2431 (33.32) 74.25 [72.24, 76.16] 71.40 [69.37, 73.34] 69.62 [67.74, 71.45] 25.75 [23.84, 27.76] 28.60 [26.66, 30.63] 30.38 [28.55, 32.26] 0.004 Region Eastern Central Western 1914 (26.24) 2597 (35.60) 2784 (38.16) 70.08 [67.77, 72.29] 74.55 [72.64, 76.37] 70.22 [68.33, 72.05] 29.92 [27.71, 32.23] 25.45 [23.63, 27.36] 29.78 [27.95, 31.67] 0.001 Residence Urban Rural 2618 (35.89) 4677 (64.11) 71.32 [69.34, 73.23] 71.99 [70.59, 73.35] 28.68 [26.77, 30.66] 28.01 [26.65, 29.41] 0.583 As mentioned earlier, a total of 7295 women aged 18 and older were considered in this study. The prevalence of hypertension in Bangladesh was 28.19%. Approximately 59% of women were not working at the time of the survey whereas only around 3% were professional and businessman. Almost half of the women were aged between 15 to 34 years while only 17.64% of them had age 55 or higher. More than half of the respondents (54.86%) had normal BMI whereas a few (6.15%) of women were obese. Most of the women (79.41%) were married while only 5.47% were never married. The percentage of women who had completed primary education (29.44%) was very close to secondary education (30.57%) whereas about half (14.08%) of them had higher level of education. It was revealed that an equal proportion of women (33.34%) belong to poor, middle class and rich families. Results Table 1 presents the frequency distribution along with the percentage distribution of each covariate as well as the weighted percentage distribution of prevalence and non-prevalence of hypertension for each category of the selected explanatory variables. This table also depicts the p-values of the chi-square tests that examine the association between hypertension status and each selected covariate. Page 5/16 Table 1 Percentage distribution of hypertension status by different explanatory variables. Variable Frequency n (%) Hypertension (%) p value No Yes Hypertension 7295 (100) 71.81 [70.66, 72.94] 28.19 [27.06, 29.34]   Occupation Not working Physical worker Professional Businessman 4275 (58.60) 2764 (37.89) 135 (1.85) 121 (1.66) 69.69 [68.22, 71.11] 74.58 [72.69, 76.38] 78.37 [69.85, 85.00] 75.54 [65.73, 83.26] 30.31 [28.89, 31.78] 25.42 [23.62, 27.31] 21.63 [15.00, 30.15] 24.46 [16.74, 34.27] < 0.001 Age 15–34 35–54 55+ 3666 (50.25) 2342 (32.10) 1287 (17.64) 87.62 [86.40, 88.75] 61.39 [59.13, 63.60] 44.46 [41.51, 47.45] 12.38 [11.25, 13.60] 38.61 [36.40, 40.87] 55.54 [52.55, 58.49] < 0.001 BMI Thin Normal Overweight Obese 1103 (15.12) 4002 (54.86) 1741 (23.87) 449 (6.15) 80.41 [77.67, 82.88] 76.18 [74.73, 77.57] 60.72 [58.05, 63.33] 53.55 [48.72, 58.31] 19.59 [17.12, 22.33] 23.82 [22.43, 25.27] 39.28 [36.67, 41.95] 46.45 [41.69, 51.28] < 0.001 Marital Status Never married Married Widowed/divorced 399 (5.47) 5793 (79.41) 1103 (15.12) 93.49 [90.21, 95.72] 74.38 [73.11, 75.60] 50.08 [46.82, 53.35] 6.51 [4.275, 9.793] 25.62 [24.40, 26.89] 49.92 [46.65, 53.18] < 0.001 Education No education Primary Secondary Higher 1890 (25.91) 2148 (29.44) 2230 (30.57) 1027 (14.08) 57.57 [55.03, 60.06] 70.78 [68.53, 72.93] 79.79 [77.96, 81.51] 84.48 [82.01, 86.67] 42.43 [39.94, 44.97] 29.22 [27.07, 31.47] 20.21 [18.49, 22.04] 15.52 [13.33, 17.99] < 0.001 Table 1 Page 6/16 Variable Frequency n (%) Hypertension (%) p value No Yes Wealth Poor Middle Rich 2432 (33.34) 2432 (33.34) 2431 (33.32) 74.25 [72.24, 76.16] 71.40 [69.37, 73.34] 69.62 [67.74, 71.45] 25.75 [23.84, 27.76] 28.60 [26.66, 30.63] 30.38 [28.55, 32.26] 0.004 Region Eastern Central Western 1914 (26.24) 2597 (35.60) 2784 (38.16) 70.08 [67.77, 72.29] 74.55 [72.64, 76.37] 70.22 [68.33, 72.05] 29.92 [27.71, 32.23] 25.45 [23.63, 27.36] 29.78 [27.95, 31.67] 0.001 Residence Urban Rural 2618 (35.89) 4677 (64.11) 71.32 [69.34, 73.23] 71.99 [70.59, 73.35] 28.68 [26.77, 30.66] 28.01 [26.65, 29.41] 0.583 As mentioned earlier, a total of 7295 women aged 18 and older were considered in this study. The prevalence of hypertension in Bangladesh was 28.19%. Results The maximum number of women resided in the western region (38.16%) whereas the minimum being in the eastern region (26.24%). Approximately two-third (64.11) of the women lived in rural areas and the remaining lived in urban areas. Chi-square test has been employed to examine whether a specific explanatory variable being associated with hypertension. From Table 1 it was found that all explanatory variables except place of residence (p- value = 0.583) had significant effect on prevalence status at 1% level of significance. Women who were unemployed at the time of the survey had the highest prevalence of hypertension (30.31%) followed by physical worker (25.42%), businessman (24.46%), and professional (21.63%). The prevalence of hypertension increased significantly (p-value < 0.001) with the increase of the women’s age. Obese women had more than twice hypertension than thin women (46.45% versus 19.59%). Almost half of widowed/divorced women had hypertension whereas married women had hypertension slightly over quarter. The prevalence of hypertension decreases significantly (p-value < 0.001) as women became more educated. Wealth and hypertension showed a positive association. Among the women of rich families, 30.38% had hypertension, whereas 25.75% and 28.60% of women of poor and middle class families Page 7/16 Page 7/16 respectively had hypertension. The prevalence of hypertension was almost equal in Eastern and Western region (29.92% and 29.78% respectively). respectively had hypertension. The prevalence of hypertension was almost equal in Eastern and Western region (29.92% and 29.78% respectively). Binary logistic regression models were employed to examine both the unadjusted and adjusted effects of occupation on hypertension. Model-I examine the effect of occupation on hypertension and Model-II observe its effect after adjusting other covariates that were found significantly associated with hypertension in the bivariate analysis. The odds ratios (OR) with p-values of Model-I and Model-II are reported in Table 2. The unadjusted model showed that occupation had a significant effect on hypertension. The findings indicated that women who did not work at the time of the survey were 1.276 times (p-value < 0.001) more likely to have hypertension than physically working women. Page 8/16 Table 2 Table 2 Determinants of hypertension from binary logistic regression models (BDHS 2017-18). Results Variable Model-I Model-II Odds Ratio p value Odds Ratio p value Intercept 0.341 < 0.001 0.364 < 0.001 Occupation Non-worker Physical worker (Ref) Professional Businessman 1.276 0.810 0.950 < 0.001 0.363 0.838 1.176 1.271 1.178 0.023 0.388 0.528 Age 15–34 35–54 (Ref) 55+     0.271 2.092 < 0.001 < 0.001 BMI Thin Normal (Ref) Overweight Obese     0.575 2.359 3.069 < 0.001 < 0.001 < 0.001 Marital Status Never married Married (Ref) Widowed/Divorced     0.658 1.399 0.087 0.001 Education No education (Ref) Primary Secondary Higher     0.917 0.827 0.619 0.326 0.042 0.001 Wealth ( )     1.062 0.457 Middle Rich Region Eastern Central (Ref) Western     1.266 1.289 0.005 0.001 All the explanatory variables were found to have significant effect on hypertension in the adjusted model except wealth index. The results showed that women unemployed were 1.176 times (p-value = 0.023) as likely to have hypertension as women who worked physically. Women aged 15–34 years were approximately 72.9% (p-value < 0.001) less likely to have hypertension whereas women aged 55 years or higher were 2.092 times (p-value < 0.001) as likely to have hypertension as women aged 35–54 years. The likelihood of hypertension was 42.5% lower (p-value < 0.001) among thin women than women with normal weight. On the contrary, overweight and obese women were 2.359 and 3.069 times (p-value <  0.001) respectively, more likely to have hypertension than normal women. Never married women had 34.2% less (p-value = 0.087) hypertension than married women while widowed/divorced women were 1.399 times (p-value = 0.001) more likely to have hypertension. The likelihoods of hypertension were 17.3% (p-value 0.042) and 38.1% (p-value 0.001) lower among women who completed secondary and higher-level of education respectively with compared to uneducated women. Women who lived in Eastern and Western region were 1.266 (p-value = 0.005) and 1.289 (p-value = 0.001) times respectively more likely to have hypertension than women of Central region. All the explanatory variables were found to have significant effect on hypertension in the adjusted model except wealth index. The results showed that women unemployed were 1.176 times (p-value = 0.023) as likely to have hypertension as women who worked physically. Women aged 15–34 years were Discussion The study was carried out to assess the role of occupation on hypertension after adjusting other demographic and socio-economic variables among the women of Bangladesh. As many studies have reported the significant effect of working status on hypertension, we designed the current study to identify how different job categories may influence hypertension among women in Bangladesh. We have observed that occupation played an important role in the prevalence of hypertension. Women who were not working had significantly higher odds of developing hypertension than those who were belonging to physically working in both the unadjusted and adjusted model. This study revealed that among 7295 women aged 18 years and older, 28.19% had hypertension. Whereas 26.4% and 7.5% women had hypertension in India and Ghana respectively [13, 22]. Our study showed that women aged 55 years or higher were more likely to have hypertension in comparison to women aged between 35 and 54 years. On the contrary, women aged 15 to 35 years had lower odds of developing hypertension compare to mid-aged women. Similar findings were observed in previous studies for both men and women [28, 29, 31]. Page 10/16 Page 10/16 Women with their weight were less likely to be hypertensive whereas overweight and obese women had higher likelihoods of occurring hypertension compared to women with normal weight. The result of our study agree with other studies in Italy, China and South Asian countries [23, 34, 35]. BMI may cause a direct positive effect on blood pressure, i.e, blood pressure increases with the increases of BMI [35]. In our analysis, never married women suffered 34.2% less from hypertension than married women. Similar results has been found among the women in Ghana [22]. Married and widowed/divorced women were suffered more from hypertension compared to men in Bhutan [36]. It is not clear enough why married and widowed or divorced women were more likely to be hypertensive, it may occur due to “vicissitudes of marriage” [22]. Taking responsibility of a family is a new experience for women and it might be a reason for hypertension. Further study in this context may bring more insight. Education had a vital role on hypertension and could be used as a potential tool to reduce the prevalence of hypertension [37]. This study was observed that hypertension had decreased gradually with a higher level of education of women. Similar results were obtained for Austrian women [38]. Discussion However, our findings were different than some other low-income countries like Ghana where illiterate or lower educated women were less suffered from hypertension than higher educated women [22]. The variation in hypertension due to geographic location was visible in USA, China, and South Africa [39– 41]. Though Bangladesh is a small country, this study confirmed that there was a significant regional effect on hypertension among women. The women residing both in the Eastern and western region had 1.3 times more hypertension than women in the Central region. Similar findings were obtained in previous work as well [26, 42]. Different socio-economic or socio-cultural structure may cause such regional difference. However, further study may clearly explain such phenomenon. Besides some important findings, this study has some limitations as well. We have basic demographic information in BDHS data, but it does not include detailed qualitative information regarding stress from different job types. This restricted us from definitely concluding job-specific contribution on prevalence of hypertension. The cross-sectional nature of the study could not allow us to check the trend of hypertension among working women which could confirm job-specific effect on hypertension. Acknowledgements We are grateful to MEASURE DHS for giving permission to use BDHS 2017-2018 data. The 1st and 2nd authors were supported by BUTEX research grants 2021-2022. Conclusion Page 11/16 Occupational hazard is well known contributor on different health problem and hypertension is also included among these problem. We found a significant role of occupation on hypertension considering both unadjusted and adjusted models. The prevalence of hypertension among Bangladeshi women was diverse for different job categories like non-worker, physical worker, professional, and businessman due to variations in stress. However, non-worker women were hypertensive approximately 18% more than physically working women. Besides the significant effect of occupation; age, BMI, marital status, education, and region were the determinants of hypertension among Bangladeshi women. The difference in the prevalence of hypertension between urban and rural areas no longer exists which require further investigation. In order to minimize the prevalence of hypertension among Bangladeshi women Page 11/16 stakeholders and government should pay attention to create and increase the facility for physical exercise. stakeholders and government should pay attention to create and increase the facility for physical exercise. Funding BUTEX research grants 2021-2022 has been approved only for conducting this research but not for publication charges. Author contributions MKI, AMFR, MEH and ZRS generated the idea for the study; MKI, AMFR, MEH and ZRS conducted the statistical analysis; MKI drafted the manuscript; AMFR and ZRS contributed in reviewing and editing the manuscript with MKI; ZRS supervised the entire work. All authors read and approved the final manuscript. Funding Abbreviations BDHS Bangladesh Demographic and Health Survey BMI Body mass index CVDs Cardiovascular diseases DBP Diastolic blood pressure NCDs Non-communicable diseases SBP Systolic blood pressure Competing interests There are no competing interests. Author details 1Department of Mathematics and Statistics, Bangladesh University of Textiles, Dhaka, Bangladesh. 2Department of Statistics, University of Dhaka, Dhaka, Bangladesh. 2Department of Statistics, University of Dhaka, Dhaka, Bangladesh. Consent for publication Not applicable Availability of data and materials Ethics approval and consent to participate Page 12/16 For the purpose of analysis, the nationally representative secondary data set BDHS 2017–2018 has been used, which was downloaded from the DHS website after taking permission from MEASURE DHS. Hence no ethics approval was needed. All methods were carried out following the relevant guidelines and regulations approved by ICF Macro Institutional Review Board and National Research Ethics Committee of the Bangladesh Medical Research Council. References 1. WHO. A global brief on Hypertension; World Health Day-2013. World Hea 2. He J, Whelton PK. Epidemiology and prevention of hypertension. Med Clin North Am. 1997;81:1077– 97. 3. Hunter PG, Chapman FA, Dhaun N. Hypertension: Current trends and future perspectives. Br J Clin Pharmacol. 2021;87:3721–36. 3. Hunter PG, Chapman FA, Dhaun N. Hypertension: Current trends and future perspectives. 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Sex differences in hypertension prevalence and control: Analysis of the 2010–2014 Korea National Health and Nutrition Examination Survey. References PLoS One. 2017;12. 22. Nyarko SH. Prevalence and Sociodemographic Determinants of Hypertension History among Women in Reproductive Age in Ghana. Int J Hypertens. 2016;2016. 22. Nyarko SH. Prevalence and Sociodemographic Determinants of Hypertension History among Women in Reproductive Age in Ghana. Int J Hypertens. 2016;2016. 23. Hossain FB, Adhikary G, Chowdhury AB, Shawon MSR. Association between body mass index (BMI) and hypertension in south Asian population: evidence from nationally-representative surveys. Clin Hypertens 2019 251. 2019;25:1–9. 23. Hossain FB, Adhikary G, Chowdhury AB, Shawon MSR. Association between body mass index (BMI) and hypertension in south Asian population: evidence from nationally-representative surveys. Clin Hypertens 2019 251. 2019;25:1–9. 24. Barua L, Faruque M, Rakhshanda S, Banik PC, Shawon RA, Mashreky SR. Baseline prevalence of high blood pressure and its predictors in a rural adult population of Bangladesh: Outcome from the application of WHO PEN interventions. J Clin Hypertens. 2021;23:2042–52. 24. Barua L, Faruque M, Rakhshanda S, Banik PC, Shawon RA, Mashreky SR. Baseline prevalence of high blood pressure and its predictors in a rural adult population of Bangladesh: Outcome from the application of WHO PEN interventions. J Clin Hypertens. 2021;23:2042–52. 25. Iqbal A, Ahsan KZ, Jamil K, Haider MM, Khan SH, Chakraborty N, et al. Demographic, socioeconomic, and biological correlates of hypertension in an adult population: evidence from the Bangladesh demographic and health survey 2017–18. BMC Public Health. 2021;21:1229. Page 14/16 Page 14/16 26. Chowdhury MAB, Islam M, Rahman J, Uddin MT, Haque MR, Uddin MJ. Changes in prevalence and risk factors of hypertension among adults in Bangladesh: An analysis of two waves of nationally representative surveys. PLoS One. 2021;16:e0259507. 27. Khan MN, Oldroyd JC, Chowdhury EK, Hossain MB, Rana J, Renzetti S, et al. Prevalence, awareness, treatment, and control of hypertension in Bangladesh: Findings from National Demographic and Health Survey, 2017–2018. J Clin Hypertens. 2021;23:1830–42. 28. Khanam R, Ahmed S, Rahman S, Kibria GM Al, Syed JRR, Khan AM, et al. Prevalence and factors associated with hypertension among adults in rural Sylhet district of Bangladesh: a cross-sectional study. BMJ Open. 2019;9:1–9. 29. Islam JY, Zaman MM, Haq SA, Ahmed S, Al- Quadir Z. Epidemiology of hypertension among Bangladeshi adults using the 2017 ACC/AHA Hypertension Clinical Practice Guidelines and Joint National Committee 7 Guidelines. J Hum Hypertens. 2018;32:668–80. 30. Rahman M, Zaman MM, Islam JY, Chowdhury J, Ahsan HN, Rahman R, et al. 40. Yin M, Augustin B, Fu Z, Yan M, Fu A, Yin P. Geographic Distributions in Hypertension Diagnosis, Measurement, Prevalence, Awareness, Treatment and Control Rates among Middle-aged and Older Adults in China. Sci Reports 2016 61. 2016;6:1–11. References Prevalence, treatment patterns, and risk factors of hypertension and pre-hypertension among Bangladeshi adults. J Hum Hypertens. 2017;32:334–48. 31. Chowdhury MAB, Uddin MJ, Haque MR, Ibrahimou B. Hypertension among adults in Bangladesh: Evidence from a national cross-sectional survey. BMC Cardiovasc Disord. 2016;16:1–10. 32. Mucci N, Giorgi G, Ceratti SDP, Fiz-Pérez J, Mucci F, Arcangeli G. Anxiety, stress-related factors, and blood pressure in young adults. Front Psychol. 2016;7 October:1–10. 33. Spruill T. Chronic Psychological Stress and Hypertension. Curr Hypertens Rep. 2013;12:10–6. 33. Spruill T. Chronic Psychological Stress and Hypertension. Curr Hypertens 34. Chen H, Zhang R, Zheng Q, Yan X, Wu S, Chen Y. Role of body mass index on long-term blood pressure variability: A cross-sectional study in a cohort of Chinese adults. BMC Public Health. 2018;18:1–8. 35. Landi F, Calvani R, Picca A, Tosato M, Martone AM, Ortolani E, et al. Body mass index is strongly associated with hypertension: Results from the longevity check-up 7 + study. Nutrients. 2018;10:1– 12. 36. Segawa HK, Uematsu H, Dorji N, Wangdi U, Dorjee C, Yangchen P, et al. Gender with marital status, cultural differences, and vulnerability to hypertension: Findings from the national survey for noncommunicable disease risk factors and mental health using WHO STEPS in Bhutan. PLoS One. 2021;16 8 August:1–17. 37. Ekezie J, Anyanwu EG, Danborno B, Anthony U. Role of urbanization on obesity, anthropometric profile and blood pressure in the Igbos of Nigeria. N Am J Med Sci. 2011;3:242–6. 37. Ekezie J, Anyanwu EG, Danborno B, Anthony U. Role of urbanization on obesity, anthropometric profile and blood pressure in the Igbos of Nigeria. N Am J Med Sci. 2011;3:242–6. 38. Kautzky-Willer A, Dorner T, Jensby A, Rieder A. Women show a closer association between educational level and hypertension or diabetes mellitus than males: A secondary analysis from the Austrian HIS. BMC Public Health. 2012;12:1–9. 38. Kautzky-Willer A, Dorner T, Jensby A, Rieder A. Women show a closer association between educational level and hypertension or diabetes mellitus than males: A secondary analysis from the Austrian HIS. BMC Public Health. 2012;12:1–9. 39. Kershaw KN, Diez Roux A V., Carnethon M, Darwin C, Goff DC, Post W, et al. Geographic variation in hypertension prevalence among blacks and whites: The multi-ethnic study of atherosclerosis. Am J Hypertens. 2010;23:46–53. Page 15/16 Page 15/16 40. Yin M, Augustin B, Fu Z, Yan M, Fu A, Yin P. 42. Shanta SS, Sayed A, Mamun A, Rana M. Prevalence and associated factors of pre-hypertension and hypertension among married women in Bangladesh. Genus Homo. 2020;4 January:66–82. 41. Wandai ME, Norris SA, Aagaard-Hansen J, Manda SOM. Geographical influence on the distribution of the prevalence of hypertension in South Africa: A multilevel analysis. Cardiovasc J Afr. 2020;31:47– 53. References Geographic Distributions in Hypertension Diagnosis, Measurement, Prevalence, Awareness, Treatment and Control Rates among Middle-aged and Older Adults in China. Sci Reports 2016 61. 2016;6:1–11. 41. Wandai ME, Norris SA, Aagaard-Hansen J, Manda SOM. Geographical influence on the distribution of the prevalence of hypertension in South Africa: A multilevel analysis. Cardiovasc J Afr. 2020;31:47– 53. 42. Shanta SS, Sayed A, Mamun A, Rana M. Prevalence and associated factors of pre-hypertension and hypertension among married women in Bangladesh. Genus Homo. 2020;4 January:66–82. 42. Shanta SS, Sayed A, Mamun A, Rana M. Prevalence and associated factors of pre-hypertension and hypertension among married women in Bangladesh. Genus Homo. 2020;4 January:66–82. Page 16/16
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Presente e Futuro da Psicologia
Psicologia
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Presente e Futuro da Psicologia Present and Future of Psychology Presente y Futuro de la Psicología Roberto Moraes Cruz Universidade Federal de Santa Catarina, SC, Brasil. Dados do infográfico do Conselho Federal de Psicologia (CFP), atualizados em 17 de outubro de 2016, mostram que, no Brasil, 282.498 psicólogos estão inscritos nos CFPs (CFP, 2016). Ou seja, rapidamente atingiremos a marca de 300.000 psicólogos brasileiros até o final de dessa década, uma das maiores do mundo. Ponto de partida para uma reflexão inicial acerca da qualidade da formação profissional e da formação continuada em Psicologia no Brasil, assim como seus desdobramentos em termos de produção e aplicação de conhecimentos psicoló- gicos relevantes e necessários à sociedade. Ou seja, sobre o presente e o futuro da profissão da Psicologia no país. Crescemos muito. Amadurecemos com a mesma rapidez? Qual a qualidade do processo de profissionalização de psicólogos no país? Na base dessas questões, é possível identificar os seguintes cenários e perspectivas. i Cursos de graduação em Psicologia são responsáveis por fornecer uma formação básica e ampla em diferentes áreas do conhecimento psicológico, desejáveis e úteis à construção de uma carreira profissional. De fato, esse é o horizonte, embora a percepção de que há lacunas no processo de formação, especialmente no âmbito da relação teoria-prática, que dificultam a inserção no mundo do trabalho seja uma queixa comum entre egressos dos cursos de Psico- logia. É possível identificar que, em números gerais, aproximadamente metade dos psicólogos brasileiros não atuam exclusivamente como psicólogos, isto é, complementam sua fonte de renda com outras atividades técnicas, acadêmicas ou administrativas, fato igualmente identifi- cado na primeira pesquisa de caráter nacional, promovido pelo CFP e publicado no livro “Quem é o psicólogo brasileiro?” (Bastos & Gomide, 1989). Quais implicações tem esse fato no processo de profissionalização dos psicólogos? i A qualidade dos processos de ensino-aprendizagem na graduação, o baixo consenso sobre quais devem ser as diretrizes curriculares orientadoras da formação de psicólogos no país, a cultura pouco sólida de formação continuada e a escassez de programas de formação de professores de Psicologia são, certamente, fatores importantes a serem considerados na discussão sobre as restrições e dificuldades enfrentadas pelos psicólogos na construção e manutenção de uma carreira profissional profícua. Psicologia: Ciência e Profissão Out/Dez. 2016 v. 36 n°4, 783-786. DOI: 10.1590/1982-3703003642016 Psicologia: Ciência e Profissão Out/Dez. 2016 v. 36 n°4, 783-786. DOI: 10.1590/1982-3703003642016 Psicologia: Ciência e Profissão Out/Dez. 2016 v. 36 n°4, 783-786. DOI: 10.1590/1982-3703003642016 Psicologia: Ciência e Profissão Out/Dez. 2016 v. 36 n°4, 783-786. DOI: 10.1590/1982-3703003642016 Presente e Futuro da Psicologia Present and Future of Psychology Presente y Futuro de la Psicología i No âmbito da formação profissional básica, verificam-se mudanças curriculares sem o devido planejamento do ensino orientado para as perspectivas de inserção do psicólogo no mundo do trabalho (prestação de serviços para as instituições e comunidade) e, muitas vezes, descoladas das Diretrizes Curriculares Nacionais (DCN) para a área da Psicologia. Há uma redução, cada vez mais acentuada nos fluxos curriculares, de disciplinas metodológicas e técnicas e voltadas à intervenção psicológica, assim como uma valorização de conteúdos programáticos e de experiências mais genéricas que, embora possam ser enriquecedores para a formação geral do cidadão (as chamadas disciplinas transversais ou temas diversos), restringem cada vez mais o ensino e o desenvolvimento de competências específicas à atuação dos psicó- logos. A carência de programas de formação de professores em Psicologia, de metodologias de programação de ensino, de avaliação de processos de ensino-aprendizagem e da inserção dos egressos dos cursos de Psicologia acentuam a generalização de opiniões sobre o que deve ser Disponível em www.scielo.br/pcp Psicologia: Ciência e Profissão Out/Dez. 2016 v. 36 n°4, 783-786. Além disso, currículos, por definição, são estru- turas programáticas que precisam estar atentas às mudanças na sociedade e no desenvolvimento da Psicologia como ciência e da profissão, sob pena de não refletirem a realidade e dificultarem o processo de inserção dos egressos dos cursos de Psicologia no mundo do trabalho e nas diferentes especialidades em Psicologia. É assim, por exemplo, que ainda perma- necem restritas, quando não ausentes nos currículos de Psicologia no Brasil, perspectivas e matérias científicas tais como Psicologia baseada em evidências, Psicofi- siologia, Neurociências, Psicoimunologia, Psicofarma- cologia, Ergonomia, Psicologia Ambiental, Psicologia do Esporte e do Exercício, Psicomotricidade, Psicologia Jurídica, Psicogerontologia, dentre outras. ensinado e como devem ser ensinados os conteúdos programáticos de matérias psicológicas e afins. i Disciplinas do denominado núcleo comum, presumivelmente aquele que mantém, em seu domínio, as chamadas disciplinas básicas para a formação do psicólogo, são menos prestigiadas na contratação de novos docentes, em detrimentos das chamadas ênfases curriculares. Assim, geralmente há uma menor estabilidade de professores no núcleo comum, cujas disciplinas são ocupadas por profes- sores menos experientes, estagiários docentes, profes- sores temporários e assemelhados. Com isso, aparen- temente sai fortalecido o espectro profissionalizante do currículo, mas que sofre influência exatamente das lacunas na formação do núcleo comum. Presente e Futuro da Psicologia Present and Future of Psychology Presente y Futuro de la Psicología Ainda assim, não é incomum, nos estágios curriculares, perceber a angústia dos alunos por não saber como agir em situ- ações típicas da intervenção profissional do psicólogo (promover a atenção e cuidados, entrevistar, adminis- trar uma anamnese, avaliar e elaborar diagnósticos, redigir documento psicológico segundo seu objeto, finalidade e contexto, elaborar e comunicar raciocínio clínico, redigir relatórios técnicos, saber atuar em equipes multiprofissionais. g g Cursos de Psicologia, por meio de suas ações e processos de ensino-aprendizagem, devem incentivar os alunos a participarem de programas de formação continuada, especialmente os de pós-graduação (lato e stricto sensu), a fim de assegurar o desenvolvimento de habilidades específicas, assim como o fortale- cimento das especialidades e o desenvolvimento técnico-científico e social da Psicologia. O domínio de assuntos e processos de intervenção específicos, para os quais os psicólogos devem estar preparados técnica e cientificamente ao longo do seu processo de profissionalização (formação básica, capacitação, treinamento), aumenta a possibilidade de prestação de serviços adequados às demandas da população e das instituições (cuidados primários, diagnóstico e tratamento de problemas saúde mental, serviços de consultoria e assessoramento em organizações públicas e privadas, avaliação de processos de apren- dizagem e programas de desenvolvimento, aplicação de conhecimentos específicos na investigação e perícia de problemas psicológicos, dentre outros).ii i No auge das críticas ao currículo mínimo da Psicologia (ao longo da década de 1990), iniciou-se um processo de desconstrução dos currículos exis- tentes, considerados, até então, excessivamente tecni- cistas, por um lado, e pouco atualizado em torno dos campos emergentes da Psicologia, por outro. De fato, o aprofundamento dessa discussão, no contexto da proliferação de cursos de Psicologia no Brasil e do advento das DCN, acelerou movimentos de mudança na organização e composição dos fluxos curricu- lares. Há que se avaliar, desde então, se as mudanças implementadas surtiram os efeitos desejados, ou seja, uma melhor formação em Psicologia e uma ampliação da inserção do psicológico no mundo do trabalho. As pistas indicam que inseguranças e fragili- dades identificadas no exercício profissional (dificul- dades de inserção no mundo do trabalho, infrações ético-profissionais centrada basicamente na comu- nicação de informação psicológica, sem o devido fundamento científico e técnico, uso das especiali- zações lato sensu como forma de complementação de lacunas na formação básica, baixo empreendedo- rismo nas chamadas áreas emergentes, dentre outras) permanecem salientes no processo de formação profissional dos psicólogos. Presente e Futuro da Psicologia Present and Future of Psychology Presente y Futuro de la Psicología Formação e certificação profissional são processos que dependem cada vez mais de mudanças no cenário científico e mercadológico. Manter, aperfeiçoar ou criar alternativas curriculares básicas, mas, ao mesmo tempo, modernas e viáveis à inserção de psicólogos no mundo do trabalho, é um desafio permanente dos cursos de Psicologia, considerando que já há algum tempo não se tem uma discussão nacional sobre a formação de psicólogos, tal como realizada em uma das mais significativas discussões sobre Formação em Psicologia, realizada no Encontro Nacional de Cursos de Psicologia, que resultou no documento denomi- nado Carta de Serra Negra (CFP, 1992). 784 Cruz, R. M. (2016). Presente e Futuro da Psicologia. Cruz, R. M. (2016). Presente e Futuro da Psicologia. no Brasil, na esteira da crítica ao currículo mínimo nacional, que definiu matérias específicas básicas e complementares (Brasil, 1962). O processo de acreditação de psicólogos deverá se generalizar nas especialidades profissionais, tendo em vista a necessidade de avaliar e certificar a qualidade de serviços oferecidos por psicológicos em seus respectivos segmentos de atuação. Com a ampliação de associa- ções e sociedades científicas e profissionais em Psico- logia, verificado nos últimos anos, acentua-se a neces- sidade de integração e tutela de princípios, conceitos e procedimentos que constituam referências às inter- venções profissionais, assim como à produção e disse- minação de conhecimentos à comunidade. O processo de acreditação, dessa forma, além de ser uma forma de alinhamento e fortalecimento de áreas científicas e dos campos de atuação profissional em Psicologia, promove o interesse pela capacitação e formação continuada dos psicólogos, o que não deve ser confundido com a fisca- lização e controle normativo do exercício profissional do psicólogo, cuja jurisdição pertence aos Conselhos Regionais e Federal de Psicologia. Formação generalista e diversidade de propostas teórico-metodológicas em Psicologia não refletem, na prática, conhecimento aplicado sobre como investigar, avaliar e intervir em fenômenos psicológicos por parte dos psicólogos. Processos de ensino básicos e comple- mentares devem ser afirmativos de condutas profissio- nais relevantes e necessárias à atuação do psicólogo. Presente e Futuro da Psicologia Present and Future of Psychology Presente y Futuro de la Psicología Pros- pectivamente, algumas diretrizes ético-metodológicas de conduta profissional dos psicólogos parecem ser consen- suais à afirmação do psicólogo no mundo do trabalho: • Produzir conhecimento sobre fenômenos e processos psicológicos que auxiliem na compreensão da realidade humana; • Manter-se atualizado técnica e cientificamente ao longo da vida profissional; Assim como o processo de acreditação, um aspecto que tem se apresentado de forma cada vez mais crescente é a revalidação de diplomas e a mobili- dade de profissionais estrangeiros em nosso país. Esse fato desencadeado pelo movimento de integração de psicólogos de países latino-americanos, iniciado pelo CFP, em parceria com entidades representativas de psicólogos de países do Mercosul, no início da década de 1990, e consolidado no Protocolo de Prin- cípios para a Formação de Psicólogos nos Países do Mercosul e Países Associados, propunha, entre outras coisas, “garantir uma formação básica comum para o reconhecimento de um psicólogo, em todos os países da região” (Schneider, 1998). • Intervir em situações e problemas que exijam conhecimento psicológico, visando superar dificuldades e promover algum benefício às pessoas; • Promover mudanças pessoais e coletivas para um estado melhor; • Procurar não causar prejuízo ou dano às pessoas ou instituições; • Guardar sigilo e confidencialidade como forma de preservar a vida, manter a relação de confiança e os melhores resultados para os usuários de seus serviços; Revalidar um diploma estrangeiro, contudo, ainda é um processo demorado e sujeito às inúmeras variações curriculares dos cursos de Psicologia, com grandes diferenças regionais e pouco alinhamento a currículos internacionais. A necessidade de organi- zação de um currículo mínimo ou de base nacional certamente retornará à ordem do dia, considerando as críticas acerca da implantação das DCN nos cursos de graduação. É perceptível que a criação da noção de núcleo comum e de ênfases curriculares voltadas à construção de competências profissionais e de habilidades técnicas e científicas, definidas nas DCN, não foi suficiente para dotar os currículos de uma identidade nacional. As DCN se propuseram a ser um conjunto de proposições básicas de orga- nização de eixos comuns aos cursos de Psicologia • Encaminhar a outros especialistas quando o limite de sua atuação tornar-se evidente; • Buscar as melhores formas de comunicação oral ou por escrito para posicionar-se como psicólogo na sociedade. Presente e Futuro da Psicologia Present and Future of Psychology Presente y Futuro de la Psicología Assim, pensar o presente e o futuro da Psicologia é, ao mesmo tempo, aprofundar a leitura dos desafios enfrentados pelos psicólogos no mundo do trabalho e afirmar diretrizes orientadoras da profissão para as próximas décadas. É preciso, antes de tudo, compreender o papel da Psicologia no mundo das ciências e no futuro 785 Psicologia: Ciência e Profissão Out/Dez. 2016 v. 36 n°4, 783-786. da humanidade para entender o seu próprio desen- volvimento (Cassepp-Borges, 2013). Significa, de fato, compreender o papel dos conhecimentos científicos acumulados na área, tornando-os acessíveis aos alunos, aperfeiçoar habilidades conceituais (para conhecer e operacionalizar processos de investigar, avaliar, intervir) e habilidades instrumentais (para utilizar os melhores recursos disponíveis no exercício profissional). Pautar novos horizontes é um desafio e uma necessidade para o presente e futuro da Psicologia. Conselho Federal de Psicologia (1992). Carta de Serra Negra. Documento síntese do Encontro Nacional dos Cursos de Psicologia: Repensando a Formação. Serra Negra (SP).i Como citar: Cruz, R. M. (2016). Presente e Futuro da Psicologia. Psicologia: Ciência e Profissão, 36(4): 783-786. doi:10.1590/1982-3703003642016 Roberto Moraes Cruz Editor-chefe Universidade Federal de Santa Catarina Cómo citar: Cruz, R. M. (2016). Presente y Futuro de la Psicología. Psicologia: Ciência e Profissão, 36(4): 783-786. doi:10.1590/1982-3703003642016 Referências Bastos, A. V. B. & Gomide, P. I. C. (1989). O psicólogo brasileiro: sua atuação e formação profissional. Psi- col. Cienc. Prof., v.9, n.1, pp.6-15. Conselho Federal de Psicologia (2016). Infográfico. A Psicologia brasileira apresentada em números. http://www2.cfp.org.br/infografico/quantos-so- mos/. Brasília. DF. Conselho Federal de Psicologia (2016). Infográfico. A Psicologia brasileira apresentada em números. http://www2.cfp.org.br/infografico/quantos-so- mos/. Brasília. DF. Brasil (1962). Ministério da Educação. Resolução de 19 de dezembro de 1962. Currículo mínimo para os cursos de Psicologia. Brasília, DF. Brasil (2004). Ministério da Educação. Parecer CNE/CES n. 62 de 19 de fevereiro de 2004. Diretrizes Curriculares Nacionais para os cursos de graduação em Psicologia. Brasília: DF. Schneider, D. R. (1998). A formação em Psicologia no Mer- cosul: perspectivas da integração latino-americana. Schneider, D. R. (1998). A formação em Psicologia no Mer- cosul: perspectivas da integração latino-americana. Temas em Psicologia, v. 6, n. 1, pp. 69-74. Temas em Psicologia, v. 6, n. 1, pp. 69-74. Cassepp-Borges, V. (2013). Desafios para o futuro da Psicologia: contribuições da Psicologia na constru- ção do conhecimento no século XXI. Psicol. Cienc. Prof. [online], vol.33, n.esp., pp. 14-23. Roberto Moraes Cruz Editor-chefe Universidade Federal de Santa Catarina Roberto Moraes Cruz Editor-chefe Universidade Federal de Santa Catarina Como citar: Cruz, R. M. (2016). Presente e Futuro da Psicologia. Psicologia: Ciência e Profissão, 36(4): 783-786. doi:10.1590/1982-3703003642016 How to cite: Cruz, R. M. (2016). Present and Future of Psychology. Psicologia: Ciência e Profissão, 36(4): 783-786. doi:10.1590/1982-3703003642016 Cómo citar: Cruz, R. M. (2016). Presente y Futuro de la Psicología. Psicologia: Ciência e Profissão, 36(4): 783-786. doi:10.1590/1982-3703003642016 Cómo citar: Cruz, R. M. (2016). Presente y Futuro de la Psicología. Psicologia: Ciência e Profissão, 36(4): 783-786. doi:10.1590/1982-3703003642016 786
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Cu(II)-Catalyzed C-N Coupling of (Hetero)aryl Halides and N-Nucleophiles Promoted by α-Benzoin Oxime
Molecules/Molecules online/Molecules annual
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Article Cu(II)-Catalyzed C-N Coupling of (Hetero)aryl Halides and N-Nucleophiles Promoted by α-Benzoin Oxime Chunling Yuan *, Lei Zhang and Yingdai Zhao Department of Medicinal Chemistry, Pharmacy School, Jinzhou Medical University, Jinzhou 121001, China; hm7835831@163.com (L.Z.); ZYd1216221530@163.com (Y.Z.) * Correspondence: yuanchunling@jzmu.edu.cn; Tel.: +86-0416-4673440 Chunling Yuan *, Lei Zhang and Yingdai Zhao Chunling Yuan *, Lei Zhang and Yingdai Zhao Department of Medicinal Chemistry, Pharmacy School, Jinzhou Medical University, Jinzhou 121001, China; hm7835831@163.com (L.Z.); ZYd1216221530@163.com (Y.Z.) * Correspondence: yuanchunling@jzmu.edu.cn; Tel.: +86-0416-4673440 Received: 29 October 2019; Accepted: 16 November 2019; Published: 18 November 2019 Abstract: We first reported the new application of a translate metal chelating ligand α-benzoin oxime for improving Cu-catalyzed C-N coupling reactions. The system could catalyse coupling reactions of (hetero)aryl halides with a wide of nucleophiles (e.g., azoles, piperidine, pyrrolidine and amino acids) in moderate to excellent yields. The protocol allows rapid access to the most common scaffolds found in FDA-approved pharmaceuticals. Keywords: α-Benzoin oxime; Copper catalyst; (Hetero)aryl halides; N-Nucleophiles; C-N Coup molecules molecules molecules Molecules 2019, 24, 4177; doi:10.3390/molecules24224177 lecules atalyzed C-N Coupling of (Hetero)aryl and N-Nucleophiles Promoted by in Oxime *, Lei Zhang and Yingdai Zhao Medicinal Chemistry, Pharmacy School, Jinzhou Medical University, Jinzhou 121001, China; .com (L.Z.); ZYd1216221530@163.com (Y.Z.) nce: yuanchunling@jzmu.edu.cn; Tel.: +86-0416-4673440 tober 2019; Accepted: 16 November 2019; Published: 18 November 2019   first reported the new application of a translate metal chelating ligand α-benzoin oxime Cu-catalyzed C-N coupling reactions. The system could catalyse coupling reactions of lides with a wide of nucleophiles (e.g., azoles, piperidine, pyrrolidine and amino acids) excellent yields. The protocol allows rapid access to the most common scaffolds found ved pharmaceuticals. Benzoin oxime; Copper catalyst; (Hetero)aryl halides; N-Nucleophiles; C-N Coupling d compounds are ubiquitous synthons in numerous natural products and functional Particularly, their most important function is as structural fragment for FDA-approved s (Figure 1), for example, antibacterial agents (Ofloxacin [3] and Pipemidic abetic drug (Repaglinide [5]), nonsteroidal anti-inflammatory drug (Celecoxib [6]), e drug (Prazosin [7]), etc. Although C-N bond-formation reactions between (hetero)aryl nucleophiles are well known, including the SNAr reactions [8], classical Ullmann-type ons [9–11], and Buchwald-Hartwig reactions [12–14]. Amongst Pd-catalyzed C-N on has been confirmed as a very useful method to build aromatic amines or 18]. However, considering to the cost and toxicity of both palladium catalyst and hine ligands, the arylation of N-nucleophiles with (hetero)aryl halides to form new l remains a significant opportunity. In recent years, Cu-catalyzed Ullmann-type attracted more and more attention due to the inexpensive catalyst and low toxicity. commercially available or novel designed ligands have been developed for promoting ed couplings of aryl halides with N-nucleophiles, few of them are effective with less rocyclic aryl chlorides. Besides, an easy-removal catalyst system is also very important ment. Amongst Pd-catalyzed C-N very useful method to build aromatic amines or he cost and toxicity of both palladium catalyst and -nucleophiles with (hetero)aryl halides to form new unity. In recent years, Cu-catalyzed Ullmann-type ion due to the inexpensive catalyst and low toxicity. designed ligands have been developed for promoting h N-nucleophiles, few of them are effective with less an easy-removal catalyst system is also very important molecules lecules atalyzed C-N Coupling of (Hetero)aryl and N-Nucleophiles Promoted by in Oxime *, Lei Zhang and Yingdai Zhao Medicinal Chemistry, Pharmacy School, Jinzhou Medical University, Jinzhou 121001, China; .com (L.Z.); ZYd1216221530@163.com (Y.Z.) nce: yuanchunling@jzmu.edu.cn; Tel.: +86-0416-4673440 tober 2019; Accepted: 16 November 2019; Published: 18 November 2019   first reported the new application of a translate metal chelating ligand α-benzoin oxime Cu-catalyzed C-N coupling reactions. The system could catalyse coupling reactions of lides with a wide of nucleophiles (e.g., azoles, piperidine, pyrrolidine and amino acids) excellent yields. The protocol allows rapid access to the most common scaffolds found ved pharmaceuticals. Benzoin oxime; Copper catalyst; (Hetero)aryl halides; N-Nucleophiles; C-N Coupling d compounds are ubiquitous synthons in numerous natural products and functional Particularly, their most important function is as structural fragment for FDA-approved s (Figure 1), for example, antibacterial agents (Ofloxacin [3] and Pipemidic abetic drug (Repaglinide [5]), nonsteroidal anti-inflammatory drug (Celecoxib [6]), e drug (Prazosin [7]), etc. Although C-N bond-formation reactions between (hetero)aryl nucleophiles are well known, including the SNAr reactions [8], classical Ullmann-type ons [9–11], and Buchwald-Hartwig reactions [12–14]. Amongst Pd-catalyzed C-N on has been confirmed as a very useful method to build aromatic amines or 18]. However, considering to the cost and toxicity of both palladium catalyst and hine ligands, the arylation of N-nucleophiles with (hetero)aryl halides to form new l remains a significant opportunity. In recent years, Cu-catalyzed Ullmann-type attracted more and more attention due to the inexpensive catalyst and low toxicity. commercially available or novel designed ligands have been developed for promoting ed couplings of aryl halides with N-nucleophiles, few of them are effective with less rocyclic aryl chlorides. Besides, an easy-removal catalyst system is also very important ment. lecules atalyzed C-N Coupling of (Hetero)aryl and N-Nucleophiles Promoted by in Oxime *, Lei Zhang and Yingdai Zhao Medicinal Chemistry, Pharmacy School, Jinzhou Medical University, Jinzhou 121001, China; .com (L.Z.); ZYd1216221530@163.com (Y.Z.) nce: yuanchunling@jzmu.edu.cn; Tel.: +86-0416-4673440 tober 2019; Accepted: 16 November 2019; Published: 18 November 2019   first reported the new application of a translate metal chelating ligand α-benzoin oxime Cu-catalyzed C-N coupling reactions. The system could catalyse coupling reactions of lides with a wide of nucleophiles (e.g., azoles, piperidine, pyrrolidine and amino acids) excellent yields. The protocol allows rapid access to the most common scaffolds found ved pharmaceuticals. Benzoin oxime; Copper catalyst; (Hetero)aryl halides; N-Nucleophiles; C-N Coupling d compounds are ubiquitous synthons in numerous natural products and functional Particularly, their most important function is as structural fragment for FDA-approved s (Figure 1), for example, antibacterial agents (Ofloxacin [3] and Pipemidic abetic drug (Repaglinide [5]), nonsteroidal anti-inflammatory drug (Celecoxib [6]), e drug (Prazosin [7]), etc. Although C-N bond-formation reactions between (hetero)aryl nucleophiles are well known, including the SNAr reactions [8], classical Ullmann-type ons [9–11], and Buchwald-Hartwig reactions [12–14]. Amongst Pd-catalyzed C-N on has been confirmed as a very useful method to build aromatic amines or 18]. However, considering to the cost and toxicity of both palladium catalyst and hine ligands, the arylation of N-nucleophiles with (hetero)aryl halides to form new l remains a significant opportunity. In recent years, Cu-catalyzed Ullmann-type attracted more and more attention due to the inexpensive catalyst and low toxicity. commercially available or novel designed ligands have been developed for promoting ed couplings of aryl halides with N-nucleophiles, few of them are effective with less rocyclic aryl chlorides. Besides, an easy-removal catalyst system is also very important ment. upling of (Hetero)aryl les Promoted by ool, Jinzhou Medical University, Jinzhou 121001, China; om (Y.Z.) l.: +86-0416-4673440 2019; Published: 18 November 2019   of a translate metal chelating ligand α-benzoin oxime tions. The system could catalyse coupling reactions of (e.g., azoles, piperidine, pyrrolidine and amino acids) ows rapid access to the most common scaffolds found (Hetero)aryl halides; N-Nucleophiles; C-N Coupling nthons in numerous natural products and functional nt function is as structural fragment for FDA-approved ntibacterial agents (Ofloxacin [3] and Pipemidic nonsteroidal anti-inflammatory drug (Celecoxib [6]), gh C-N bond-formation reactions between (hetero)aryl luding the SNAr reactions [8], classical Ullmann-type wig reactions [12–14]. 2. Results and Discussion To initiate our studies 2. Results and Discussions , py p in DMF. Regrettably, coupling product 3a was obtained in 12% yield, along with unreacted starting material (Table 1, entry 1). The use of Cu powder and Cu(OTf)2 as catalyst in DMF delivered 3a in low yields (entries 2, 3). Consistently, Cu(II) gluconate and Cu2(OH)2CO3 did not provide the desired product (entries 4, 5), only a trace of 3a was observed. In the event, 10 mol% Cu(OAc)2 enabled the coupling of 2-bromoanisole and pyrrole to provide the desired product in 53% yield (entry 6). Attempts to improve the yield through changing the base were successful, with K3PO4 proving to be optimal in terms of yield (entries 6–11). Furthermore, with dioxane, toluene, DCE, H2O as solvents (entries 12–16), the yield was lower than that in DMF. Fortunately, DMSO gave significantly better results, and 3a was obtained in a much-improved 90% yield. Meanwhile, lowering the temperature to 60 °C decreased the yield and led incomplete conversion (entry 17). A significant amount of coupling product was observed when using BO as ligand, comparing with the corresponding control experiments (entries 12 and 18). The scalability of this protocol was also tested through synthesis of the 3a on a 4.4 g scale (entry 19). Additionally, the BO-promoted C-N reaction was also carried out under air, 3a was obtained in 61% yield, indicating that this catalytic system was To initiate our studies, 2-bromoanisole was treated with pyrrole in the presence of 0.28 mol CuI in DMF. Regrettably, coupling product 3a was obtained in 12% yield, along with unreacted starting material (Table 1, entry 1). The use of Cu powder and Cu(OTf)2 as catalyst in DMF delivered 3a in low yields (entries 2, 3). Consistently, Cu(II) gluconate and Cu2(OH)2CO3 did not provide the desired product (entries 4, 5), only a trace of 3a was observed. In the event, 10 mol% Cu(OAc)2 enabled the coupling of 2-bromoanisole and pyrrole to provide the desired product in 53% yield (entry 6). Attempts to improve the yield through changing the base were successful, with K3PO4 proving to be optimal in terms of yield (entries 6–11). Furthermore, with dioxane, toluene, DCE, H2O as solvents (entries 12–16), the yield was lower than that in DMF. Fortunately, DMSO gave significantly better results, and 3a was obtained in a much-improved 90% yield. Meanwhile, lowering the temperature to 60 ◦C decreased the yield and led incomplete conversion (entry 17). 1. Introduction N-Arylated compounds are ubiquitous synthons in numerous natural products and functional molecules [1,2]. Particularly, their most important function is as structural fragment for FDA-approved pharmaceuticals (Figure 1), for example, antibacterial agents (Ofloxacin [3] and Pipemidic acid [4]), antidiabetic drug (Repaglinide [5]), nonsteroidal anti-inflammatory drug (Celecoxib [6]), antihypertensive drug (Prazosin [7]), etc. Although C-N bond-formation reactions between (hetero)aryl halides and N-nucleophiles are well known, including the SNAr reactions [8], classical Ullmann-type coupling reactions [9–11], and Buchwald-Hartwig reactions [12–14]. Amongst Pd-catalyzed C-N coupling reaction has been confirmed as a very useful method to build aromatic amines or arylazoles [15–18]. However, considering to the cost and toxicity of both palladium catalyst and auxiliary phosphine ligands, the arylation of N-nucleophiles with (hetero)aryl halides to form new C-N bonds still remains a significant opportunity. In recent years, Cu-catalyzed Ullmann-type couplings have attracted more and more attention due to the inexpensive catalyst and low toxicity. Although many commercially available or novel designed ligands have been developed for promoting copper-catalyzed couplings of aryl halides with N-nucleophiles, few of them are effective with less inactivated heterocyclic aryl chlorides. Besides, an easy-removal catalyst system is also very important for the environment. Molecules 2019, 24, 4177; doi:10.3390/molecules24224177 www.mdpi.com/journal/molecules www.mdpi.com/journal/molecules 2 of 12 Molecules 2019, 24, 4177 Figure 1. Chemical structures of selected pharmaceuticals containing the N-arylated core. Figure 1. Chemical structures of selected pharmaceuticals containing the N-arylated core. Figure 1. Chemical structures of selected pharmaceuticals containing the N-arylated core. Figure 1. Chemical structures of selected pharmaceuticals containing the N-arylated core. During the past few years, Ma and co-workers have reported that the oxalic diamide ligands are powerful ligands for the copper-catalyzed couplings [19–21]. In the presence of ligands, the reaction temperature and catalyst loading could be significantly decreased while the yields were increased [22–24]. Following these studies, a number of bidentate ligands were reported for the synthesis of N-arylated compounds. On the other hand, α-benzoin oxime (BO) is a common ligand, which is usually applied to During the past few years, Ma and co-workers have reported that the oxalic diamide ligands are powerful ligands for the copper-catalyzed couplings [19–21]. In the presence of ligands, the reaction temperature and catalyst loading could be significantly decreased while the yields were increased [22–24]. Following these studies, a number of bidentate ligands were reported for the synthesis of N-arylated compounds. 1. Introduction , ( ) g , y pp inspect and measure copper, molybdenum, and tungsten [25,26], and also as a chelating agent for extracting antimony, vanadium, tungsten [27]. However, the use of BO in improving the Cu-catalyzed Ullmann style reactions is never reported. Herein, it was found that BO could be used in the direct couplings of the (hetero)aryl halides with N-nucleophiles. The reaction allowed rapid access to N-arylated compounds, the most common scaffolds found in FDA-approved pharmaceuticals. These reactions were occurred at mild temperature (80 °C), with employing (hetero)aryl halides, nucleophiles (e.g., azoles, piperidine, pyrrolidine and amino acids) and inexpensive catalysts, and affording high yields. Importantly, this process was general with respect to both the (hetero)aryl halides and nucleophiles, including the use of secondary amines and amino acids. On the other hand, α-benzoin oxime (BO) is a common ligand, which is usually applied to inspect and measure copper, molybdenum, and tungsten [25,26], and also as a chelating agent for extracting antimony, vanadium, tungsten [27]. However, the use of BO in improving the Cu-catalyzed Ullmann style reactions is never reported. Herein, it was found that BO could be used in the direct couplings of the (hetero)aryl halides with N-nucleophiles. The reaction allowed rapid access to N-arylated compounds, the most common scaffolds found in FDA-approved pharmaceuticals. These reactions were occurred at mild temperature (80 ◦C), with employing (hetero)aryl halides, nucleophiles (e.g., azoles, piperidine, pyrrolidine and amino acids) and inexpensive catalysts, and affording high yields. Importantly, this process was general with respect to both the (hetero)aryl halides and nucleophiles, including the use of secondary amines and amino acids. 2. Results and Discussion To initiate our studies 2. Results and Discussions c Almost no reaction was observed by TLC. d 60 ◦C. e Without BO. f The loading of 2-bromoanisole was 28.1 mmol. g The reaction was carried out under air. Red bond indicated cleavage bond; blue bond indicated formed bond; and BO was α-benzoin oxime. Isolated yield with column chromatography. c Almost no reaction was observed by TLC. d 60 °C. e Without BO. f The loading of 2-bromoanisole was 28.1 mmol. g The reaction was carried out under air. Red bond indicated cleavage bond; blue bond indicated formed bond; and BO was α-benzoin oxime. Reaction conditions: 2 Bromoanisole (2.81 mmol), pyrrole (3.37 mmol), Cu source (0.28 mmol), BO (0.28 mmol), solvent (4 mL), base (5.62 mmol), under Ar, at 80 °C, unless otherwise noted for 8 h. b Isolated yield with column chromatography. c Almost no reaction was observed by TLC. d 60 °C. e Without BO. f The loading of 2-bromoanisole was 28.1 mmol. g The reaction was carried out under air. Red bond indicated cleavage bond; blue bond indicated formed bond; and BO was α-benzoin a Reaction conditions: 2-Bromoanisole (2.81 mmol), pyrrole (3.37 mmol), Cu source (0.28 mmol), BO (0.28 mmol), solvent (4 mL), base (5.62 mmol), under Ar, at 80 ◦C, unless otherwise noted for 8 h. b Isolated yield with column chromatography. c Almost no reaction was observed by TLC. d 60 ◦C. e Without BO. f The loading of 2-bromoanisole was 28.1 mmol. g The reaction was carried out under air. Red bond indicated cleavage bond; blue bond indicated formed bond; and BO was α-benzoin oxime. With optimized conditions in hand, we set out to evaluate the scope of (hetero)aryl halides that would participate in this transformation (Table 2). The reaction tolerated a diverse array of functional groups on the (hetero)aryl halides, including methoxy (3a), aldehyde (3c), carboxyl (3d), amino (3e), ketone (3f), ester (3g, 3h), cyano (3i). Electronic properties of the (hetero)aryl halides were evaluated by introducing electron-withdrawing and electron-donating groups on the aryl moiety. Although electron-poor (hetero)aryl halides (e.g., 3c, 3d, 3i) underwent coupling faster than electron rich ones (3a, 3e), the desired products were successfully obtained in all cases. Unfortunately, chlorobenzene couldn’t provide the product 3b. By changing which heteroaryl With optimized conditions in hand, we set out to evaluate the scope of (hetero)aryl halides that would participate in this transformation (Table 2). 2. Results and Discussion To initiate our studies 2. Results and Discussions Br + Cu cources (10 mol%) BO (10 mol%) Base (2 equiv.) Solvent, Ar, 80 oC N OH N HO BO HN OMe OMe 3a 1 K2CO3 DMF 12 2 K2CO3 DMF 15 3 K2CO3 DMF 22 4 K2CO3 DMF Trace 5 K2CO3 DMF Trace 6 K2CO3 DMF 53 7 Cs2CO3 DMF 60 8 K3PO4 DMF 72 9 NaHCO3 DMF 0 c 10 Et3N DMF 0 c 11 DMF Trace 12 DMSO 90 13 Dioxane 43 14 DCE Trace 15 Toluene 0 c 16 H2O 0 c 17 DMSO 70 d 18 DMSO 15 e 19 DMSO 90 f CuI Cu powder Cu(OTf)2 gluconat Cu(II) e Cu2(OH)2CO3 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 t-BuOK 3PO4 K 3PO4 K 3PO4 K 3PO4 K 3PO4 K 3PO4 K 3 N N N N Me H N OH O NH COOH O OEt O L1 L2 L3 L4 L5 0% trace trace 55% 42% a Reaction conditions: 2-Bromoanisole (2.81 mmol), pyrrole (3.37 mmol), Cu source (0.28 mmol), BO (0.28 mmol), solvent (4 mL), base (5.62 mmol), under Ar, at 80 °C, unless otherwise noted for 8 h. b Isolated yield with column chromatography. c Almost no reaction was observed by TLC. d 60 °C. e Without BO. f The loading of 2-bromoanisole was 28.1 mmol. g The reaction was carried out under air. Red bond indicated cleavage bond; blue bond indicated formed bond; and BO was α-benzoin oxime a Reaction conditions: 2-Bromoanisole (2.81 mmol), pyrrole (3.37 mmol), Cu source (0.28 mmol), BO (0.28 mmol), solvent (4 mL), base (5.62 mmol), under Ar, at 80 ◦C, unless otherwise noted for 8 h. b Isolated yield with column chromatography. c Almost no reaction was observed by TLC. d 60 ◦C. e Without BO. f The loading of 2-bromoanisole was 28.1 mmol. g The reaction was carried out under air. Red bond indicated cleavage bond; blue bond indicated formed bond; and BO was α-benzoin oxime. Table 1. Identification of reaction condition a. Table 1. Identification of reaction condition a. may be effective when chelating with Cu(I). 2. Results and Discussion To initiate our studies 2. Results and Discussions Br + Cu cources (10 mol%) BO (10 mol%) Base (2 equiv.) Solvent, Ar, 80 oC N OH N HO BO HN OMe OMe 3a 1 K2CO3 DMF 12 2 K2CO3 DMF 15 3 K2CO3 DMF 22 4 K2CO3 DMF Trace 5 K2CO3 DMF Trace 6 K2CO3 DMF 53 7 Cs2CO3 DMF 60 8 K3PO4 DMF 72 9 NaHCO3 DMF 0 c 10 Et3N DMF 0 c 11 DMF Trace 12 DMSO 90 13 Dioxane 43 14 DCE Trace 15 Toluene 0 c 16 H2O 0 c 17 DMSO 70 d 18 DMSO 15 e 19 DMSO 90 f CuI Cu powder Cu(OTf)2 gluconat Cu(II) e Cu2(OH)2CO3 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 t-BuOK 3PO4 K 3PO4 K 3PO4 K 3PO4 K 3PO4 K 3PO4 K 3 N N N N Me H N OH O NH COOH O OEt O L1 L2 L3 L4 L5 0% trace trace 55% 42% a Reaction conditions: 2-Bromoanisole (2.81 mmol), pyrrole (3.37 mmol), Cu source (0.28 mmol), BO (0.28 mmol), solvent (4 mL), base (5.62 mmol), under Ar, at 80 °C, unless otherwise noted for 8 h. b Isolated yield with column chromatography. c Almost no reaction was observed by TLC. d 60 °C. e Without BO. f The loading of 2-bromoanisole was 28.1 mmol. g The reaction was carried out under air. Red bond indicated cleavage bond; blue bond indicated formed bond; and BO was α-benzoin oxime. 2. Results and Discussion To initiate our studies 2. Results and Discussions Entry Cu Sources Base Solvent Yield (%) b 1 CuI K2CO3 DMF 12 2 Cu powder K2CO3 DMF 15 3 Cu(OTf)2 K2CO3 DMF 22 4 Cu(II) gluconate K2CO3 DMF Trace 5 Cu2(OH)2CO3 K2CO3 DMF Trace 6 Cu(OAc)2 K2CO3 DMF 53 7 Cu(OAc)2 Cs2CO3 DMF 60 8 Cu(OAc)2 K3PO4 DMF 72 9 Cu(OAc)2 NaHCO3 DMF 0 c 10 Cu(OAc)2 Et3N DMF 0 c 11 Cu(OAc)2 t-BuOK DMF Trace 12 Cu(OAc)2 K3PO4 DMSO 90 13 Cu(OAc)2 K3PO4 Dioxane 43 14 Cu(OAc)2 K3PO4 DCE Trace 15 Cu(OAc)2 K3PO4 Toluene 0 c 16 Cu(OAc)2 K3PO4 H2O 0 c 17 Cu(OAc)2 K3PO4 DMSO 70 d 18 Cu(OAc)2 K3PO4 DMSO 15 e 19 Cu(OAc)2 K3PO4 DMSO 90 f 20 Cu(OAc)2 K3PO4 DMSO 61 g Br + Cu cources (10 mol%) BO (10 mol%) Base (2 equiv.) Solvent, Ar, 80 oC N OH N HO BO HN OMe OMe 3a 1 K2CO3 DMF 12 2 K2CO3 DMF 15 3 K2CO3 DMF 22 4 K2CO3 DMF Trace 5 K2CO3 DMF Trace 6 K2CO3 DMF 53 7 Cs2CO3 DMF 60 8 K3PO4 DMF 72 9 NaHCO3 DMF 0 c 10 Et3N DMF 0 c 11 DMF Trace 12 DMSO 90 13 Dioxane 43 14 DCE Trace 15 Toluene 0 c 16 H2O 0 c 17 DMSO 70 d 18 DMSO 15 e 19 DMSO 90 f CuI Cu powder Cu(OTf)2 gluconat Cu(II) e Cu2(OH)2CO3 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 t-BuOK 3PO4 K 3PO4 K 3PO4 K 3PO4 K 3PO4 K 3PO4 K 3 N N N N Me H N OH O NH COOH O OEt O L1 L2 L3 L4 L5 0% trace trace 55% 42% a Reaction conditions: 2-Bromoanisole (2.81 mmol), pyrrole (3.37 mmol), Cu source (0.28 mmol), BO (0.28 mmol), solvent (4 mL), base (5.62 mmol), under Ar, at 80 °C, unless otherwise noted for 8 h. b Isolated yield with column chromatography. c Almost no reaction was observed by TLC. d 60 °C. e Without BO. f The loading of 2-bromoanisole was 28.1 mmol. g The reaction was carried out under air. Red bond indicated cleavage bond; blue bond indicated formed bond; and BO was α-benzoin i a Reaction conditions: 2-Bromoanisole (2.81 mmol), pyrrole (3.37 mmol), Cu source (0.28 mmol), BO (0.28 mmol), solvent (4 mL), base (5.62 mmol), under Ar, at 80 ◦C, unless otherwise noted for 8 h. b Isolated yield with column chromatography. 2. Results and Discussion To initiate our studies 2. Results and Discussions A significant amount of coupling product was observed when using BO as ligand, comparing with the corresponding control experiments (entries 12 and 18). The scalability of this protocol was also tested through synthesis of the 3a on a 4.4 g scale (entry 19). Additionally, the BO-promoted C-N reaction was also carried out under air, 3a was obtained in 61% yield, indicating that this catalytic system was sensitive with air (entry 20). Finally, we checked other ligands (including N,N-, N,O-, O,O-type bidentate ligands) that were applied in Cu-catalyzed 3 of 12 12 Molecules 2019, 24, 4177 Molecules 2019, 24, x N-arylation reactions, and discovered that only L3 and L4 could furnish 3a in moderate yields under standard condition. It was reasoned that these reported ligands may be effective when chelating with Cu(I). sensitive with air (entry 20). Finally, we checked other ligands (including N,N-, N,O-, O,O-type bidentate ligands) that were applied in Cu-catalyzed N-arylation reactions, and discovered that only L3 and L4 could furnish 3a in moderate yields under standard condition. It was reasoned that these reported ligands may be effective when chelating with Cu(I). sensitive with air (entry 20). Finally, we checked other ligands (including N,N-, N,O-, O,O-type bidentate ligands) that were applied in Cu-catalyzed N-arylation reactions, and discovered that only L3 and L4 could furnish 3a in moderate yields under standard condition. It was reasoned that h d li d b ff i h h l i i h C (I) Table 1. Identification of reaction condition a. Table 1. Identification of reaction condition a. 2. Results and Discussion To initiate our studies 2. Results and Discussions Br + Cu cources (10 mol%) BO (10 mol%) Base (2 equiv.) Solvent, Ar, 80 oC N OH N HO BO HN OMe OMe 3a 1 K2CO3 DMF 12 2 K2CO3 DMF 15 3 K2CO3 DMF 22 4 K2CO3 DMF Trace 5 K2CO3 DMF Trace 6 K2CO3 DMF 53 7 Cs2CO3 DMF 60 8 K3PO4 DMF 72 9 NaHCO3 DMF 0 c 10 Et3N DMF 0 c 11 DMF Trace 12 DMSO 90 13 Dioxane 43 14 DCE Trace 15 Toluene 0 c 16 H2O 0 c 17 DMSO 70 d 18 DMSO 15 e 19 DMSO 90 f CuI Cu powder Cu(OTf)2 gluconat Cu(II) e Cu2(OH)2CO3 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 Cu(OAc)2 t-BuOK 3PO4 K 3PO4 K 3PO4 K 3PO4 K 3PO4 K 3PO4 K 3 N N N N Me H N OH O NH COOH O OEt O L1 L2 L3 L4 L5 0% trace trace 55% 42% a Reaction conditions: 2-Bromoanisole (2.81 mmol), pyrrole (3.37 mmol), Cu source (0.28 mmol), BO (0.28 mmol), solvent (4 mL), base (5.62 mmol), under Ar, at 80 °C, unless otherwise noted for 8 h. b Isolated yield with column chromatography. c Almost no reaction was observed by TLC. d 60 °C. e Without BO. f The loading of 2-bromoanisole was 28.1 mmol. g The reaction was carried out under air. Red bond indicated cleavage bond; blue bond indicated formed bond; and BO was α-benzoin oxime. Entry Cu Sources Base Solvent Yield (%) b 1 CuI K2CO3 DMF 12 2 Cu powder K2CO3 DMF 15 3 Cu(OTf)2 K2CO3 DMF 22 4 Cu(II) gluconate K2CO3 DMF Trace 5 Cu2(OH)2CO3 K2CO3 DMF Trace 6 Cu(OAc)2 K2CO3 DMF 53 7 Cu(OAc)2 Cs2CO3 DMF 60 8 Cu(OAc)2 K3PO4 DMF 72 9 Cu(OAc)2 NaHCO3 DMF 0 c 10 Cu(OAc)2 Et3N DMF 0 c 11 Cu(OAc)2 t-BuOK DMF Trace 12 Cu(OAc)2 K3PO4 DMSO 90 13 Cu(OAc)2 K3PO4 Dioxane 43 14 Cu(OAc)2 K3PO4 DCE Trace 15 Cu(OAc)2 K3PO4 Toluene 0 c 16 Cu(OAc)2 K3PO4 H2O 0 c 17 Cu(OAc)2 K3PO4 DMSO 70 d 18 Cu(OAc)2 K3PO4 DMSO 15 e 19 Cu(OAc)2 K3PO4 DMSO 90 f 20 Cu(OAc)2 K3PO4 DMSO 61 g these reported ligands may be effective when chelating with Cu(I). Table 1. Identification of reaction condition a. Table 2. C-N Coupling reactions of substituted aryl compounds with pyrrole or azoles a,b. sterically encumbered 2-methylimidazole also reacted without incidents (3l, 3n). Z Y X R Heteroaryl-NH + Cu(OAc)2 (10 mol%) BO (10 mol%) K3PO4 (2 equiv.) DMSO, Ar, 80 oC Z Y N R Heteroaryl OHC N N N N CO2H N N 3a: X=Br 88% 3b X B 94% NH2 3 X B 95% 3d X B 96% 3 X B 85% N N Y, Z = CH or N X=Br or Cl OMe (OAc)2 (10 mol%) (10 mol%) PO4 (2 equiv.) SO, Ar, 80 oC Z Y N R Heteroaryl Y, Z = CH or N X=Br or Cl , X=Br or Cl 3a: X=Br, 88% N OMe 2019 24 OHC N N 3c: X=Br, 95% N N NH2 3e: X=Br, 85%4 o N N CO2H 3d: X=Br, 96% 3b: X=Br, 94% X=Cl, 0% N 3a: X=Br, 88% 2019 24 3a: X=Br, 88% 2019 24 3d: X=Br, 96% 3e: X=Br, 85%4 3d: X=Br, 96% 3c: X=Br, 95% 3b: X=Br, 94% X=Cl, 0% N N CN 3i: X=Br, 98% although the yiel hout incidents (3 N N 3j: X=Cl, 84% 4 o ld was decreased l, 3n). 3i: X=Br, 98% out incidents (3 MeO2C azole als 3j: X=Cl, 84% 3i: X=Br, 98% 3f: X=Br, 90% 3g: X=Br, 91% 3h: X=Br, 93% N N N N 3m: X=Cl, 83% u(OAc)2 (10 mol%) O (10 mol%) 3PO4 (2 equiv.) MSO, Ar, 80 oC N N N N Me 3n: X=Cl, 80% Z Y N R N N N Me 3l: X=Cl, 85% Heteroaryl-NH + 3k: X=Cl, 88% Y, Z = CH o X=Br or Cl 3n: X=Cl, 80% 3n: X=Cl, 8 3m: X=Cl, 83% , , Table 3. C-N Coupling reactions of substituted aryl compounds with amines a,b a Reactions conducted on (hetero)aryl halides (2.81 mmol), pyrrole or azoles (3.37 mmol), Cu(OAc)2 (0.28 mmol), BO (0.28 mmol), K3PO4 (5.62 mmol), DMSO (4 mL) under Ar at 80 ◦C for 8~10 h. b Isolated yield with column chromatography. Table 2. C-N Coupling reactions of substituted aryl compounds with pyrrole or azoles a,b. sterically encumbered 2-methylimidazole also reacted without incidents (3l, 3n). OHC N N N N CO2H N N NH2 N N OMe N N N N N N N N N N N N N N NEt H N W Z Y X R + Cu(OAc)2 (10 mol%) BO (10 mol%) K3PO4 (2 equiv.) DMSO, Ar, 80 oC W Z Y NRR' R W, Y, Z = CH or N X=Br or Cl OMe OMe 4a: X=Cl, 87% 4b: X=Cl, 88% 4c: X=Cl, 85% 4d: X=Cl, 86% 4g: X=Br, 80% 4h: X=Br, 82% NHRR' In order to explore the feasibility of this approach, cyclic secondary amines (piperidine and pyrrolidine), acyclic secondary amine (diethylamine), aliphatic primary amine (ethanolamine) were examined to achieve the desired coupling (Table 3). First, the reactions were carried out by using 2-chloropyridine as starting material. To our delight, the corresponding coupling product 4a and 4b were obtained in 87% and 88% yield, respectively. We also attempted the coupling of 4-chloropyridine to form 4c and 4d. In both cases, the products were obtained in good yields. Both electron-rich (4g) and electron-poor (4i) aryl bromides participated equally well in the reaction. The reaction was effective in the presence of unprotected polar functional groups such as alcohol. It was encouraged that the ethanolamine substitute could provide 4l in 90% yield; the lower yield was due to incomplete conversion of the starting material. N N N N Me O N N N N EtO2C MeO2C N N N N X=Cl, 0% 3f: X=Br, 90% 3g: X=Br, 91% CN N N N N N N N N Me N N N N 3j: X=Cl, 84% 3k: X=Cl, 88% 3l: X=Cl, 85% 3m: X=Cl, 83% 3n: X=Cl, 80% 3h: X=Br, 93% 3i: X=Br, 98% Me Table 3 C N Coupling reactions of substituted aryl compounds with amines a b OHC OHC N N O2N 4e: X=Cl, 88% 4f: X=Cl, 87% 4i: X=Br, 89% 4j: X=Br, 90% 4k: X=Br, 92% 4l: X=Br, 90% In order to explore the feasibility of this approach, cyclic secondary amines (piperidine and pyrrolidine), acyclic secondary amine (diethylamine), aliphatic primary amine (ethanolamine) were examined to achieve the desired coupling (Table 3). First, the reactions were carried out by using 2-chloropyridine as starting material. To our delight, the corresponding coupling product 4a and 4b were obtained in 87% and 88% yield, respectively. We also attempted the coupling of 4-chloropyridine to form 4c and 4d. Table 2. C-N Coupling reactions of substituted aryl compounds with pyrrole or azoles a,b. sterically encumbered 2-methylimidazole also reacted without incidents (3l, 3n). Table 2. C-N Coupling reactions of substituted aryl compounds with pyrrole or azoles a,b re sterically encumbered 2-methylimidazole also reacted without incidents (3l, 3n). N N N N Me Z Y X R Heteroaryl-NH + Cu(OAc)2 (10 mol%) BO (10 mol%) K3PO4 (2 equiv.) DMSO, Ar, 80 oC Z Y N R Heteroaryl OHC N N N N CO2H N N O N N N N EtO2C MeO2C N N N N 3a: X=Br, 88% 3b: X=Br, 94% X=Cl, 0% NH2 3c: X=Br, 95% 3d: X=Br, 96% 3e: X=Br, 85% 3f: X=Br, 90% 3g: X=Br, 91% CN N N N N N N N N Me N N N N N N 3j: X=Cl, 84% 3k: X=Cl, 88% 3l: X=Cl, 85% 3m: X=Cl, 83% 3n: X=Cl, 80% 3h: X=Br, 93% 3i: X=Br, 98% Y, Z = CH or N X=Br or Cl Me OMe Table 3. C-N Coupling reactions of substituted aryl compounds with amines a,b a Reactions conducted on (hetero)aryl halides (2.81 mmol), pyrrole or azoles (3.37 mmol), Cu(OAc)2 (0.28 mmol), BO (0.28 mmol), K3PO4 (5.62 mmol), DMSO (4 mL) under Ar at 80 ◦C for 8~10 h. b Isolated yield with column chromatography. Molecules 2019, 24, x 4 of 12 chlorides were employed, couplings were smoothly proceeded, although the yield was decreased. A more sterically encumbered 2-methylimidazole also reacted without incidents (3l, 3n). Table 2. C-N Coupling reactions of substituted aryl compounds with pyrrole or azoles a,b. sterically encumbered 2-methylimidazole also reacted without incidents (3l, 3n). In both cases, the products were obtained in good yields. Both electron-rich (4g) and electron-poor (4i) aryl bromides participated equally well in the reaction. The reaction was effective in the presence of unprotected polar functional groups such as alcohol. It was Table 3. C-N Coupling reactions of substituted aryl compounds with amines a,b. N N N N N N OHC N N N N OHC N N N N N N N N NEt2 H N OH O2N W Z Y X R + Cu(OAc)2 (10 mol%) BO (10 mol%) K3PO4 (2 equiv.) DMSO, Ar, 80 oC W Z Y NRR' R W, Y, Z = CH or N X=Br or Cl OMe OMe 4a: X=Cl, 87% 4b: X=Cl, 88% 4c: X=Cl, 85% 4d: X=Cl, 86% 4e: X=Cl, 88% 4f: X=Cl, 87% 4g: X=Br, 80% 4h: X=Br, 82% 4i: X=Br, 89% 4j: X=Br, 90% 4k: X=Br, 92% 4l: X=Br, 90% NHRR' a Reactions conducted on (hetero)aryl halides (2.81 mmol), amines (3.37 mmol), Cu(OAc)2 (0.28 mmol), BO (0.28 mmol), K3PO4 (5.62 mmol), DMSO (4 mL) under Ar at 80 ◦C for 8 h. b Isolated yield with column chromatography. HC OHC N N O2N Table 3. C-N Coupling reactions of substituted aryl compounds with amines a,b. HC OHC N N O2N Table 3. C-N Coupling reactions of substituted aryl compounds with amines a,b. r, 89% 4j: X Br, 9 W Z Y X R + W, Y, Z = CH or N X=Br or Cl N n order to explore the feasibility of this approach, cyclic idine), acyclic secondary amine (diethylamine), aliphatic p ned to achieve the desired coupling (Table 3). First, the r ropyridine as starting material. To our delight, the corresp N N N N N N N N 4a: X=Cl, 87% 4b: X=Cl, 88% 4c: X=Cl, 85% 4d: X=Cl, 86% he feasibility of this approach, cyclic ndary amine (diethylamine), aliphatic p desired coupling (Table 3). First, the r ng material. To our delight, the corresp N N N N N N X=Cl, 88% 4c: X=Cl, 85% 4d: X=Cl, 86% pproach, cyclic ine), aliphatic 3). 2. Results and Discussion To initiate our studies 2. Results and Discussions The reaction tolerated a diverse array of functional groups on the (hetero)aryl halides, including methoxy (3a), aldehyde (3c), carboxyl (3d), amino (3e), ketone (3f), ester (3g, 3h), cyano (3i). Electronic properties of the (hetero)aryl halides were evaluated by introducing electron-withdrawing and electron-donating groups on the aryl moiety. Although electron-poor (hetero)aryl halides (e.g., 3c, 3d, 3i) underwent coupling faster than electron rich ones (3a, 3e), the desired products were successfully obtained in all cases. Unfortunately, chlorobenzene couldn’t provide the product 3b. By changing which heteroaryl With optimized conditions in hand, we set out to evaluate the scope of (hetero)aryl halides that would participate in this transformation (Table 2). The reaction tolerated a diverse array of functional groups on the (hetero)aryl halides, including methoxy (3a), aldehyde (3c), carboxyl (3d), amino (3e), ketone (3f), ester (3g, 3h), cyano (3i). Electronic properties of the (hetero)aryl halides were evaluated by introducing electron-withdrawing and electron-donating groups on the aryl moiety. Although electron-poor (hetero)aryl halides (e.g., 3c, 3d, 3i) underwent coupling faster than electron rich ones (3a, 3e), the desired products were successfully obtained in all cases. Unfortunately, chlorobenzene couldn’t provide the product 3b. By changing which heteroaryl chlorides were employed, couplings were smoothly proceeded, although the yield was decreased. A more sterically encumbered 2-methylimidazole also reacted without incidents (3l, 3n). 4 of 12 12 Molecules 2019, 24, 4177 Molecules 2019, 24, x a Reactions conducted on (hetero)aryl halides (2.81 mmol), amines (3.37 mmol), Cu(OAc)2 (0.28 mmol), BO (0.28 mmol), K3PO4 (5.62 mmol), DMSO (4 mL) under Ar at 80 ◦C for 8 h. b Isolated yield with column chromatography. Table 2. C-N Coupling reactions of substituted aryl compounds with pyrrole or azoles a,b. sterically encumbered 2-methylimidazole also reacted without incidents (3l, 3n). C-N Coupling reactions of substituted aryl compounds with amino acids/esters a,b. Table 4. C-N Coupling reactions of substituted aryl compounds with amino acids/esters a,b. Table 4. C-N Coupling reactions of substituted aryl compounds with amino acids/esters a,b. Table 4. C-N Coupling reactions of substituted aryl compounds with amino acids/esters a,b. Z Y X R Amino Acid/Ester + Cu(OAc)2 (10 mol%) BO (10 mol%) K3PO4 (2 equiv.) DMSO, Ar, 80 oC Z Y NHR1 R Y, Z = CH or N X=Br or Cl iPr OtBu O NH H N OH O O2N O OH HN Me O OH NH iPr OH O NH N N NH OH O MeS N N 5a: X=Br, 90% 5b: X=Br, 92% 5c: X=Br, 88% 5d: X=Br, 90% 5e: X=Cl, 86% 5f: X=Cl, 87% a Reactions conducted on (hetero)aryl halides (2.81 mmol), amino acids/esters (3.37 mmol), Cu(OAc)2 a Reactions conducted on (hetero)aryl halides (2.81 mmol), amino acids/esters (3.37 mmol), Cu(OAc)2 (0.28 mmol), BO 0.28 mmol), K3PO4 (5.62 mmol), DMSO (4 mL) under Ar at 80 ◦C for 8 h. b Isolated yield with column chromatography. a Reactions conducted on (hetero)aryl halides (2 81 mmol) amino acids/esters (3 37 mmol) Cu(OAc)2 a Reactions conducted on (hetero)aryl halides (2.81 mmol), amino acids/esters (3.37 mmol), Cu(OAc)2 (0.28 mmol), BO 0.28 mmol), K3PO4 (5.62 mmol), DMSO (4 mL) under Ar at 80 ◦C for 8 h. b Isolated yield with column chromatography. (0.28 mmol), BO 0.28 mmol), K3PO4 (5.62 mmol), DMSO (4 mL) under Ar at 80 C for 8 h. Isolated yield with column chromatography. Although N-heterocycle electrophiles were the primary focus of this study, amino acids-based electrophiles were also evaluated (Table 4). Amino acids underwent coupling to afford corresponding products in moderate yields. Although the yields were modest, it was noted that these reactions were conducted under the conditions developed for the 2-bromoanisole with minimal reoptimization. Substrates bearing either electron-withdrawing (5b) or electron-donating groups (5c) on the (hetero)aryl halides coupled with high yields. Introduction of an ester group into amino acid was also tolerated (5a). Finally, we were pleased to find that our method was not limited The ligand BO has been reported to be used as a metal chelating agent [28], which is a typical N,O-ligand. Thus, it was believed that the Cu-catalyzed couplings could process in a homogeneous manner due to the formation of Cu-benzoinoxime complex. Table 2. C-N Coupling reactions of substituted aryl compounds with pyrrole or azoles a,b. sterically encumbered 2-methylimidazole also reacted without incidents (3l, 3n). First, the r ght, the corresp N N 4d: X=Cl, 86% n order to explore the feasibi idine), acyclic secondary amin ned to achieve the desired co ropyridine as starting materia N N N N 4a: X=Cl, 87% 4b: X=Cl, 88% c secondary a primary amin reactions wer ponding coup N OMe 4g: X=Br, 80% ore the feasib econdary ami the desired co arting materia N N 4b: X=Cl, 88% mines (piperid ne (ethanolamin e carried out b ling product 4a N OMe 4h: X=Br, 82% ght, the corres 4d: X=Cl, 86% dine as starting material. To our delight, the corre X=Cl, 87% 4b: X=Cl, 88% 4c: X=Cl, 85% 4d: X=Cl, 86% ponding coup 4g: X=Br, 80% pyridine as st 4a: X=Cl, 87% ling product 4 4h: X=Br, 82% obtained in 87% and 88% oropyridine to form 4c and 4d. I on-rich (4g) and electron-poor ( on was effective in the presence N OHC OHC N 4i: X=Br, 89% 4j: X=Br, 90% re obtained in 87% and 88% yield, respectively. We hloropyridine to form 4c and 4d. In both cases, the products ctron-rich (4g) and electron-poor (4i) aryl bromides participa ction was effective in the presence of unprotected polar funct N OHC OHC N N N N N N N 4e: X=Cl, 88% 4f: X=Cl, 87% 4i: X=Br, 89% 4j: X=Br, 90% 5 of 12 Molecules 2019, 24, 4177 Although N-heterocycle electrophiles were the primary focus of this study, amino acids-based electrophiles were also evaluated (Table 4). Amino acids underwent coupling to afford corresponding products in moderate yields. Although the yields were modest, it was noted that these reactions were conducted under the conditions developed for the 2-bromoanisole with minimal reoptimization. Substrates bearing either electron-withdrawing (5b) or electron-donating groups (5c) on the (hetero)aryl halides coupled with high yields. Introduction of an ester group into amino acid was also tolerated (5a). Finally, we were pleased to find that our method was not limited to 2-chloropyrimidine. Using 2-chloropyrimidine as the substrate led to the formation of 5e, 5f in 86%, 87% yield, respectively. Molecules 2019, 24, x 5 of 12 encouraged that the ethanolamine substitute could provide 4l in 90% yield; the lower yield was due to incomplete conversion of the starting material. Table 4. C-N Coupling reactions of substituted aryl compounds with amino acids/esters a,b. Table 4. C-N Coupling reactions of substituted aryl compounds with amino acids/esters a,b. Table 4. 3. Materials and Method 3. Materials and Methods All of the starting materials, reagents, and solvents are commercially available and used without further purification. Melting points were determined with a X-4 apparatus (Beijing Taike Instrument Co., Ltd., Beijing, China) and were uncorrected. The nuclear magnetic resonance (NMR) spectra were recorded on a Bruker (Bruker Technology Co., Ltd., Karlsruhe, Germany) 400 MHz spectrometer in CDCl3 or DMSO-d6 using tetramethylsilane (TMS) as an internal standard. Electrospray ionization mass spectrometry (MS (ESI)) analyses were recorded in an Agilent 1100 Series MSD Trap SL (Santa Clara, CA, USA). The reactions were monitored by thin-layer chromatography (TLC: HG/T2354-92, GF254), and compounds were visualized on TLC with UV light (Gongyi Yuhua Instrument Co., Ltd, Zhengzhou, China). General Procedure for Catalytic Experiments All of the starting materials, reagents, and solvents are commercially available and used without further purification. Melting points were determined with a X-4 apparatus (Beijing Taike Instrument Co., Ltd., Beijing, China) and were uncorrected. The nuclear magnetic resonance (NMR) spectra were recorded on a Bruker (Bruker Technology Co., Ltd., Karlsruhe, Germany) 400 MHz spectrometer in CDCl3 or DMSO-d6 using tetramethylsilane (TMS) as an internal standard. Electrospray ionization mass spectrometry (MS (ESI)) analyses were recorded in an Agilent 1100 Series MSD Trap SL (Santa Clara, CA, USA). The reactions were monitored by thin-layer chromatography (TLC: HG/T2354-92, GF254), and compounds were visualized on TLC with UV light (Gongyi Yuhua Instrument Co., Ltd, Zhengzhou, China). Table 2. C-N Coupling reactions of substituted aryl compounds with pyrrole or azoles a,b. sterically encumbered 2-methylimidazole also reacted without incidents (3l, 3n). Herein, it was proposed that the possible mechanism for the couplings might run via a prototypical Cu (I)/Cu (III) catalytic cycle. [29] As shown in Figure 2, the catalytic cycle initiated from a Cu complex (A). Then, coordinated copper species (B) were produced via oxidative addition of an ArX and A. Ligand exchange was subsequently occurred between A and N-heterocycles to form intermediate C, which could be converted to D in the presence of base. The N-arylazole was obtained by final reductive elimination of D. 6 of 12 Molecules 2019, 24, 4177 Ph O Cu N Ph OH H B-HX ArX A B D C Ar-N-Het B NH-Het Ph O Cu N Ph OH H Ar X Ph O Cu N Ph OH H Ar X NH-Het Ph O Cu N Ph OH H Ar N-Het Figure 2. Proposed mechanism for the couplings of (hetero)aryl halides with N-containing heterocycles. NH-Het represented N-hetero nucleophiles; X was bromine or chloride. Figure 2. Proposed mechanism for the couplings of (hetero)aryl halides with N-containing heterocycles. NH-Het represented N-hetero nucleophiles; X was bromine or chloride. Ar-N-Het ArX B Figure 2. Proposed mechanism for the couplings of (hetero)aryl halides with N-containing heterocycles. NH-Het represented N-hetero nucleophiles; X was bromine or chloride. Figure 2. Proposed mechanism for the couplings of (hetero)aryl halides with N-containing heterocycles. NH-Het represented N-hetero nucleophiles; X was bromine or chloride. To a solution of (hetero)aryl halide General Procedure for Catalytic Experiments 1H-NMR (400 MHz, CDCl3) δ (ppm): 11.40 (1H, br), 8.05–8.02 (1H, dd, J = 7.8 Hz, 1.2 Hz), 7.76–7.74 (2H, m), 7.62–7.58 (1H, m), 7.49–7.40 (2H, m), 6.48 (1H, s). MS (ESI) m/z: 189.06 [M + H]+. 2-(1H-Pyrazol-1-yl)aniline (3e) [34]: brown oil (0.38 g, 85%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.74–7.71 (2H, m), 7.19–7.13 (2H, m), 6.85–6.76 (2H, m), 6.44 (1H, t, J = 2.0 Hz), 4.63 (2H, br). MS (ESI) m/z: 160.09 [M + H]+. 2-(1H-Pyrazol-1-yl)aniline (3e) [34]: brown oil (0.38 g, 85%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.74–7.71 (2H, m), 7.19–7.13 (2H, m), 6.85–6.76 (2H, m), 6.44 (1H, t, J = 2.0 Hz), 4.63 (2H, br). MS (ESI) m/z: 160.09 [M + H]+. 1-(4-(1H-Pyrazol-1-yl)phenyl)ethan-1-one (3f) [35]: yellow oil (0.47 g, 90%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.08–8.06 (2H, m), 8.02 (1H, d, J = 2.5 Hz), 7.83–7.81 (2H, m), 7.78 (1H, d, J = 1.4 Hz), 6.53 (1H, t, J = 2.0 Hz), 2.63 (3H, s). MS (ESI) m/z: 187.09 [M + H]+. 1-(4-(1H-Pyrazol-1-yl)phenyl)ethan-1-one (3f) [35]: yellow oil (0.47 g, 90%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.08–8.06 (2H, m), 8.02 (1H, d, J = 2.5 Hz), 7.83–7.81 (2H, m), 7.78 (1H, d, J = 1.4 Hz), 6.53 (1H, t, J = 2.0 Hz), 2.63 (3H, s). MS (ESI) m/z: 187.09 [M + H]+. Ethyl 3-(1H-pyrazol-1-yl)benzoate (3g) [36]: yellow liquid (0.55 g, 91%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.31 (1H, t, J = 1.8 Hz), 7.99 (1H, d, J = 2.4 Hz), 7.97–7.93 (2H, m), 7.74 (1H, d, J = 1.6 Hz), 7.53 (1H, t, J = 8.0 Hz), 6.49 (1H, t, J = 2.0 Hz), 4.44–4.38 (2H, q, J = 14.3 Hz, 7.2 Hz), 1.41 (3H, t, J = 7.1 Hz). MS (ESI) m/z: 217.10 [M + H]+. Methyl 4-(1H-pyrazol-1-yl)benzoate (3h) [37]: white solid (0.53 g, 93%), m.p. 103–105 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.15–8.12 (2H, m), 8.00 (1H, d, J = 2.4 Hz), 7.80–7.77 (2H, m), 7.76 (1H, d, J = 1.4 Hz), 6.51 (1H, t, J = 2.1 Hz), 3.93 (3H, s). MS (ESI) m/z: 203.11 [M + H]+. Methyl 4-(1H-pyrazol-1-yl)benzoate (3h) [37]: white solid (0.53 g, 93%), m.p. 103–105 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.15–8.12 (2H, m), 8.00 (1H, d, J = 2.4 Hz), 7.80–7.77 (2H, m), 7.76 (1H, d, J = 1.4 Hz), 6.51 (1H, t, J = 2.1 Hz), 3.93 (3H, s). To a solution of (hetero)aryl halide General Procedure for Catalytic Experiments MS (ESI) m/z: 203.11 [M + H]+. 2-(1H-Pyrazol-1-yl)benzonitrile (3i) [38]: yellow oil (0.47 g, 98%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.15 (1H, d, J = 2.5 Hz), 7.81–7.79 (3H, m), 7.77 (1H, d, J = 1.3 Hz), 7.72–7.68 (1H, m), 6.55 (1H, t, J = 2.1 Hz). MS (ESI) m/z: 170.07 [M + H]+. 2-(1H-Pyrazol-1-yl)benzonitrile (3i) [38]: yellow oil (0.47 g, 98%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.15 (1H, d, J = 2.5 Hz), 7.81–7.79 (3H, m), 7.77 (1H, d, J = 1.3 Hz), 7.72–7.68 (1H, m), 6.55 (1H, t, J = 2.1 Hz). MS (ESI) m/z: 170.07 [M + H]+. 2-(1H-Pyrrol-1-yl)pyridine (3j) [39]: colorless oil (0.34 g, 84%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.43–8.42 (1H, m), 7.75–7.71 (1H, m), 7.52 (2H, t, J = 2.3 Hz), 7.32 (1H, d, J = 8.3 Hz), 7.11–7.08 (1H, m), 6.36 (2H, t, J = 2.3 Hz). MS (ESI) m/z: 145.04 [M + H]+. 2-(1H-Pyrrol-1-yl)pyridine (3j) [39]: colorless oil (0.34 g, 84%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.43–8.42 (1H, m), 7.75–7.71 (1H, m), 7.52 (2H, t, J = 2.3 Hz), 7.32 (1H, d, J = 8.3 Hz), 7.11–7.08 (1H, m), 6.36 (2H, t, J = 2.3 Hz). MS (ESI) m/z: 145.04 [M + H]+. 2-(1H-Imidazol-1-yl)pyridine (3k) [40]: white solid (0.36 g, 88%), m.p. 40–41 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.50–8.48 (1H, m), 8.35 (1H, s), 7.85–7.80 (1H, m), 7.65 (1H, t, J = 1.3 Hz), 7.37–7.35 (1H, m), 7.27–7.23 (1H, m), 7.20 (1H, s). MS (ESI) m/z: 146.08 [M + H]+. 2-(1H-Imidazol-1-yl)pyridine (3k) [40]: white solid (0.36 g, 88%), m.p. 40–41 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.50–8.48 (1H, m), 8.35 (1H, s), 7.85–7.80 (1H, m), 7.65 (1H, t, J = 1.3 Hz), 7.37–7.35 (1H, m), 7.27–7.23 (1H, m), 7.20 (1H, s). MS (ESI) m/z: 146.08 [M + H]+. 2-(2-Methyl-1H-imidazol-1-yl)pyridine (3l) [41]: colorless oil (0.38 g, 85%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.56–8.54 (1H, m), 7.86–7.82 (1H, m), 7.32–7.29 (2H, m), 7.28 (1H, d, J = 1.5 Hz), 7.02 (1H, d, J = 1.5 Hz), 7.02 (1H, d, J = 1.4 Hz), 2.59 (3H, s). MS (ESI) m/z: 160.09 [M + H]+. 2-(2-Methyl-1H-imidazol-1-yl)pyridine (3l) [41]: colorless oil (0.38 g, 85%). To a solution of (hetero)aryl halide General Procedure for Catalytic Experiments K3PO4 (5.62 mmol) in DMSO (4 mL), were added Cu(OAc)2 (0.28 mmol). The flask was evacuated and backfilled with argon for three times. The resulting suspension was heated in a 80 °C oil bath with stirring for the indicated time. The reactor was cooled to r.t., the flask was opened to air and the reaction mixture was poured into water (20 mL), extracted with ethyl acetate (20 mL × 3), and organic layer was washed with water (20 mL × 2) and once with brine (25 mL), dried over magnesium sulfate and concentrated in vacuo. The product was purified by column chromatography on silica gel using petroleum ether and ethyl acetate as eluent. 1-(2-Methoxyphenyl)-1H-pyrrole (3a) [30]: colorless oil (0.43 g, 88%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.30–7.23 (2H, m), 7.03–6.98 (4H, m), 6.30 (2H, t, J = 2.2 Hz), 3.82 (3H, s). MS (ESI) m/z: 174.11 [M + H]+, To a solution of (hetero)aryl halide (2.81 mmol), N-nucleophile (3.37 mmol), BO (0.28 mmol), K3PO4 (5.62 mmol) in DMSO (4 mL), were added Cu(OAc)2 (0.28 mmol). The flask was evacuated and backfilled with argon for three times. The resulting suspension was heated in a 80 ◦C oil bath with stirring for the indicated time. The reactor was cooled to r.t., the flask was opened to air and the reaction mixture was poured into water (20 mL), extracted with ethyl acetate (20 mL × 3), and organic layer was washed with water (20 mL × 2) and once with brine (25 mL), dried over magnesium sulfate and concentrated in vacuo. The product was purified by column chromatography on silica gel using petroleum ether and ethyl acetate as eluent. see supplementary material. 1-(2-Methoxyphenyl)-1H-pyrrole (3a) [30]: colorless oil (0.43 g, 88%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.30–7.23 (2H, m), 7.03–6.98 (4H, m), 6.30 (2H, t, J = 2.2 Hz), 3.82 (3H, s). MS (ESI) m/z: 174.11 [M + H]+, see Supplementary Materials. see supplementary material. 1-(2-Methoxyphenyl)-1H-pyrrole (3a) [30]: colorless oil (0.43 g, 88%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.30–7.23 (2H, m), 7.03–6.98 (4H, m), 6.30 (2H, t, J = 2.2 Hz), 3.82 (3H, s). MS (ESI) m/z: 174.11 [M + H]+, see Supplementary Materials. 7 of 12 7 of 12 Molecules 2019, 24, 4177 1-Phenyl-1H-pyrrole (3b) [31]: white solid (0.38 g, 94%). m.p. 60–62 C. To a solution of (hetero)aryl halide General Procedure for Catalytic Experiments 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.43–7.37 (4H, m), 7.25–7.21 (1H, m), 7.08 (2H, t, J = 2.2 Hz), 6.34 (2H, t, J = 2.2 Hz). MS (ESI) m/z: 144.04 [M + H]+. 1-Phenyl-1H-pyrrole (3b) [31]: white solid (0.38 g, 94%). m.p. 60–62 C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.43–7.37 (4H, m), 7.25–7.21 (1H, m), 7.08 (2H, t, J = 2.2 Hz), 6.34 (2H, t, J = 2.2 Hz). MS (ESI) m/z: 144.04 [M + H]+. 1-Phenyl-1H-pyrrole (3b) [31]: white solid (0.38 g, 94%). m.p. 60–62 C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.43–7.37 (4H, m), 7.25–7.21 (1H, m), 7.08 (2H, t, J = 2.2 Hz), 6.34 (2H, t, J = 2.2 Hz). MS (ESI) m/z: 144.04 [M + H]+. 3-(1H-Pyrazol-1-yl)benzaldehyde (3c) [32]: offwhite solid (0.46 g, 95%), m.p. 28–30 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 10.08 (1H, s), 8.19 (1H, t, J = 1.8 Hz), 8.05–8.02 (2H, m), 7.81–7.76 (2H, m), 7.64 (1H, t, J = 7.9 Hz), 6.52 (1H, t, J = 2.1 Hz). MS (ESI) m/z: 173.08 [M + H]+. 3-(1H-Pyrazol-1-yl)benzaldehyde (3c) [32]: offwhite solid (0.46 g, 95%), m.p. 28–30 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 10.08 (1H, s), 8.19 (1H, t, J = 1.8 Hz), 8.05–8.02 (2H, m), 7.81–7.76 (2H, m), 7.64 (1H, t, J = 7.9 Hz), 6.52 (1H, t, J = 2.1 Hz). MS (ESI) m/z: 173.08 [M + H]+. 3-(1H-Pyrazol-1-yl)benzaldehyde (3c) [32]: offwhite solid (0.46 g, 95%), m.p. 28–30 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 10.08 (1H, s), 8.19 (1H, t, J = 1.8 Hz), 8.05–8.02 (2H, m), 7.81–7.76 (2H, m), 7.64 (1H, t, J = 7.9 Hz), 6.52 (1H, t, J = 2.1 Hz). MS (ESI) m/z: 173.08 [M + H]+. 2-(1H-Pyrazol-1-yl)benzoic acid (3d) [33]: white solid (0.51 g, 96%), m.p. 128–129 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 11.40 (1H, br), 8.05–8.02 (1H, dd, J = 7.8 Hz, 1.2 Hz), 7.76–7.74 (2H, m), 7.62–7.58 (1H, m), 7.49–7.40 (2H, m), 6.48 (1H, s). MS (ESI) m/z: 189.06 [M + H]+. 2-(1H-Pyrazol-1-yl)benzoic acid (3d) [33]: white solid (0.51 g, 96%), m.p. 128–129 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 11.40 (1H, br), 8.05–8.02 (1H, dd, J = 7.8 Hz, 1.2 Hz), 7.76–7.74 (2H, m), 7.62–7.58 (1H, m), 7.49–7.40 (2H, m), 6.48 (1H, s). MS (ESI) m/z: 189.06 [M + H]+. 2-(1H-Pyrazol-1-yl)benzoic acid (3d) [33]: white solid (0.51 g, 96%), m.p. 128–129 ◦C. To a solution of (hetero)aryl halide General Procedure for Catalytic Experiments 1-(2-Methoxyphenyl)pyrrolidine (4g) [48]: colorless oil (0.40 g, 80%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 6.90–6.81 (4H, m), 3.83 (3H, s), 3.30–3.27 (4H, m), 1.95–1.91 (4H, m). MS (ESI) m/z: 178.16 [M + H]+. 1-(2-Methoxyphenyl)piperidine (4h) [49]: colorless oil (0.44 g, 82%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 6.99–6.84 (4H, m), 3.86 (3H, s), 2.99–2.97 (4H, m), 1.78–1.54 (6H, m). MS (ESI) m/z: 192.17 [M + H]+. 4-(Pyrrolidin-1-yl)benzaldehyde (4i) [50]: white solid (0.44 g, 89%), m.p. 83–85 ◦C.1H-NMR (400 MHz, CDCl3) δ (ppm): 9.72 (1H, s), 7.73 (2H, d, J = 8.8 Hz), 6.58 (2H, d, J = 8.8 Hz), 3.40–3.37 (4H, m), 2.08–2.02 (4H, m). MS (ESI) m/z: 176.13 [M + H]+. 4-(Piperidin-1-yl)benzaldehyde (4j) [51]: white solid (0.48 g, 90%). m.p. 63–64 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 9.75 (1H, s), 7.75–7.71 (2H, m), 6.91 (2H, d, J = 8.9 Hz), 3.41–3.40 (4H, m), 1.68 (6H, s). MS (ESI) m/z: 190.16 [M + H]+. 4-(Piperidin-1-yl)benzaldehyde (4j) [51]: white solid (0.48 g, 90%). m.p. 63–64 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 9.75 (1H, s), 7.75–7.71 (2H, m), 6.91 (2H, d, J = 8.9 Hz), 3.41–3.40 (4H, m), 1.68 (6H, s). MS (ESI) m/z: 190.16 [M + H]+. N,N-Diethylaniline (4k) [52]: yellow liquid (0.39 g, 92%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.23–7.18 (2H, m), 6.69–6.61 (3H, m), 3.37–3.32 (4H, q, J = 14.1 Hz, 7.0 Hz), 1.15 (6H, t, J = 7.1 Hz). MS (ESI) m/z: 150.12 [M + H]+. N,N-Diethylaniline (4k) [52]: yellow liquid (0.39 g, 92%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.23–7.18 (2H, m), 6.69–6.61 (3H, m), 3.37–3.32 (4H, q, J = 14.1 Hz, 7.0 Hz), 1.15 (6H, t, J = 7.1 Hz). MS (ESI) m/z: 150.12 [M + H]+. 2-((4-Nitrophenyl)amino)ethan-1-ol (4l) [53]: yellow solid (0.46 g, 90%). m.p. 110–111 ◦C.1H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.05 (2H, d, J = 9.2 Hz), 7.35 (1H, t, J = 5.0 Hz), 6.73 (2H, d, J = 9.2 Hz), 4.86 (1H, t, J = 5.4 Hz), 3.65–3.61 (2H, q, J = 11.2 Hz, 5.6 Hz), 3.31–3.27 (2H, q, J = 11.4 Hz, 5.7 Hz). MS (ESI) m/z: 183.08 [M + H]+. tert-Butyl phenyl-D-valinate (5a) [54]: white solid (0.63 g, 90%), m.p. 64–66 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 7.17–7.13 (2H, m), 6.72–6.69 (1H, m), 6.64–6.62 (2H, m), 4.12 (1H, br), 3.75 (1H, d, J = 5.3 Hz), 2.15–2.04 (1H, m), 1.42 (9H, s), 1.05–1.01 (6H, m). To a solution of (hetero)aryl halide General Procedure for Catalytic Experiments 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.56–8.54 (1H, m), 7.86–7.82 (1H, m), 7.32–7.29 (2H, m), 7.28 (1H, d, J = 1.5 Hz), 7.02 (1H, d, J = 1.5 Hz), 7.02 (1H, d, J = 1.4 Hz), 2.59 (3H, s). MS (ESI) m/z: 160.09 [M + H]+. 2-(1H-Imidazol-1-yl)pyrimidine (3m) [42]: white solid (0.34 g, 83%), m.p. 120–122 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.71 (2H, d, J = 4.8 Hz), 8.64 (1H, s), 7.90 (1H, s), 7.21 (1H, t, J = 4.8 Hz), 7.18 (1H, s). MS (ESI) m/z: 147.06 [M + H]+. 2-(1H-Imidazol-1-yl)pyrimidine (3m) [42]: white solid (0.34 g, 83%), m.p. 120–122 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.71 (2H, d, J = 4.8 Hz), 8.64 (1H, s), 7.90 (1H, s), 7.21 (1H, t, J = 4.8 Hz), 7.18 (1H, s). MS (ESI) m/z: 147.06 [M + H]+. 2-(2-Methyl-1H-imidazol-1-yl)pyrimidine (3n) [43]: white solid (0.36 g, 80%), m.p. 90–92 ◦C.1H-NMR (400 MHz, CDCl3) δ (ppm): 8.72 (2H, d, J = 4.8 Hz), 7.86 (1H, d, J = 1.4 Hz), 7.18 (1H, t, J = 4.8 Hz), 6.97 (1H, d, J = 1.3 Hz), 2.82 (3H, s). MS (ESI) m/z: 161.10 [M + H]+. 2-(2-Methyl-1H-imidazol-1-yl)pyrimidine (3n) [43]: white solid (0.36 g, 80%), m.p. 90–92 ◦C.1H-NMR (400 MHz, CDCl3) δ (ppm): 8.72 (2H, d, J = 4.8 Hz), 7.86 (1H, d, J = 1.4 Hz), 7.18 (1H, t, J = 4.8 Hz), 6.97 (1H, d, J = 1.3 Hz), 2.82 (3H, s). MS (ESI) m/z: 161.10 [M + H]+. 2-(2-Methyl-1H-imidazol-1-yl)pyrimidine (3n) [43]: white solid (0.36 g, 80%), m.p. 90–92 ◦C.1H-NMR (400 MHz, CDCl3) δ (ppm): 8.72 (2H, d, J = 4.8 Hz), 7.86 (1H, d, J = 1.4 Hz), 7.18 (1H, t, J = 4.8 Hz), 6.97 (1H, d, J = 1.3 Hz), 2.82 (3H, s). MS (ESI) m/z: 161.10 [M + H]+. 2-(Piperidin-1-yl)pyridine (4a) [44]: colorless oil (0.40 g, 87%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.16–8.15 (1H, m), 7.44–7.39 (1H, m), 6.63 (1H, d, J = 8.6 Hz), 6.54–6.51 (1H, m), 3.52 (4H, d, J = 4.9 Hz), 1.62 (6H, s). MS (ESI) m/z: 163.13 [M + H]+. 2-(Piperidin-1-yl)pyridine (4a) [44]: colorless oil (0.40 g, 87%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.16–8.15 (1H, m), 7.44–7.39 (1H, m), 6.63 (1H, d, J = 8.6 Hz), 6.54–6.51 (1H, m), 3.52 (4H, d, J = 4.9 Hz), 1.62 (6H, s). MS (ESI) m/z: 163.13 [M + H]+. To a solution of (hetero)aryl halide General Procedure for Catalytic Experiments 8 of 12 Molecules 2019, 24, 4177 2-(Pyrrolidin-1-yl)pyridine (4b) [45]: colorless oil (0.37 g, 88%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.16–8.14 (1H, m), 7.44–7.39 (1H, m), 6.51–6.48 (1H, m), 6.35 (1H, d, J = 8.5 Hz), 3.46–3.43 (4H, m), 2.02–1.98 (4H, m). MS (ESI) m/z: 149.12 [M + H]+. 2-(Pyrrolidin-1-yl)pyridine (4b) [45]: colorless oil (0.37 g, 88%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.16–8.14 (1H, m), 7.44–7.39 (1H, m), 6.51–6.48 (1H, m), 6.35 (1H, d, J = 8.5 Hz), 3.46–3.43 (4H, m), 2.02–1.98 (4H, m). MS (ESI) m/z: 149.12 [M + H]+. 4-(Piperidin-1-yl)pyridine (4c) [46]: colorless oil (0.39 g, 85%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.22–8.20 (2H, q, J = 5.1 Hz, 1.6 Hz), 6.62–6.61 (2H, q, J = 5.1 Hz, 1.6 Hz), 3.31 (4H, d, J = 4.9 Hz), 1.63 (6H, s). MS (ESI) m/z: 163.13 [M + H]+. 4-(Piperidin-1-yl)pyridine (4c) [46]: colorless oil (0.39 g, 85%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.22–8.20 (2H, q, J = 5.1 Hz, 1.6 Hz), 6.62–6.61 (2H, q, J = 5.1 Hz, 1.6 Hz), 3.31 (4H, d, J = 4.9 Hz), 1.63 (6H, s). MS (ESI) m/z: 163.13 [M + H]+. 4-(Pyrrolidin-1-yl)pyridine (4d) [44]: colorless oil (0.36 g, 86%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.18 (2H, d, J = 4.9 Hz), 6.36 (2H, d, J = 4.9 Hz), 3.30–3.27 (4H, m), 2.03–1.99 (4H, m). MS (ESI) m/z: 149.09 [M + H]+. 2-(Pyrrolidin-1-yl)pyrimidine (4e) [47]: colorless oil (0.37 g, 88%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.32 (2H, d, J = 4.8 Hz), 6.45 (1H, t, J = 4.8 Hz), 3.59–3.56 (4H, m), 2.02–1.98 (4H, m). MS (ESI) m/z: 150.11 [M + H]+. 2-(Pyrrolidin-1-yl)pyrimidine (4e) [47]: colorless oil (0.37 g, 88%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.32 (2H, d, J = 4.8 Hz), 6.45 (1H, t, J = 4.8 Hz), 3.59–3.56 (4H, m), 2.02–1.98 (4H, m). MS (ESI) m/z: 150.11 [M + H]+. 2-(Piperidin-1-yl)pyrimidine (4f) [47]: colorless oil (0.40 g, 87%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.30 (2H, t, J = 5.6 Hz), 6.42 (1H, t, J = 5.6 Hz), 3.81–3.78 (4H, m), 1.72–1.59 (6H, m). MS (ESI) m/z: 164.14 [M + H]+. 2-(Piperidin-1-yl)pyrimidine (4f) [47]: colorless oil (0.40 g, 87%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.30 (2H, t, J = 5.6 Hz), 6.42 (1H, t, J = 5.6 Hz), 3.81–3.78 (4H, m), 1.72–1.59 (6H, m). MS (ESI) m/z: 164.14 [M + H]+. To a solution of (hetero)aryl halide General Procedure for Catalytic Experiments MS (ESI) m/z: 250.16 [M + H]+. (4-Nitrophenyl)glycine (5b) [55]: brown solid (0.51 g, 92%), 224–226 ◦C. 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.02 (2H, d, J = 9.0 Hz), 7.44 (1H, t, J = 5.6 Hz), 6.66 (2H, d, J = 9.1 Hz), 3.98 (2H, d, J = 6.0 Hz). MS (ESI) m/z: 197.08 [M + H]+. (4-Nitrophenyl)glycine (5b) [55]: brown solid (0.51 g, 92%), 224–226 ◦C. 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.02 (2H, d, J = 9.0 Hz), 7.44 (1H, t, J = 5.6 Hz), 6.66 (2H, d, J = 9.1 Hz), 3.98 (2H, d, J = 6.0 Hz). MS (ESI) m/z: 197.08 [M + H]+. Phenyl-D-phenylalanine (5c) [56]: white solid (0.60 g, 88%), m.p. 173–176 ◦C .1H-NMR (400 MHz, CDCl3) δ (ppm): 7.33–7.17 (7H, m), 6.80 (1H, t, J = 7.3 Hz), 6.62 (2H, d, J = 7.8 Hz), 4.32 (1H, t, J = 5.8 Hz), 3.30–3.10 (2H, m). MS (ESI) m/z: 242.11 [M + H]+. Phenyl-L-alanine (5d) [57]: white solid (0.42 g, 90%), m.p. 133–135 ◦C. 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 7.06 (2H, t, J = 7.8 Hz), 6.56–6.53 (3H, m), 3.95–3.89 (1H, q, J = 14.0 Hz, 7.0 Hz), 1.37 (3H, d, J = 7.0 Hz). MS (ESI) m/z: 166.07 [M + H]+. 9 of 12 Molecules 2019, 24, 4177 Pyrimidin-2-yl-D-valine (5e): white solid (0.47 g, 86%), 113–115 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 11.24 (1H, br), 8.25 (2H, s), 7.22 (1H, d, J = 8.1 Hz), 6.55 (1H, t, J = 4.9 Hz), 4.62–4.59 (1H, q, J = 13.1 Hz, 5.0 Hz), 2.37–2.29 (1H, m), 1.06 (6H, t, J = 7.2 Hz). MS (ESI) m/z: 196.16 [M + H]+, 218.11 [M + H]+. 13 C-NMR (100 MHz, CDCl3) δ (ppm): 176.0, 161.2, 110.4, 59.4, 31.0, 18.9, 18.2. Pyrimidin-2-yl-D-valine (5e): white solid (0.47 g, 86%), 113–115 ◦C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 11.24 (1H, br), 8.25 (2H, s), 7.22 (1H, d, J = 8.1 Hz), 6.55 (1H, t, J = 4.9 Hz), 4.62–4.59 (1H, q, J = 13.1 Hz, 5.0 Hz), 2.37–2.29 (1H, m), 1.06 (6H, t, J = 7.2 Hz). MS (ESI) m/z: 196.16 [M + H]+, 218.11 [M + H]+. 13 C-NMR (100 MHz, CDCl3) δ (ppm): 176.0, 161.2, 110.4, 59.4, 31.0, 18.9, 18.2. Pyrimidin-2-ylmethionine (5f): white solid (0.56 g, 87%). 4. Conclusions In summary, a highly effective coupling reaction has been developed for the preparation of N-aryl compounds. This transformation occurs with good to excellent yields. A variety of substituted (hetero)aryl halides can be used as electrophiles, and azoles, piperidine, pyrrolidine, and amino acids, etc. function as nucleophiles. The key to this discovery was the identification of benzoin oxime ligand that can promote the (hetero)aryl halides to the corresponding N-arylation compounds. Efforts to apply our Cu-based system to other catalytic reactions and to expand the scope of the N-nucleophiles to other classes of nucleophiles are currently underway in our laboratory. Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/22/4177/s1. Copies of 1H NMR and MS for known compounds and copies of 13C NMR for new compounds. Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/22/4177/s1. Copies of 1H NMR and MS for known compounds and copies of 13C NMR for new compounds. Author Contributions: Conceptualization, C.Y.; Methodology, C.Y. and L.Z.; Software, C.Y.; Formal Analysis, Y.Z. and L.Z.; Writing-Original Draft Preparation, C.Y.; Writing-Review and Editing, C.Y. and Y.Z.; Project Administration, C.Y. Author Contributions: Conceptualization, C.Y.; Methodology, C.Y. and L.Z.; Software, C.Y.; Formal Analysis, Y.Z. and L.Z.; Writing-Original Draft Preparation, C.Y.; Writing-Review and Editing, C.Y. and Y.Z.; Project Administration, C.Y. Funding: This research was funded by Liaoning Provincial Natural Science Foundation of China, grant number “2019-BS-102”; College Students Innovation Training Program of Liaoning Province, grant number “201910160017” and “The APC was funded by Liaoning Provincial Natural Science Foundation of China”. Funding: This research was funded by Liaoning Provincial Natural Science Foundation of China, grant number “2019-BS-102”; College Students Innovation Training Program of Liaoning Province, grant number “201910160017” and “The APC was funded by Liaoning Provincial Natural Science Foundation of China”. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. To a solution of (hetero)aryl halide General Procedure for Catalytic Experiments 1H-NMR (400 MHz, CDCl3) δ (ppm): 13.32 (1H, br), 8.29 (2H, br), 7.92 (1H, d, J = 6.6 Hz), 6.62 (1H, t, J = 4.9 Hz), 4.88–4.84 (1H, q, J = 12.2 Hz, 6.1 Hz), 2.70–2.64 (2H, m), 2.37–2.22 (2H, m), 2.11 (3H, s). MS (ESI) m/z: 228.13 [M + H]+. 13 C-NMR (100 MHz, CDCl3) δ (ppm): 175.8, 160.3, 110.3, 53.7, 31.9, 30.0, 15.4. References 1. Roughley, S.D.; Jordan, A.M. The medicinal chemist’s toolbox: An analysis of reactions used in the pursuit of drug candidates. J. Med. 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[CrossRef] Sample Availability: Samples of the compounds are not available from the authors. Sample Availability: Samples of the compounds are not available from the authors. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Nuova serie / New series n. 04 - 2020 Nuova serie / New series n. 04 - 2020 Rivista internazionale di architettura e paesaggio alpino / Revue internationale d’architecture et de paysage dans les Alpes / Internationale Zeitschrift für Alpine Architektur und Landschaft / Revija za alpsko arhitekturo in pokrajino / International journal of alpine architecture and landscape a a ps o a te tu o po aj o te at o a jou a o a p e a c tectu e a d a dscape Per una nuova abitabilità delle Alpi. Architetture per il welfare e la rigenerazione For a new inhabitability of the Alps. Architectures for welfare and regeneration / Pour une nouvelle habitabilité des Alpes. Architectures pour le welfare et la régénération / Für eine neue Bewohnbarkeit in den Alpen. Architekturen für Wohlfahrt und Regeneration / Za novo bivalnost v Alpah, arhitekture za dobrobit in regeneracijo 1. Temi Futuro e rigenerazione Marco Bussone 2. Esperienze Dorferneuerung zwischen Erhalten und Gestalten / 39 Il rinnovamento dei villaggi: tra conservazione e progettazione Armando Ruinelli, Gion A. Caminada, Ludmila Seifert Marginalità e memoria come valori progettuali nell’esperienza 53 di Gion A. Caminada a Vrin / Marginality and memory as planning values in Gion A. Caminada’s Vrin experience Valerio Botta La costruzione dell’abitabilità in Val Bregaglia nel XX secolo / 61 The construction of habitability in Val Bregaglia in the 20th century Armando Ruinelli, Anna Innocenti Indice dei contenuti Contents Editoriale / Editorial 8 1. Temi Sulla centralità di spazio e territorio nel progetto di rigenerazione 13 delle montagne e delle aree interne / On the centrality of space and territory in the project of regeneration of mountains and internal areas Antonio De Rossi, Laura Mascino I servizi nelle Alpi italiane: quali e dove? Idee per uno scenario 19 post-pandemico / Facilities in the Italian Alps: which ones and where? Ideas for a post-pandemic scenario Giuseppe Dematteis Manifesto di Camaldoli per una nuova centralità della montagna 22 Alla ricerca della distanza perduta. Rigenerare luoghi, persone 27 e immaginari del riabitare alpino / In search of the lost distance. Regenerating places, people and images related to Alpine reinhabitation Filippo Barbera, Andrea Membretti Futuro e rigenerazione 34 Marco Bussone 2. Esperienze Dorferneuerung zwischen Erhalten und Gestalten / 39 Il rinnovamento dei villaggi: tra conservazione e progettazione Armando Ruinelli, Gion A. Caminada, Ludmila Seifert Marginalità e memoria come valori progettuali nell’esperienza 53 di Gion A. Caminada a Vrin / Marginality and memory as planning values in Gion A. Caminada’s Vrin experience Valerio Botta La costruzione dell’abitabilità in Val Bregaglia nel XX secolo / 61 The construction of habitability in Val Bregaglia in the 20th century Armando Ruinelli, Anna Innocenti Editoriale / Editorial 8 Editoriale / Editorial 2. Esperienze 71 Valades ousitanes, architettura e rigenerazione / Valades ousitanes, architecture and regeneration Antonio De Rossi, Laura Mascino Antonio De Rossi, Laura Mascino Ostana e Topolò: hardware, software e welfare nelle comunità 79 di “ritorno” / Ostana and Topolò: hardware, software and welfare in “return” communities Margherita Valcanover “Senza mostrare i muscoli”: i progetti di rigenerazione di 87 Tao+C e AZL nel solco della “prosperosa società” della Cina contemporanea / “Without flexing one’s muscles”: the regeneration projects of Tao+C and AZL in the wake of the “prosperous society” of contemporary China Edoardo Bruno, Dalila Tondo L’archipel Butor. Une régénération, par la culture, d’un village 95 soumis à la métropolisation genevoise / The Butor archipelago. A regeneration, through culture, of a village subject to the metropolisation of Geneva Arnaud Dutheil Pratiche e progettualità di rigenerazione e welfare: il “Premio 105 triennale Giulio Andreolli – Fare paesaggio” / Actions and projects of regeneration and welfare: the “Premio triennale Giulio Andreolli – Fare paesaggio” Giorgio Tecilla Architetture e strategie per il welfare. Il caso di Brunico in 113 Val Pusteria / Welfare architectures and strategies. The Bruneck case in Val Pusteria Eleonora Gabbarini Arhitektura oživlja / Architecture revives 121 Kristina Dešman, Maja Ivanič Si Crans-Montana meurt. Soigner le corps malade d’une station / 131 If Crans-Montana dies. Taking care of the ailing body of a tourist resort Patrick Giromini Infra-strutture comunitarie. L’essere e il farsi dei luoghi / 139 Community infra-structures. The being and the making of places Giovanni Teneggi Arhitektura oživlja / Architecture revives Kristina Dešman, Maja Ivanič Ludmila Seifert Born in Soglio in 1954, he is an FAS architect. In 2000, he founded the studio Ruinelli Associati Architetti SIA with Fernando Giovanoli. Art historian, she has been Head of business of the association Bündner Heimatschutz (Homeland Protection of the Canton of Grisons) since 2011. In 2018, she received a recognition award from the canton of Grisons government for her work on the architectural history of the canton and her commitment to building culture issues as managing director of the association Bündner Heimatschutz. Il rinnovamento dei villaggi: tra conservazione e progettazione The renovation of the villages: between conservation and design They are high-profile supporters of an architecture developed from existing struc­ tures, and both of them think of their work as a social task to a considerable degree: they are Armando Ruinelli (* 1954), who has been running an architectural office in Soglio (Bregaglia) since 1982, and Gion A. Caminada (* 1957), who has been work­ ing as an architect, town planner and construction consultant in Vrin (Lumnezia) since the beginning of the 1990s. After their “years of apprenticeship” in Zurich, they both returned to their home villages, where not only did they carry out projects, but they also got involved in community issues. Initially, they both worked exclusively in their home valleys, but today they are known and sought after beyond their coun­ try’s borders. The interest in the future of the peripheral Alpine valleys has remained a central aspect of their work. A conversation about village regeneration, townscape protection and political com­ mitment, recorded by Ludmila Seifert, Managing Director of the association Bünd­ ner Heimatschutz (Homeland Protection of the canton of Grisons). Gion A. Caminada Born in Vrin (Switzerland) in 1957. He trained as a carpenter/joiner and attended the Kunstgewerbeschule (Arts and Crafts School). He graduated at the Swiss Federal Institute of Technology (ETH) in Zurich, and he then founded his own architectural office in Vrin. He has been professor of Architectural Design at the ETH Zurich since 1998. Dorferneuerung zwischen Erhalten und Gestalten Il rinnovamento dei villaggi: tra conservazione e progettazione The renovation of the villages: between conservation and design Il rinnovamento dei villaggi: tra conservazione e progettazione The renovation of the villages: between conservation and design Vorherige Seite / In apertura Vrin, Graubünden / Vrin, Grigioni (foto Gion A. Caminada). Keywords Caminada, Ruinelli, preservation, design, project, Vrin, Soglio, Switzerland Doi: 10.30682/aa2004e 40 Ludmila Seifert: Qualche anno fa, molte voci di spicco hanno chiesto sempre più insistentemente la progressiva riforestazione delle aree montane che prevedesse anche l’abbandono dei villaggi tradizio­ nali abitati dai contadini di montagna. Oggi si parla di rinnovare questi villaggi, dando quindi natural­ mente per scontato che essi abbiano ancora il dirit­ to di esistere. Perché dovrebbero essere conservati? Armando Ruinelli: Io non credo vi sia l’obbligo di conservare i villaggi. Solo perché qualcosa esiste o è bello non significa che debba essere conservato. Ha bisogno anche di una funzione. Dobbiamo ab­ bandonare l’idea di una tradizionale comunità di villaggio costituita da un gran numero di famiglie “del posto” con bambini. In questi villaggi oggi ci sono troppo pochi “nativi”, bisogna aprirsi a nuo­ vi scenari. Circa il 70% della popolazione svizzera vive nei centri urbani – nelle città e nelle loro im­ mediate vicinanze, nell’agglomerato urbano. Agli abitanti delle città possiamo offrire un rifugio in cui possano trovare un luogo di lavoro e di vita tem­ poraneo, in quanto “residenti part-time”, per così dire. Non solo per le vacanze, ma per settimane, o addirittura mesi. Ogni tanto nei villaggi si trasferi­ scono persone provenienti perfino da altre città. Si tratta spesso di personalità particolari e interessan­ ti, dotate di spirito critico, spesso anticonformisti, artisti, che portano con sé anche nuove prospetti­ ve, contribuendo così a modo loro al rinnovamen­ to del villaggio. Per noi “nativi” il villaggio è la no­ stra casa, qui abbiamo le nostre radici, il che rende vivere in questi luoghi più tollerabile. Ma a volte è molto difficile per chi viene da fuori. Dobbiamo aiutare queste persone a integrarsi, il che richiede, tuttavia, una comprensione reciproca. Nelle valli montane c’è spesso una certa riluttanza ad aprir­ si agli estranei, nella convinzione di sapere già cosa sia meglio per se stessi, ma questo è vero solo in parte. Le influenze esterne ci arricchiscono, favo­ riscono la rigenerazione. Possiamo sapere davve­ ro che cosa vogliamo solo se ci confrontiamo an­ che con “l’altro”. Gion A Caminada: Le ragioni per la conserva Ludmila Seifert: Vor Jahren wurden prominen­ te Stimmen laut, die eine weitgehende Vergan­ dung der Berggebiete fordern – der Aufgabe der alten Bergbauerndörfer inklusive. Heute sprechen wir über Dorferneuerung, gehen also wie selbst­ verständlich davon aus, dass unsere Dörfer eine Existenzberechtigung haben. Keywords Warum soll man sie erhalten? Armando Ruinelli: Ich sehe keinen grundsätzli­ chen Zwang zum Erhalt. Nur weil etwas existiert oder gar schön ist, heisst das noch nicht, dass es auch erhalten werden muss. Es braucht eine Funk­ tion. Wir müssen uns von der Vorstellung einer tra­ ditionellen Dorfgemeinschaft mit einer Vielzahl von «einheimischen» Familien mit Kindern verab­ schieden. Es gibt zu wenig ortsansässige «Einheimi­ sche». Man muss sich anderen Ideen gegenüber öff­ nen. Rund 70% der Schweizer Bevölkerung lebt in den Zentren – in Städten und darum herum, in der Agglomeration. Wir können den Städtern ein Refu­ gium bieten, wo sie, als «Part-time-Einwohner» so­ zusagen, temporären Wohn- und Arbeitssitz neh­ men können. Nicht nur ferienhalber, sondern für Wochen oder gar Monate. Ab und zu ziehen Leu­ te auch tatsächlich von auswärts zu. Das sind oft besondere, interessante Persönlichkeiten mit einem kritischen Geist, die auch neue Sichtweisen einbrin­ gen, also auf ihre Weise zu einer Dorferneuerung beitragen. Aussteiger, Künstler. Für uns «Eingebo­ rene» ist das Dorf Heimat, wir haben hier unsere Wurzeln, die uns das Leben im Dorf gut aushalten lassen. Aber für Auswärtige ist das zuweilen sehr schwierig. Wir müssen ihnen helfen, sich zu inte­ grieren, was allerdings ein gegenseitiges Verständ­ nis bedingt. In den Bergtälern herrscht oft ein Wi­ derwille, sich dem Fremden gegenüber zu öffnen, in der Meinung, man wisse schon selber, was gut für einen ist. Aber das ist nur bedingt richtig. Einflüs­ se von aussen sind bereichernd, sie helfen der Re­ generation. Man kann ja nur wissen, was man sel­ ber will, wenn man sich auch mit dem «Anderen» auseinandersetzt. Gi A C i d Di G ü d fü di E h l Gion A. Caminada: Le ragioni per la conserva­ zione dei villaggi devono essere continuamente ri­ definite. Trent’anni fa, a Vrin, abbiamo cercato di attuare il modello di 500 persone di Peter Rieder, Gion A. Caminada: Die Gründe für die Erhal­ tung der Dörfer gilt es immer wieder neu zu de­ finieren. Vor dreissig Jahren haben wir in Vrin versucht, das 500-Seelen-Modell von Peter Rie­ 41 secondo cui il funzionamento di un villaggio dipen­ de da un numero minimo di abitanti. L’obiettivo di questo modello non è stato raggiunto, e il suo falli­ mento mi ha portato a sviluppare una visione com­ pletamente diversa della questione. Considero un privilegio poter vivere in un posto dove quasi nes­ suno vuole abitare. Abb. 1 / Fig. 1 Stallumnutzung in Soglio, Graubünden / Riqualificazione stalla a Soglio, Grigioni (foto © Armando Ruinelli). Abb. 2 / Fig. 2 Aussicht von Soglio, Graubünden / Veduta di Soglio, Grigioni (foto Ralph Feiner). Keywords Invece che di quantità, dovremmo iniziare a parlare di qualità, in relazione a un’inten­ zione specifica. Credo che, indipendentemente dal­ le dimensioni, in un villaggio ci sia bisogno di idee su misura per le sue esigenze. Idee in cui le persone si identifichino, a cui prendano parte, che possano celebrare. Questo genera qualità della vita, e perciò è necessario esplorare le possibilità offerte da una piccola comunità. der umzusetzen, welches das Funktionieren ei­ nes Dorfes von einer bestimmten Mindest-Bevöl­ kerungsanzahl abhängig macht. Das Ziel wurde nicht erreicht. Dieses Scheitern hat bei mir zu ei­ ner radikal anderen Sicht geführt. Ich empfinde es als Privileg, an einem Ort zu wohnen, in dem fast niemand wohnen will. Statt von Quantitäten sollten wir anfangen, von Qualitäten zu sprechen. Qualität in Bezug auf eine bestimmte Intention. Ich den­ ke, es braucht, unabhängig von der Grösse, mass­ geschneiderte Ideen für ein Dorf. Ideen, mit denen sich die Leute identifizieren, an denen sie teilha­ ben, die sie zelebrieren können. Das generiert Le­ bensqualität. Man muss die Möglichkeiten einer kleinen Gemeinschaft ausloten. LS: Ma il modello di Rieder era anche un model­ lo economico, basato sulla questione fondamentale LS: Aber das Rieder’sche Modell war ja auch ein ökonomisches Modell, ausgehend von der Frage, 1 1 42 2 2 unter welchen Bedingungen ein Dorf wirtschaftlich überleben kann. delle condizioni in cui un villaggio può essere eco­ nomicamente sostenibile. unter welchen Bedingungen ein Dorf wirtschaftlich überleben kann. GC: Ja, natürlich, aber da haben die Gemeinde­ fusionen eine Erleichterung gebracht. Ich war ur­ sprünglich gegen den Zusammenschluss. Ich hatte den Verdacht einer aufkommenden Verantwor­ tungslosigkeit gegenüber dem, was wir in den letz­ ten Jahrzehnten erarbeitet hatten. Jetzt, da die Fu­ sion erfolgt ist, sehe ich durchaus Vorteile. Die GC: Sì, certo, ma a tal proposito le fusioni tra co­ muni hanno portato un po’ di sollievo. Inizialmen­ te ero contrario alla fusione, mi preoccupava la cre­ scente mancanza di responsabilità verso quello che abbiamo progettato nel corso degli ultimi decenni. Ora che la fusione è avvenuta, ne vedo anche i van­ taggi. Nelle piccole comunità di un tempo le diffi­ GC: Ja, natürlich, aber da haben die Gemeinde­ fusionen eine Erleichterung gebracht. Ich war ur­ sprünglich gegen den Zusammenschluss. Ich hatte den Verdacht einer aufkommenden Verantwor­ tungslosigkeit gegenüber dem, was wir in den letz­ ten Jahrzehnten erarbeitet hatten. Jetzt, da die Fu­ sion erfolgt ist, sehe ich durchaus Vorteile. Keywords Die Ora che la fusione è avvenuta, ne vedo anche i van­ taggi. Nelle piccole comunità di un tempo le diffi­ 43 ehemaligen Kleingemeinden wurden finanziell entlastet. Vor der Fusion wollten viele Vriner ihr Wasser unbedingt ins Kraftwerk nach Vals über­ leiten, um von den Wasserzinsen zu profitieren. Heute ist das kein Thema mehr. Ohne das ökono­ mische Damoklesschwert im Nacken ist man freier und kann andere Möglichkeiten, die für das gute Lebensgefühl des Dorfes wichtig sind, in Betracht ziehen. coltà economiche si sono alleviate. Prima della fu­ sione, molti cittadini a Vrin volevano fare deviare la propria acqua verso la centrale idroelettrica di Vals per poter beneficiare delle imposte sul consu­ mo idrico. Oggi non succede più. Senza la spada di Damocle delle finanze sul capo, si è più liberi e si possono prendere in considerazione altre opzioni, che sono importanti per raggiungere un buono stile di vita nel villaggio. AR: Anch’io sono stato a lungo contrario alla fusio­ ne, ma in effetti le piccole comunità sono state sol­ levate dall’onere di dover funzionare in modo indi­ pendente. Non è solo una questione economica, ma anche politica, come la nomina del consiglio comu­ nale o del comitato direttivo locale. Alla fine, è stato un processo molto complicato. AR: Ich war auch lange gegen die Fusion. Aber den kleinen Gemeinden hat man die Last genommen, eigenständig funktionieren zu müssen. Es geht nicht nur um die Finanzen, sondern auch um die Poli­ tik, um die Besetzung des Gemeinderats oder des Gemeindepräsidiums zum Beispiel. Das war am Schluss ganz schwierig. GC: Da quando c’è stata la fusione dei comuni, le persone prestano anche maggiore attenzione alle differenze tra i singoli villaggi. È da qui che dob­ biamo iniziare: bisogna identificare le differen­ ze e rafforzarle. Ad esempio, ciò che è possibile a Vrin non lo è a Morissen – e viceversa. Se si sfruttano le risorse e le qualità specifiche del sin­ golo villaggio si ottengono effetti positivi sull’inte­ ra comunità. GC: Seit der Fusion achtet man auch ein bisschen mehr auf die Unterschiede zwischen den einzelnen Dörfern. Da gilt es anzusetzen: die Differenzen he­ rauszuschälen und diese zu stärken. Was ist zum Beispiel in Vrin möglich, was in Morissen nicht möglich ist – und umgekehrt. Wenn man auf den spezifischen Ressourcen und Qualitäten des jewei­ ligen Dorfes aufbaut, wirkt sich das positiv auf die ganze Gemeinde aus. Keywords AR: Al contempo, è assolutamente necessario im­ pedire lo sviluppo di monoculture. Prendiamo ad esempio le gallerie d’arte o le cliniche, che “occu­ pano” diversi villaggi. Per quanto questi approc­ ci possano essere interessanti, esiste sempre il ri­ schio della monopolizzazione. In quanto residente locale, se non si fa parte della sfera di influenza di questi business si è fuori dai giochi. A Soglio, fi­ nora, solo le case più semplici e le stalle sono sta­ te vendute a terzi, le “briciole”, per così dire. Ora c’è in palio “il piatto forte”, i maestosi palazzi del­ la famiglia von Salis. Uno è appena stato vendu­ to e in questo momento è in fase di estesa ricon­ versione – per fortuna, sembra che stia accadendo qualcosa che potrebbe essere interessante per l’in­ tero villaggio. Ma proviamo ad immaginare l’ar­ rivo di una galleria d’arte esterna o, per quanto mi riguarda, anche di una clinica, che investe mi­ lioni negli altri palazzi. Allora conterà solo il “tu­ rismo” dell’arte o della salute, e il resto non avrà più alcun valore. AR: Dabei sollte man unbedingt verhindern, dass Monokulturen entstehen. Nehmen wir das Beispiel der Kunstgalerien oder Gesundheitszentren, die da und dort ein Dorf quasi «besetzen». So interessant diese Ansätze auch sein mögen, es besteht da im­ mer die Gefahr der Monopolisierung. Als Einheimi­ scher ist man dann entweder im Dunstkreis dieses Betriebs – oder man ist weg vom Fenster. In Soglio hat man bisher nur die etwas einfacheren Häuser und die Ställe an Auswärtige verkauft, sozusagen die «Plastikbecher». Jetzt aber steht das «Tafelsil­ ber» zur Disposition, die herrschaftlichen Palazzi der Familie von Salis. Gerade eben wurde eines ver­ äussert und das wird jetzt aufwändig umgebaut – glücklicherweise scheint da etwas zu passieren, das auch für das Dorf interessant werden könnte. Aber stellen wir uns vor, da kommt eine auswärtige Ga­ lerie oder von mir aus, eine Klinik und buttert in die restlichen Palazzi Millionen rein. Dann zählt da nur noch die Kunst oder der Gesundheits «touris­ mus» – und der Rest ist nichts mehr wert. LS: Beim Erneuern stellt sich ja immer auch die Frage nach dem Umgang mit dem histo­ rischen Bestand. Wie ist Euer Verhältnis zum Ortsbildschutz? LS: Quando si rinnova, c’è sempre la questione di come gestire il patrimonio edilizio esistente. Qual è il vostro rapporto con la tutela del paesaggio abita­ to locale? Keywords In que sto modo si evita la discussione critica. LS: Beh, però è comprensibile. Non tutti son davvero portati a lavorare su edifici storici e, del re sto pochissimi interventi sono pensati minuziosa mente e realizzati con criterio. Perciò è più facile im porre norme vincolanti applicabili a tutti i casi. D punto di vista estetico e della cultura edilizia, la ca renza di risorse economiche del passato aveva i suo vantaggi La carenza di materiali e forme dovut AR: Ich denke auch, dass das Streben nach Veränderung im Sinne von Verbesserung der menschlichen Psyche eigen ist – dass sich das (vermeintlich) Bessere dann häufig nicht unbe­ dingt als solches erweist, ist eine andere Sache. Aber den Erneuerungswillen braucht es unbe­ dingt. Das museale Bewahren ist kein zukunfts­ trächtiger Ansatz. Sonst haben wir eben die zwei Situationen, die Städte, die sich erneuern, gesell­ schaftlich, politisch und auch architektonisch, und die Dörfer, die im status quo verharren bis zur Leblosigkeit. q ,i p GC: Ma questo atteggiamento non preclude affat­ to la conservazione. Entrambi cerchiamo costante­ mente di esplorare il potenziale di ciò che già c’è, semplicemente non bisognerebbe restringere troppo il concetto di conservazione. Oggi celebriamo la storia perché la troviamo bella, interessante, ma significa davvero qualcosa per noi? Ad esempio, i dipinti murali religiosi che erano stati coperti in un determinato periodo storico per volere di una certa posizione religiosa, oggi vengono riportati alla luce, senza che esista però nessun legame reale tra noi ed essi – a scopo puramente decorativo, per così dire. Non ha molto senso. Quando lavoro su un’a­ bitazione storica di valore mi rendo costantemen­ te conto di quanto poco coraggio mostrino le istitu­ zioni quando si tratta di approvare, o anche solo di prendere in considerazione, interventi significativi. In questi casi, con la scusa della conservazione dei monumenti, viene semplicemente proibita qualsiasi cosa. Così è troppo semplice, secondo me. In que­ sto modo si evita la discussione critica. GC: Aber diese Haltung schliesst ja das Erhalten überhaupt nicht aus. Uns beiden geht es ja im­ mer auch um das Ausloten des Potenzials dessen, was da ist. Man darf den Schutzgedanken ein­ fach nicht zu eng fassen. Heute zelebriert man die Geschichte, weil man sie schön und interessanter findet. Aber bedeutet sie uns wirklich etwas? Keywords GC: Der Gedanke des «Erhalts» ist für das Berg­ dorf relativ neu. Der Bergbauer hat immer die Überwindung des Hier und Jetzt gesucht, er war immer bestrebt, seine – grundsätzlich als ungenü­ gend empfundene – Situation zu verbessern. Die­ se Mentalität hat sich nicht geändert. Der Vriner will nach wie vor nicht nur erhalten. Das wollen GC: L’idea di “conservare” è relativamente nuova per il villaggio montano. Il contadino di montagna ha sempre cercato di affrontare il qui e ora, provan­ do continuamente a migliorare la propria condizio­ ne, che è sempre stata percepita come fondamen­ talmente insoddisfacente. Questa mentalità non è cambiata. L’abitante di Vrin non vuole solo conser­ 44 vare. Solo chi prova un certo distacco dalle cose vuo­ le farlo, chi percepisce una perdita, ma “dall’ester­ no”, per così dire, e basandosi su questo sentimento, prende le dovute precauzioni. Il desiderio di miglio­ rare ciò che si ha, con la prospettiva di raggiungere una sorta di paradiso immaginario, uno spazio ver­ so cui tendere, mi sembra uno stato d’animo interes­ sante e stimolante nel contesto di queste nuove re­ altà. La domanda che mi sorge spontanea è: come possiamo conciliare la “protezione” in senso stretto, in un’ottica di conservazione del patrimonio archi­ tettonico, e lo “sviluppo continuo”, spontaneo e a ogni costo, con qualcosa di nuovo, che rispetti l’esi­ stente e allo stesso tempo lo sviluppi costantemente? AR: Penso anch’io che la ricerca del cambiamento, inteso come miglioramento, appartenga natural­ mente alla mente umana. Che ciò che appare pre­ sumibilmente migliore finisca poi, in molti casi, per non esserlo, è un’altra questione, ma la volontà di rinnovamento è assolutamente necessaria. La con­ servazione in stile museale non è un approccio pro­ mettente per il futuro. Si creerebbero altrimenti due situazioni distinte: da una parte le città si rinnove­ rebbero dal punto di vista sociale, politico e archi­ tettonico, mentre dall’altra i villaggi resterebbero bloccati nello status quo, fino a scomparire. f einzig jene, die in einer gewissen Distanz zu den Dingen stehen, die quasi «von aussen» einen Ver­ lust realisieren und aus dieser Empfindung her­ aus Gegensteuer geben. Der Wunsch nach Über­ windung dessen, was man hat im Hinblick auf ein imaginiertes Paradies, einen Sehnsuchtsraum finde ich als Geisteshaltung spannend und un­ ter den neuen Wirklichkeiten herausfordernd. Keywords Für mich stellt sich die Frage: Wie können wir das «Schützen» im strengen, denkmalpflegeri­ schen Sinn und das unreflektierte «Weiterentwi­ ckeln» um jeden Preis vereinen zu etwas Neuem, das den Bestand respektiert und kontinuierlich weiterbaut. vare. Solo chi prova un certo distacco dalle cose vuo le farlo, chi percepisce una perdita, ma “dall’este no”, per così dire, e basandosi su questo sentiment prende le dovute precauzioni. Il desiderio di miglio rare ciò che si ha, con la prospettiva di raggiunger una sorta di paradiso immaginario, uno spazio ve so cui tendere, mi sembra uno stato d’animo intere sante e stimolante nel contesto di queste nuove re altà. La domanda che mi sorge spontanea è: com possiamo conciliare la “protezione” in senso strett in un’ottica di conservazione del patrimonio arch tettonico, e lo “sviluppo continuo”, spontaneo e ogni costo, con qualcosa di nuovo, che rispetti l’es stente e allo stesso tempo lo sviluppi costantemente AR: Penso anch’io che la ricerca del cambiament inteso come miglioramento, appartenga natura mente alla mente umana. Che ciò che appare pre sumibilmente migliore finisca poi, in molti casi, pe non esserlo, è un’altra questione, ma la volontà d rinnovamento è assolutamente necessaria. La con servazione in stile museale non è un approccio pro mettente per il futuro. Si creerebbero altrimenti du situazioni distinte: da una parte le città si rinnove rebbero dal punto di vista sociale, politico e arch tettonico, mentre dall’altra i villaggi resterebber bloccati nello status quo, fino a scomparire. GC: Ma questo atteggiamento non preclude affa to la conservazione. Entrambi cerchiamo costante mente di esplorare il potenziale di ciò che già c’ semplicemente non bisognerebbe restringere tropp il concetto di conservazione. Oggi celebriamo storia perché la troviamo bella, interessante, m significa davvero qualcosa per noi? Ad esempio, dipinti murali religiosi che erano stati coperti in u determinato periodo storico per volere di una cert posizione religiosa, oggi vengono riportati alla luc senza che esista però nessun legame reale tra no ed essi – a scopo puramente decorativo, per co dire. Non ha molto senso. Quando lavoro su un’a bitazione storica di valore mi rendo costantemen te conto di quanto poco coraggio mostrino le istitu zioni quando si tratta di approvare, o anche solo d prendere in considerazione, interventi significativ In questi casi, con la scusa della conservazione d monumenti, viene semplicemente proibita qualsia cosa. Così è troppo semplice, secondo me. 3 3 Keywords So bringt man zum Beispiel religiöse Wandmalerei­ en, die zu einem bestimmten Zeitpunkt aus einer bestimmten religiösen Haltung heraus zugedeckt worden sind, wieder zum Vorschein, ohne dass man dazu einen ernsthaften Bezug hätte – sozusa­ gen als Dekoration. Das macht wenig Sinn. Wenn ich an einem bedeutenden historischen Haus ar­ beite, erlebe ich immer wieder, wie mutlos sich die Behörden geben, wenn es darum geht, prägnan­ te Eingriffe zuzulassen – ja überhaupt zu beurtei­ len. Da wird mit dem Hinweis auf den Denkmal­ schutz schnell mal einfach alles verboten. Das ist mir zu simpel. So entzieht man sich der kritischen Auseinandersetzung. LS: Beh, però è comprensibile. Non tutti sono davvero portati a lavorare su edifici storici e, del re­ sto pochissimi interventi sono pensati minuziosa­ mente e realizzati con criterio. Perciò è più facile im­ porre norme vincolanti applicabili a tutti i casi. Dal punto di vista estetico e della cultura edilizia, la ca­ renza di risorse economiche del passato aveva i suoi vantaggi. La carenza di materiali e forme, dovuta alle condizioni economiche e sociali, e la stringen­ te necessità di lavorare con la topografia e non con­ LS: Das ist aber ja verständlich. Nicht jeder ist wirklich begabt im Weiterbauen am Bestand. Die wenigsten Eingriffe sind sorgfältig durchdacht und sensibel gemacht. Da ist es einfacher, man stellt ver­ bindliche Regeln auf, die dann halt für alle gel­ ten. Vom ästhetischen, baukulturellen Standpunkt aus hatte die Mangelwirtschaft der Vergangenheit ja ihre Vorteile. Die ökonomisch und auch gesell­ schaftlich bedingte Reduktion von Material und Form, die unbedingte Notwendigkeit, mit der To­ 45 bb 3 / g 3 Atelierhaus in Soglio, Graubünden, Ruinelli Associati AG Architetti SIA (Schweizerischer Ingenieur- und Architektenverein) / Casa Atelier a Soglio, Grigioni, Ruinelli Associati Architetti (foto © Armando Ruinelli). Abb. 4 / Fig. 4 Totenstube (Stiva da morts), Vrin, Graubünden, Gion A. Caminada / Stiva da morts, Vrin, Grigioni, Gion A. Caminada (foto Lucia Degonda). Abb. 3 / Fig. 3 Abb. 3 / Fig. 3 Atelierhaus in Soglio, Graubünden, Ruinelli Associati AG Architetti SIA (Schweizerischer Ingenieur- und Architektenverein) / Casa Atelier a Soglio, Grigioni, Ruinelli Associati Architetti (foto © Armando Ruinelli). pografie zu arbeiten und nicht gegen sie – das al­ les hat Ortsbilder hervorgebracht, die wir heute als charismatisch, als kraftvoll, als stark empfin­ den. Heute hingegen ist überall alles möglich. Die­ ses Unspezifische hat dem Gesicht unserer Dör­ fer nicht unbedingt gutgetan. Da geht es um die grundsätzliche Frage: Wie bringt man Ortsbild­ schutz und Ortserneuerung zusammen, wie findet man einen Ausgleich zwischen Bewahren und Ge­ tro di essa, hanno permesso la creazione dei paesag­ gi abitati locali che oggi consideriamo carismatici, dotati di forza e di grande potenziale. Oggi, al con­ trario, tutto è possibile ovunque e questa mancan­ za di specificità non ha sempre fatto bene all’esteti­ ca dei nostri villaggi. L’interrogativo fondamentale è: come possiamo coniugare la tutela dell’immagine del paesaggio abitato con il rinnovamento del luogo, trovare un equilibrio tra conservazione e progetta­ 46 4 4 stalten? Schutzbestimmungen sind ja nur der Ver­ such, die potentiell zerstörerischen Auswirkungen der Eingriffe einzudämmen. zione? Dopotutto, le norme sulla conservazione non sono altro che un tentativo di contenere gli effetti potenzialmente distruttivi degli interventi. GC: Ja, eine Mindestgesetzgebung braucht es natürlich… GC: Ja, eine Mindestgesetzgebung braucht es natürlich… GC: Ja, eine Mindestgesetzgebung braucht es natürlich… GC: Sì, ovviamente una legislazione minima è necessaria... AR: … aber Gesetze können eben auch Gutes ver­ hindern. Erneuerung hat auch mit zeitgenössischer Architektur zu tun. Ich meine, dass man viel mehr differenzieren sollte. Es gibt Sachen, die man un­ bedingt erhalten muss, aber es gibt auch in histo­ rischen Dörfern Situationen, die man verändern können sollte. Wichtig ist natürlich, dass das, was AR: …ma le leggi possono anche ostacolare cose buone. Il rinnovamento dovrebbe avere a che fare anche con l’architettura contemporanea. Penso che si dovrebbe operare una distinzione molto più net­ ta: ci sono cose che è assolutamente necessario pre­ servare, ma anche situazioni, nei centri storici, che dovrebbero poter essere modificate. Sarebbe anche AR: … aber Gesetze können eben auch Gutes ver­ hindern. Erneuerung hat auch mit zeitgenössischer Architektur zu tun. Ich meine, dass man viel mehr differenzieren sollte. Es gibt Sachen, die man un­ bedingt erhalten muss, aber es gibt auch in histo­ rischen Dörfern Situationen, die man verändern können sollte. Abb. 3 / Fig. 3 Wichtig ist natürlich, dass das, was 47 an die Stelle des Alten tritt, mindestens ebenso gut, womöglich besser ist. Und dafür, da gebe ich Dir Recht, besteht keine Garantie. In der Regel wird es schlechter. Das Problem ist, dass das Bauen zur Investition geworden ist. Früher war es eine Not­ wendigkeit. Sobald Geld ins Spiel kommt, rutscht Dir das aus der Hand. Das kann sich katastrophal auswirken. importante, ovviamente, che ciò che prende il posto del vecchio fosse almeno di pari valore e, se possibile, migliore di quest’ultimo. E in questo sono d’accordo con te, non ci sono garanzie, di solito il nuovo risul­ ta peggiore. Il problema è che costruire è diventato un investimento, mentre una volta era una necessi­ tà. Quando entra in gioco il denaro, sfugge tutto di mano e le conseguenze possono essere catastrofiche. LS E i i i l li à? LS: Gibt es Mittel, Qualität zu gewährleisten? AR: Mi viene in mente principalmente lo strumen­ to della consulenza progettuale da parte di un comi­ tato consultivo di progettazione composto da esper­ ti esterni, che possano esprimere valutazioni obiettive ed esigere qualità nella progettazione, senza l’influen­ za di condizionamenti personali. A lungo termine, questo può portare a buoni risultati, anche se, ovvia­ mente, si può intervenire solo in modo puntuale. AR: Ich denke da vor allem an das Instrument der Gestaltungsberatung durch einen Gestaltungsbei­ rat aus auswärtigen Fachleuten, die unabhängig von persönlichen Verflechtungen etwas sachlich be­ urteilen und Qualität einfordern können. Langfris­ tig bewährt sich das, auch wenn natürlich auch nur punktuell interveniert werden kann. p GC: Auch mit Quartierplänen kann man viel errei­ chen. Aber die Sache ist hochkomplex und Regulie­ rungen haben ihre Grenzen. Die Hoffnung ist, dass man gut gebildete Leute mit einer klaren Geistes­ haltung hat, die bereit sind, sich einzumischen. In Vrin, da haben wir damals das Baugesetz neu ge­ schrieben. Dieses konnte nach der Gemeindefusion nicht in das übergeordnete kommunale Baugesetz integriert werden. Nun werden uns die Baugesuche zur Beurteilung vorgelegt und so können wir das Baugeschehen direkt beeinflussen. Aber so was pas­ siert nicht von allein, das braucht konstantes En­ gagement – über Jahre hinweg. Man muss mit den Einwohnern, den Behörden und den politischen Amtsträgern in einen ernst gemeinten Dialog tre­ ten und mit ihnen argumentieren. Dann kann man etwas bewirken. In Graubünden haben wir eini­ ge ausserordentlich gute Architekten. Abb. 3 / Fig. 3 Das Problem ist nur, dass sich die meisten von ihnen nicht ex­ ponieren und politisch engagieren. Sie sind mehr oder weniger auf ihre Objekte fixiert, wollen «schö­ ne» Häuser bauen. Das ist schon einmal sehr gut. Auch die Mehrzahl der abgehenden Architekten der ETH, wo ich lehre, sind fähig, schöne Dinge zu entwerfen. Das ist aber zu wenig und führt noch zu keinem guten Ort. Ich denke, wir müssen den Be­ griff «Schönheit» um weitere Komponente ergän­ zen. Schönheit liegt tief unter der Oberfläche. Aber zurück zu Deiner Frage: Ich denke, mit ehrlich ge­ meintem, persönlichem Engagement lässt sich et­ was bewirken. GC: Anche con i piani di vicinato si può ottenere molto, ma la questione è molto complessa e le nor­ mative hanno i loro limiti. La speranza è che ci si­ ano persone ben istruite, con una mentalità aperta e pronte a mettersi in gioco. A Vrin, all’epoca, ab­ biamo scritto la nuova normativa edilizia. Tuttavia, dopo la fusione delle comunità, non è stato possibile integrarla alla legge sull’edilizia comunale di livel­ lo superiore. Oggi ci vengono sottoposte richieste di costruzione per una valutazione, quindi possia­ mo influenzare direttamente il processo di proget­ tazione, ma tutto questo non succede per caso, ri­ chiede uno sforzo costante nel corso degli anni. Bisogna avviare un dialogo serio con i residenti, le autorità e i funzionari politici, solo questo può fare la differenza. Ci sono alcuni architetti davvero bra­ vi nei Grigioni. Il problema è che molti di loro non si espongono e non sono politicamente impegnati. Sono tutti più o meno concentrati sui loro proget­ ti, vogliono costruire case “belle” e questo è già un punto di partenza molto positivo. La maggior par­ te degli architetti che escono dall’ETH di Zurigo, dove insegno, è in grado di progettare cose belle, ma questo non è ancora abbastanza e non porta da nessuna parte. Penso sia necessario arricchire la de­ finizione di “bellezza” di altri attributi, la vera bel­ lezza si trova molto sotto la superficie. Tornando alla tua domanda: penso che con un coinvolgimen­ to personale profondamente sincero si possano ot­ tenere risultati. AR: Was Du ansprichst ist das Monte-Carasso-Mo­ dell von Luigi Snozzi. Es braucht vor Ort eine Per­ sönlichkeit – eine Art «despota illuminato» – der sagt, wo’s langgeht. Da und dort sind auf diese Wei­ se interessante Geschichten entstanden – politisch, städtebaulich, architektonisch. Abb. 3 / Fig. 3 Daneben wird an den meisten Orten einfach vor sich hingebaut. Auf eine gewisse Weise sind wir auch privilegiert, dass man uns überhaupt einbezieht und gewisse Prozes­ se mitgestalten lässt. Das hängt immer zusammen AR: Ti riferisci al modello Monte Carasso di Luigi Snozzi. C’è bisogno di una personalità in loco, di una sorta di “despota illuminato”, che sappia in che dire­ zione andare. In questo modo qua e là sono successe cose interessanti – dal punto di vista politico, in termi­ ni di pianificazione urbana e a livello architettonico. Allo stesso tempo, nella maggior parte dei villaggi si continua semplicemente a costruire. In un certo sen­ so, abbiamo il privilegio di essere perlomeno coinvolti e di poter prendere parte ad alcuni processi. In que­ AR: Ti riferisci al modello Monte Carasso di Luigi Snozzi. C’è bisogno di una personalità in loco, di una sorta di “despota illuminato”, che sappia in che dire­ zione andare. In questo modo qua e là sono successe cose interessanti – dal punto di vista politico, in termi­ ni di pianificazione urbana e a livello architettonico. Allo stesso tempo, nella maggior parte dei villaggi si continua semplicemente a costruire. In un certo sen­ so, abbiamo il privilegio di essere perlomeno coinvolti e di poter prendere parte ad alcuni processi. In que­ 48 mit ein paar wenigen Leuten, die sagen: Wir pro­ bieren das mal ein bisschen anders. sti casi, si tratta sempre di poche persone che dico­ no: “proviamo a fare le cose in modo un po’ diverso”. LS: Ci sono alcuni luoghi che hanno la fortuna di avere persone simili, mentre altri vanno semplice­ mente avanti senza un piano preciso. LS: Dann hat man ein paar Orte, die das Glück ha­ ben, solche Leute zu haben und die andern wurs­ teln beliebig vor sich hin. LS: Dann hat man ein paar Orte, die das Glück ha­ ben, solche Leute zu haben und die andern wurs­ teln beliebig vor sich hin. GC: Ja, aber wir können auch ausserhalb unseres ei­ genen Dorfes wirken, wenn wir in fremde Situatio­ nen eintauchen und versuchen, diese zu verstehen. Voraussetzung ist natürlich, dass man ein Gegen­ über hat, das initiativ und offen ist. In Valendas zum Beispiel, da gab es eine Gruppierung, die das vor sich hin dümpelnde Dorf unbedingt weiterbringen wollte – und da haben neben mir viele Auswärtige mitgemacht, um das in gute Bahnen zu lenken. Abb. 3 / Fig. 3 Die Initiative von innen mit der Intervention von aus­ sen, das ist eine vielversprechende Mischung. In Di­ sentis, wo ich für das Benediktiner-Kloster gearbei­ tet habe, konnte ich auch Ideen hineinbringen, die über den Einzelbau hinausgehen. Im letzten Semes­ ter habe ich zusammen mit meinen Studenten eini­ ge Überlegungen zur anstehenden Ortsplanung der fusionierten Gemeinde Ilanz entwickelt – da hat­ ten wir als Gegenüber eine sensitive Gemeindeprä­ sidentin, die selber ausgebildete Architektin ist. Es ging darum, für die einzelnen Fraktionen der fusio­ nierten Grossgemeinde unterschiedliche Ideen her­ auszuarbeiten. Ideen entstehen über die Wahrneh­ mung, das ist nichts Abstraktes, das man losgelöst vom Kontext einfach hineinschmeissen kann. Man muss erkennen, was vorhanden ist und erspüren, was sich daraus entwickeln könnte. Sich einbringen zu wollen, verlangt von einem selber eine ehrliche Grundhaltung. Ich muss am Anfang mit Bestimmt­ heit sagen können: Ich habe die Leute gerne, ich respektiere die Differenzen, die unterschiedlichen Sichtweisen. Damit muss man zurechtkommen. Man kann nicht von aussen kommen und den Men­ schen vor Ort wie ein Missionar die eigenen Ideen überstülpen. Dann mögen Dich die Leute nicht. Und Du bist auf verlorenem Posten. GC: Sì, però possiamo fare qualcosa anche al di fuori dei nostri villaggi, quando ci troviamo in si­ tuazioni diverse e proviamo a comprenderle. Il pre­ requisito, naturalmente, è la presenza di un inter­ locutore proattivo e disponibile. A Valendas, ad esempio, c’era un gruppo che voleva assolutamen­ te far progredire il villaggio, ma non riusciva ad andare avanti, era bloccato. Molti esterni, tra cui io, hanno collaborato per fare andare le cose nel­ la giusta direzione. Un’iniziativa che nasce dall’in­ terno e un intervento esterno possono creare una combinazione vincente. A Disentis, dove ho lavo­ rato all’abbazia benedettina, ho potuto proporre idee che andassero oltre il singolo edificio. Lo scor­ so semestre ho sviluppato con i miei studenti alcu­ ne idee sull’imminente progettazione urbanistica della comunità di Ilanz – lì il nostro interlocutore era una sindaca attenta e con una formazione da architetto alle spalle. L’obiettivo era elaborare idee diverse per le singole frazioni della grande comu­ nità nata dalla fusione. Le idee nascono dalla per­ cezione, non sono qualcosa di astratto, che si può semplicemente mettere sul tavolo, indipendente­ mente dal contesto. È necessario prima di tutto ri­ conoscere quello che già esiste, per poi essere in grado di percepire ciò che potrebbe essere svilup­ pato. Abb. 3 / Fig. 3 Inoltre, il coinvolgimento in questi progetti richiede un atteggiamento onesto. All’inizio devo poter dire con convinzione: mi piacciono queste persone, rispetto le loro differenze e i loro punti di vista. Bisogna prima di tutto saper accettare que­ sto. Non puoi venire da fuori e imporre le tue idee alla gente del luogo, come farebbe un missionario. Non piaceresti alle persone e sarebbe una causa persa in partenza. LS: Dall’introduzione della legge sulle seconde case, all’inizio del 2016, le cose sono cambiate nei Grigioni, e immaginare prospettive di sviluppo ba­ sate su un atteggiamento idealistico o filosofico è di­ ventato più difficile. La legge proibisce ai comuni con oltre il 20% di seconde case – la maggioran­ za dei Grigioni – di costruire ulteriori abitazioni se­ condarie. Al fine di mitigare in qualche modo le conseguenze di questa legge in area montana sono state introdotte varie eccezioni, nell’interesse della tutela del paesaggio abitato locale. Ad esempio, gli edifici storici che per la legge precedente non erano destinati ad uso residenziale, ora possono essere ri­ convertiti in seconde case se considerati “caratteri­ stici del paesaggio abitato” e se la loro conservazio­ ne non può essere garantita in nessun altro modo. Gli edifici sono considerati “caratteristici del pae­ LS: Seit Anfang 2016, seit der Einführung des Zweitwohnungsgesetzes, haben wir in Graubünden eine neue Situation, die es nicht unbedingt leich­ ter macht, Entwicklungsperspektiven aus einer ide­ ellen oder philosophischen Grundhaltung heraus zu entwickeln. Das Gesetz verbietet Gemeinden mit mehr als 20% Zweitwohnungen – das sind in Graubünden die meisten – weitere Zweitwohnun­ gen zu bauen. Um die Auswirkungen im Bergge­ biet etwas abzuschwächen, wurden unter Vorschie­ bung von Ortsbildschutz-Interessen verschiedene Ausnahmen geschaffen. So dürfen historische Bau­ ten, die altrechtlich nicht als Wohnbauten dienten, in Zweitwohnungen umgenutzt werden, wenn sie «ortsbildprägend» sind und ihr Erhalt anders nicht gesichert werden kann. Als «ortsbildprägend» gel­ ten Gebäude, «die durch ihre Lage und Gestalt we­ 49 5 sentlich zur erhaltenswerten Qualität des Ortsbildes und zur Identität des Ortes beitragen.» Dazu gehö­ ren bei uns vor allem die Stallscheunen. Sie machen in Graubünden rund die Hälfte des Gebäudebe­ standes innerhalb der historischen Siedlungskerne aus. Durch den Strukturwandel in der Landwirt­ schaft sind sie ihrer ursprünglichen Funktion entle­ digt – und bieten nun bauwilligen Feriengästen eine Möglichkeit, sich ihren Traum vom eigenen Ferien­ haus trotz Zweitwohnungsbaustopp doch noch zu erfüllen. Abb. 5 / Fig. 5 Clavau in Vrin, Graubünden / Clavau a Vrin, Grigioni (foto Lucia Degonda). Abb. 6 / Fig. 6 Ställe Sut Vitg, Vrin, Graubünden, Gion A. Caminada / Stalle Sut Vitg, Vrin, Grigioni, Gion A. Caminada (foto Lucia Degonda). Abb. 3 / Fig. 3 Allerdings darf ein ortsbildprägender Bau bei der Umnutzung in seinem Schutzwert nicht be­ einträchtigt werden; «insbesondere die äussere Er­ scheinung und die bauliche Grundstruktur des Gebäudes» müssen, so will es das Gesetz, «im We­ sentlichen unverändert bleiben.» Auf einem land­ wirtschaftlichen Ökonomiebau übertragen heisst dies: Er muss seine historische Funktion nach aus­ sen abbilden, obwohl er nicht mehr zur Lagerung des Winterfutters gebraucht und auch keinem Vieh mehr als Unterstand dient, sondern als behagliche Behausung für Menschen einer Wohlstandsgesell­ schaft eingerichtet ist. saggio abitato”, se “per posizione e forma contribu­ iscono in modo significativo alla qualità del paesag­ gio abitato e all’identità del luogo da preservare”. Nei nostri territori questo riguarda soprattutto le stalle. Nei Grigioni, le stalle costituiscono circa la metà del patrimonio edilizio esistente all’interno dei centri storici. A causa del cambiamento strutturale nell’agricoltura, le stalle sono state private della loro funzione originale, e oggi offrono ai turisti che vo­ gliono costruire qualcosa l’opportunità di realizzare il sogno di una casa per le vacanze, nonostante il di­ vieto di costruzione di ulteriori abitazioni seconda­ rie. Il restauro di un edificio non può danneggiarne, tuttavia, il valore estetico per il paesaggio abitato lo­ cale. La legge specifica che: “In particolare, l’aspet­ to esteriore e la struttura di base dell’edificio devo­ no rimanere sostanzialmente inalterati”. Nel caso di un edificio ad uso agricolo, significa che l’aspetto esteriore deve rifletterne la funzione storica, anche se non è più utilizzato per conservare i foraggi in­ vernali o come riparo per il bestiame e oggi, al con­ trario, gli interni sono arredati per fungere da con­ fortevole abitazione per i benestanti. AR: Ja, das ist tatsächlich verheerend. Die meisten Gemeinden tendieren dazu, im Rahmen der Ge­ AR: Sì, è davvero un disastro. La maggior parte dei comuni tende ad includere in questa categoria il nu­ 50 6 samtschau, die für die Bezeichnung der ortsbild­ prägenden Bauten in der Grundordnung gefordert wird, möglichst viele der Dorfställe in diese Kate­ gorie aufzunehmen. Bei einem Umbau ist der Ar­ chitekt dann gezwungen, sich an den Leitfaden der Denkmalpflege zu halten, der eben darauf abzielt, dass das Gebäude auch nach dem Umbau das Bild eines Stalls vermittelt. Da gibt es kaum Spielraum. Das verhindert jede intellektuelle Auseinanderset­ zung mit der Umnutzungs-Thematik. Kulturell und architektonisch ist das nicht erstrebenswert, da so jede Form von Innovation verhindert wird. Abb. 3 / Fig. 3 Es wird eine Scheinwelt aufrechterhalten, die in Wirk­ lichkeit gar nicht mehr existiert. mero più alto possibile di stalle del proprio villag­ gio, all’interno del quadro complessivo per la de­ signazione degli edifici ritenuti caratteristici del paesaggio abitato locale. In caso di ristrutturazio­ ne, l’architetto è quindi costretto a aderire alle li­ nee guida per il restauro di edifici storici, volte ap­ punto a garantire che l’edificio mantenga l’aspetto di una stalla anche dopo la ristrutturazione. Non c’è margine di manovra, e così si impedisce qual­ siasi scambio di idee sul tema della riconversione. Dal punto di vista culturale e architettonico, que­ sto atteggiamento non è auspicabile, poiché ostaco­ la qualsiasi forma di innovazione. Preserva un mon­ do illusorio che in realtà non esiste più. GC: Auch im Lugnez ist man seitens der Gemein­ de bestrebt, möglichst viele Dorfställe als ortsbild­ prägend einzustufen. Für die Stallbesitzer ist das natürlich verlockend, weil damit scheinbar «wert­ lose» Gebäude zur Kapitalanlage mutieren. In GC: Anche il comune di Lumnezia sta cercando di classificare il maggior numero possibile di stalle come edifici caratteristici del paesaggio abitato lo­ cale. Ovviamente, è un’opportunità allettante per i proprietari, perché edifici apparentemente “privi di 51 valore” diventano un investimento. A Vrin, provo a contrastare questa situazione informando la gen­ te sulle sue conseguenze. Queste stalle sono mol­ to più che semplici scenografie sottoutilizzate per il paesaggio abitato locale, convertibili in seconde case. Devono essere riconosciute come patrimonio dei villaggi montani che le città non conoscono. Nel centro storico del villaggio, più densamente costru­ ito, le stalle sono piccoli distanziatori tra le case pri­ vate e una sorta di terzo spazio tra quello dichia­ ratamente pubblico e quello strettamente privato. Il villaggio è costituito da moltissime piccole unità, composte da abitazione, stalla e lo spazio che le di­ vide. Quando queste unità sono autonome, diven­ tano forti e sono in grado di relazionarsi tra loro. In questo senso, le stalle fanno parte anche dei conte­ sti circostanti. Possiamo considerare le stalle come stanze semplici, regolari e senza uno scopo preci­ so, che possono assumere usi e significati diversi a seconda della situazione, delle esigenze e delle cir­ costanze. Se tutte le stalle (e le abitazioni) vengono vendute a terzi in cambio di denaro facile, finiamo per perdere tutto questo. Abb. 3 / Fig. 3 Una volta che la stalla è stata classificata come “caratteristica del paesaggio abitato”, anche il residente del luogo deve rispetta­ re le rigide normative di conservazione secondo cui, ad esempio, la riconversione di una stalla in un ga­ rage o un’officina non è più consentita. Di fatto, vie­ ne impedito lo sviluppo di unità con funzioni indi­ spensabili in luoghi isolati come questi. Vrin versuche ich Gegensteuer zu geben, indem ich die Leute über die Folgen aufkläre. Diese Stäl­ le sind weit mehr als bloss unternutzte Kulissen für das Ortsbild, die in Zweitwohnungen umge­ baut werden können. Man muss sie als ein Privi­ leg der Dörfer erkennen, das die Stadt nicht kennt. Im dichten Dorfkern sind sie kleine Abstandhal­ ter zwischen den privaten Wohnhäusern und eine Art dritter Raum zwischen dem eindeutig öffentli­ chen und dem rein privaten Raum. Das Dorf setzt sich zusammen aus lauter kleinen Ensembles beste­ hend aus Haus und Stall und dem Raum dazwi­ schen. Wenn die Ensembles in sich abgeschlossen sind, dann wird’s stark, dann können sie zueinander in Beziehung treten. In diesem Sinne sind die Ställe Teil von nachbarschaftlichen Kontexten. Wir kön­ nen die Ställe als einfache, unkomplizierte Räume ohne festgeschriebenen Zweck betrachten, die je nach Situation, Bedürfnissen und Umständen un­ terschiedliche Aufgaben und Bedeutungen erhal­ ten können. Das verlieren wir, wenn wir alle Stäl­ le (und Häuser) nur des schnellen Geldes wegen an Auswärtige verkaufen. Ist der Stall einmal «orts­ bildprägend» eingestuft, muss auch der Einheimi­ sche sich an die rigiden Umbauvorschriften halten. Dann liegt auch ein Umbau in eine Garage oder eine Werkstatt nicht mehr drin. Eine Entwicklung in Richtung von Ensembles mit Entitäten, die für den abgelegenen Ort unabdingbar sind, ist nicht mehr möglich. AR: Auch der Ersatz des Stalles durch ein Wohn­ haus nicht. Letztlich verhindert das eine differen­ zierte Auseinandersetzung mit der Entwicklung un­ serer Dörfer. In unseren eng strukturierten, wenig bevölkerten Dörfern beschäftigt mich das Thema des Entdichtens. Vielleicht kann man da und dort einen Stall, statt ihn zu einer Zweitwohnung umzu­ bauen zu einer Dachterrasse umnutzen oder einen Garten oder einen kleinen Platz, um ein bisschen Raum und Luft zu schaffen. Das ist dann nicht mehr möglich. AR: Non sarà più concesso neanche trasforma­ re la stalla in un’abitazione e, in definitiva, que­ sto ostacola un approccio differenziato allo svilup­ po dei nostri villaggi. Abb. 3 / Fig. 3 Qui vivono poche persone, ma la struttura dell’abitato è molto densa, e io mi occupo del tema del diradamento urbano. Per fare un esempio, invece di riconvertire una stalla in una seconda casa, potrebbe essere possibile tra­ sformarla in una terrazza, un giardino o un picco­ lo luogo che crei spazio e aria. Tutto questo non sarà più possibile. GC: Ja, auch hier gibt es viel Überzeugungsarbeit zu leisten. Aber es macht uns Spass, weiter dranzu­ bleiben. Und nach uns kommen hoffentlich andere, denen die Zukunft unserer Dörfer nicht gleichgütig ist – auch in architektonischer Hinsicht. GC: Sì, anche in questo caso sarà necessario un grande sforzo di persuasione. Ma ci appassiona ve­ dere come andrà a finire. E speriamo che chi verrà dopo di noi non sarà indifferente al futuro dei nostri villaggi – anche dal punto di vista architettonico.
https://openalex.org/W2897599536
https://jurnalketerapianfisik.com/index.php/jpt/article/download/97/66
Indonesian
null
Pengaruh Workplace Stretching Exercise Terhadap Kebosanan Belajar Dan Kelelahan Belajar Mahasiswa Poltekkes Surakarta
Jurnal keterapian fisik
2,017
cc-by-sa
4,318
Kata Kunci : Workplace Stretching Exercise, Kebosanan, Kelelahan Belajar yang telah ditetapkan, metode pembelajaran yang bersifat monoton, sarana dan prasarana dalam proses pembelajaran yang tidak sesuai dengan antropometri peserta didik serta tidak adanya istirahat berupa istirahat aktif yang dilakukan peserta didik dalam proses pembelajaran di kelas. Dilihat dari luaran proses pembelajaran ternyata dampak yang ditimbulkan dapat mempengaruhi ketelitian kecepatan dan konstansi kerja peserta didik yang pada akhirnya kualitas proses pembelajaran bisa terganggu (Sutajaya, 2006). Salah satu upaya untuk mencapai kondisi tersebut adalah dengan pemberian Workplace Stretching-Exercise yang telah ditetapkan, metode pembelajaran yang bersifat monoton, sarana dan prasarana dalam proses pembelajaran yang tidak sesuai dengan antropometri peserta didik serta tidak adanya istirahat berupa istirahat aktif yang dilakukan peserta didik dalam proses pembelajaran di kelas. Dilihat dari luaran proses pembelajaran ternyata dampak yang ditimbulkan dapat mempengaruhi ketelitian kecepatan dan konstansi kerja peserta didik yang pada akhirnya kualitas proses pembelajaran bisa terganggu (Sutajaya, 2006). Salah satu upaya untuk mencapai kondisi tersebut adalah dengan pemberian Workplace Stretching-Exercise eywords : Workplace Stretching Exercise, Boredom, Fatique Learning Keywords : Workplace Stretching Exercise, Boredom, Fatique Learning Abstrak : Workplace Stretching Exercise, Kebosanan, Kelelahan Belajar. Penelitian ini bertujuan untuk mengetahui pengaruh Workplace Stretching Exercise terhadap kebosanan dan kelelahan belajar mahasiswa kampus I Politeknik Kesehatan kemenkes Surakarta. Pengambilan sampel dilakukan dengan teknik purposive sampling dengan jumlah 86 responden. Berdasarkan uji paired sample t-test diperoleh hasil p-value sebesar 0.001 (p<0.005). Kesimpulan hasil penelitian ini adalah ada pengaruh Workplace Stretching Exercise terhadap kebosanan dan kelelahan belajar mahasiswa kampus I Politeknik Kesehatan Kemenkes Surakarta. PENGARUH WORKPLACE STRETCHING EXERCISE TERHADAP KEBOSANAN BELAJAR DAN KELELAHAN BELAJAR MAHASISWA POLTEKKES SURAKARTA Lis Sarwi Hastuti, Rina Kurnia Kementerian Kesehatan Politeknik Kesehatan Surakarta Jurusan Okupasi Terapi Abstract : Workplace Stretching Exercise, Boredom, Fatique Learning. This study aims to reveal the impact of Workplace Stretching Exercise to boredom and fatique learning of student in campus I health Polytechnic of Surakarta. Sampling collection was done by purposive sampling technique with 86 respondents.The result of paired samples t-test was obtained p value = 0.001 (p<0.005). Conclusions results of this research is there is the influence of Workplace Stretching Exercise to the boredom and fatique learning student in campus I Health Polytechnic of Surakarta. Keywords : Workplace Stretching Exercise, Boredom, Fatique Learning PENDAHULUAN Proses pembelajaran melibatkan berbagai kegiatan dan tindakan yang perlu dilakukan oleh peserta didik. Pembelajaran adalah suatu proses interaksi antara pengajar/ guru/ dosen dengan peserta didik. Tercapainya tujuan pembelajaran ditandai oleh tingkat penguasaan kemampuan dan pembentukan kepribadian. Pada proses pembelajaran yang melibatkan aktivitas fisik dan mental dapat menimbulkan kebosanan dan kelelahan belajar. Hal ini dapat disebabkan oleh waktu yang digunakan pengajar dalam proses pembelajaran melebihi jadwal pelajaran 116 Lis Sarwi Hastuti, Pengaruh Workplace Stretching Exercise 117 (WSE) yang didesain dengan prinsip gerakan stretching (peregangan otot) dan iringan musik instrumental tempo sedang. Peregangan otot yaitu usaha untuk memperpanjang otot sehingga mengakibatkan otot menjadi rileks dan lentur (Nelson & Kokkonen, 2007). terdapat sistem aktivasi (bersifat simpatis) dan inhibisi (bersifat parasimpatis). Istilah kelelahan biasanya menunjukkan kondisi yang berbeda-beda dari setiap individu, tetapi semuanya bermuara pada kehilangan efisiensi dan penurunan kapasitas kerja serta ketahanan tubuh (Tarwaka, 2011). Gejala-gejala kelelahan antara lain adalah penurunan kesiagaan dan perhatian, perlambatan kecepatan reaksi, penurunan dan hambatan persepsi, depresi, kurang tenaga, dan kehilangan inisiatif (Setyawati, 2011). Penelitian ini bertujuan untuk mengetahui pengaruh pemberian workplace stretching exercise terhadap kebosanan dan kelelahan mahasiswa Politeknik Kesehatan Surakarta. Pembelajaran adalah proses berlangsungnya belajar mengajar di kelas yang merupakan inti dari kegiatan pendidikan di sekolah. Sedangkan pelaksanaan proses belajar mengajar dapat dilakukan sebagai interaksi antara pengajaran dengan pelajaran dalam menyampaikan bahan pelajaran kepada siswa untuk mencapai tujuan pembelajaran (Rahayu, 2010). Workplace Stretching-Exercise (WSE) merupakan bentuk latihan yang didesain dengan prinsip gerakan stretching (peregangan otot) dan iringan musik instrumental tempo sedang. Peregangan otot yaitu usaha untuk memperpanjang otot sehingga mengakibatkan otot menjadi rileks dan lentur (Nelson & Kokkonen, 2007). Tinjauan fisiologis terhadap manfaat Workplace Stretching-Exercise (WSE) secara umum dapat dijelaskan sebagai berikut, sifat kerja otot secara garis besar dibedakan menjadi dua, yaitu antagonis dan sinergis. Sifat kerja otot antagonis adalah dua kumpulan otot atau lebih yang cara kerjanya berlawanan untuk menghasilkan suatu koordinasi gerak, artinya jika satu kelompok otot berkontraksi, maka kelompok otot lain mengalami relaksasi. Sifat kerja otot sinergis adalah dua kelompok otot atau lebih yang cara kerjanya selaras untuk menghasilkan suatu gerakan. Untuk menghasilkan gerakan yang baik, satu kelompok otot tidak dapat bekerja sendiri, namun harus bekerjasama dengan kumpulan otot yang lain (Guyton, 2006). Stretching dapat diberikan pada otot yang sedang berkontraksi maupun relaksasi Kebosanan adalah ungkapan tidak enak dari perasaan tidak menyenangkan, perasaan lelah yang menguras seluruh minat dan tenaga (Anoraga, 1998). PENDAHULUAN Anastasi (1989) berpendapat bahwa sumber kebosanan dapat berasal dari individu dan lingkungan. Karakteristik orang berbeda-beda sehingga setiap orang memiliki kerentanan yang berbeda-beda pula terhadap kebosanan sekalipun melakukan kegiatan yang sama. Dampak dari kebosanan antara lain adalah timbulnya rasa kesal, lemas, lelah dan berkurangnya kewaspadaan (Kroemer dan Grandjean, 2000; Pulat, 1992; Kroemer dkk, 1994). Kelelahan merupakan suatu mekanisme perlindungan tubuh agar tubuh terhindar dari kerusakan lebih lanjut sehingga terjadi pemulihan setelah istirahat. Kelelahan diatur secara sentral oleh otak. Pada susunan saraf pusat 118 Jurnal Keterapian Fisik, Volume 2, No 2,November 2017, hlm 75-125 Tabel 1 Karakteristik Responden Penelitian Data Kontinu Data N Min Max Mean SD Usia 86 18 24 19,15 0,69 IMT 86 15 27,3 19,70 2,40 Lama belajar/hari 86 0 4 1,43 0,72 Lama istirahat malam 86 4 9 6,48 1,05 Jarak tempat tinggal ke kampus 86 0,1 25 7,53 9,41 b. Karakteristik Data Kategorikal 1) Jenis Kelamin tergantung sifat kerja otot tersebut (Nelson & Kokkonen, 2007). tergantung sifat kerja otot tersebut (Nelson & Kokkonen, 2007). Tabel 1 Karakteristik Responden Penelitian b. Karakteristik Data Kategorikal 1) Jenis Kelamin Responden dalam penelitian ini terdiri atas mahasiswa Kampus I dengan jenis kelamin laki-laki dan perempuan. Berikut adalah data responden berdasarkan jenis kelamin. Tabel 2 Pengambilan data penelitian dengan judul pengaruh workplace stretching exercise terhadap kebosanan dan kelelahan belajar mahasiswa di Kampus I Poltekkes Surakarta dilakukan di Kampus I Poltekkes Surakarta yang terdiri dari 3 (tiga) jurusan yaitu : Jurusan Keperawatan, Jurusan Akupuntur dan Jurusan Terapi Wicara. Pengambilan data diawali dengan melakukan perijinan selanjutnya proses pengambilan data dilakukan dengan mahasiswa mengisi kuesioner kebosanan dan kelelahan berupa pada pemeriksaan awal yaitu sebelum diberikan intervensi Workplace Stretching-Exercise, pemberian intervensi Workplace Stretching-Exercise kemudian mengisi kuesioner kebosanan dan kelelahan setelah mendapatkan intervensi. Analisis Univariat Karakteristik Responden Penelitian Berdasarkan Jenis Kelamin Jenis Kelamin n % Laki-laki 13 15,1 Perempuan 73 84,9 Jumlah 86 100 Berdasarkan data yang disajikan pada tabel 2, pada 86 responden penelitian diketahui bahwa perbandingan laki-laki dibanding perempuan adalah 1:6. 2) Alat Transportasi yang Digunakan 2) Alat Transportasi yang Digunakan Hasil karateristik responden penelitian berdasarkan alat transportasi yang digunakan dapat dilihat pada tabel 3 sebagai berikut : Hasil karateristik responden penelitian berdasarkan alat transportasi yang digunakan dapat dilihat pada tabel 3 sebagai berikut : Analisis Univariat Digunakan Digunakan Alat Transportasi n % Jalan kaki 9 10,5 Sepeda motor 74 86 Antar jemput 3 3,5 Jumlah 86 100 Hasil penelitian menunjukkan bahwa mayoritas responden penelitian METODE PENELITIAN Desain penelitian yang digunakan adalah quasi eksperimental “One groups pre and post test design”. Pada penelitian ini sebelum dan setelah pemberian Workplace Stretching-Exercise (WSE) akan dilakukan penilaian terhadap kebosanan dan kelelahan. Populasi dalam penelitian ini adalah seluruh mahasiswa Kampus I Politeknik Kesehatan Surakarta. Pengambilan sampel dengan teknik purposive sampling, jumlah sampel dalam penelitian ini adalah 86 orang. Tabel 3 Karakteristik Responden Penelitian Berdasarkan Alat Transportasi yang Karakteristik Responden Penelitian Berdasarkan Alat Transportasi yang a. Karakteristik Data Kontinu n l n n u n t n n n g a Karakteristik Responden Penelitian Berdasarkan Rutinitas Olahraga Rutin Olahraga n % Ya 23 26,7 Tidak 63 73,3 Jumlah 86 100 Hasil penelitian menunjukkan bahwa responden lebih banyak yang tidak berolahraga yaitu sebanyak 63 (73,3%). c. Kebosanan dan Kelelahan Tabel 7 Hasil rerata kebosanan dan kelelahan responden penelitian sebelum dan sesudah intervensi Data Mean Pre test kebosanan 67,77 Post test kebosanan 35,74 Pre test kelelahan 60,12 Post test kelelahan 38,42 Analisis Bivariat Tabel 8 Hasil Uji paired-sample t test Data Nilai Signifikansi (p value) Pre – post test kebosanan 0.001 0.001 Pre-post test kelelahan PEMBAHASAN Kebosanan dalam proses pembelajaran ditandai dengan berkurangnya perhatian peserta didik terhadap materi yang sedang dibahas atau peserta didik mengalami kesulitan dalam mempertahankan perhatiannya pada tugas yang sedang dilaksanakan. Kondisi seperti ini sering menyertai peserta didik pada proses pembelajaran jikalau jam belajar dialokasikan pada siang hari dan terlebih 3) Konflik Psikososial Hasil karakteristik responden penelitian berdasarkan konflik psikososial dapat dilihat pada tabel 4 sebagai berikut : Tabel 4 Karakteristik Responden Penelitian Berdasarkan Konflik Psikososial Konflik Psikososial n % Ya 5 5,8 Tidak 81 94,2 Jumlah 86 100 Berdasarkan Konflik Psikososial Konflik Psikososial n % Ya 5 5,8 Tidak 81 94,2 Jumlah 86 100 Hasil penelitian menunjukkan bahwa mayoritas responden penelitian tidak memiliki konflik sosial yaitu sebanyak 81 (94,2%). 4) Kebiasaan Merokok Hasil karakteristik responden berdasarkan kebiasaan merokok dapat dilihat pada tabel 5 sebagai berikut : Tabel 5 Karakteristik Responden Peneitian Berdasarkan Lama Belajar/Hari Kebiasaan Merokok n % Ya 1 1,2 Tidak 85 98,8 Jumlah 86 100 Hasil penelitian menunjukkan bahwa mayoritas responden penelitian a. Karakteristik Data Kontinu g Alat Transportasi n % Jalan kaki 9 10,5 Sepeda motor 74 86 Antar jemput 3 3,5 Jumlah 86 100 Hasil statistik deskriptif data kontinu yang berupa usia, IMT, lama belajar/ hari, lama istirahat malam, jarak tempat tinggal ke kampus, dapat dilihat pada tabel 1 sebagai berikut : Hasil penelitian menunjukkan bahwa mayoritas responden penelitian Lis Sarwi Hastuti, Pengaruh Workplace Stretching Exercise 119 menggunakan alat transportasi sepeda motor yaitu sebanyak 64 (86%). 3) Konflik Psikososial Hasil karakteristik responden penelitian berdasarkan konflik psikososial dapat dilihat pada tabel 4 sebagai berikut : Tabel 4 Karakteristik Responden Penelitian Berdasarkan Konflik Psikososial Konflik Psikososial n % Ya 5 5,8 Tidak 81 94,2 Jumlah 86 100 Hasil penelitian menunjukkan bahwa mayoritas responden penelitian tidak memiliki konflik sosial yaitu sebanyak 81 (94,2%). 4) Kebiasaan Merokok Hasil karakteristik responden berdasarkan kebiasaan merokok dapat dilihat pada tabel 5 sebagai berikut : Tabel 5 Karakteristik Responden Peneitian Berdasarkan Lama Belajar/Hari Kebiasaan Merokok n % Ya 1 1,2 Tidak 85 98,8 Jumlah 86 100 Hasil penelitian menunjukkan bahwa mayoritas responden penelitian tidak merokok yaitu sebanyak 85 (98,8%). 5) Rutinitas Olahraga Hasil karateristik responden penelitian berdasarkan olahraga yang dilakukan secara rutin dapat dilihat pada tabel 6 sebagai berikut : Tabel 6 Karakteristik Responden Penelitian Berdasarkan Rutinitas Olahraga Rutin Olahraga n % Ya 23 26,7 Tidak 63 73,3 Jumlah 86 100 Hasil penelitian menunjukkan bahwa responden lebih banyak yang tidak berolahraga yaitu sebanyak 63 (73,3%). c. Kebosanan dan Kelelahan Tabel 7 Hasil rerata kebosanan dan kelelahan responden penelitian sebelum dan sesudah intervensi Data Mean Pre test kebosanan 67,77 Post test kebosanan 35,74 Pre test kelelahan 60,12 Post test kelelahan 38,42 Analisis Bivariat Tabel 8 Hasil Uji paired-sample t test Data Nilai Signifikansi (p value) Pre – post test kebosanan 0.001 0.001 Pre-post test kelelahan PEMBAHASAN Kebosanan dalam proses pembelajaran ditandai dengan berkurangnya perhatian peserta didik terhadap materi yang sedang dibahas atau peserta didik mengalami kesulitan dalam mempertahankan perhatiannya pada tugas yang sedang dilaksanakan. Kondisi seperti ini sering menyertai peserta didik pada proses pembelajaran jikalau jam belajar dialokasikan pada siang hari dan terlebih lagi pengajar kurang mampu mengelola k l d b ik Jik k di i menggunakan alat transportasi sepeda motor yaitu sebanyak 64 (86%). PEMBAHASAN Kebosanan Hasil penelitian menunjukkan bahwa mayoritas responden penelitian tidak merokok yaitu sebanyak 85 (98,8%). Kebosanan dalam proses pembelajaran ditandai dengan berkurangnya perhatian peserta didik terhadap materi yang sedang dibahas atau peserta didik mengalami kesulitan dalam mempertahankan perhatiannya pada tugas yang sedang dilaksanakan. Kondisi seperti ini sering menyertai peserta didik pada proses pembelajaran jikalau jam belajar dialokasikan pada siang hari dan terlebih lagi pengajar kurang mampu mengelola kelas dengan baik. Jika kondisi yang membosankan tersebut berkepanjangan, Hasil karateristik responden penelitian berdasarkan olahraga yang dilakukan secara rutin dapat dilihat pada tabel 6 sebagai berikut : 120 Jurnal Keterapian Fisik, Volume 2, No 2,November 2017, hlm 75-125 akan muncul perasaan gelisah,ingin menghindar dari aktivitas tersebut dan menurunnya motivasi untuk belajar. Dalam hal ini Kroemer dan Grandjean (2000) menjelaskan bahwa kebosanan bisa terjadi disebabkan oleh stimulasi yang rendah, tuntutan fisik dan mental yang rendah yang mengakibatkan stimulasi yang kecil pada daerah kesadaran di otak. Konsekuensinya sistem limbic akan terpengaruh dan reaksi dari organisme secara keseluruhan akan menurun. sedang adalah 92-104 langkah/menit. Jenis musik ini termasuk musik relaksasi yang memiliki beberapa manfaat antara lain membuat tubuh dan pikiran lebih santai, meningkatkan perasaan senang dan mengatasi rasa bosan dan lelah dalam bekerja. Musik tempo sedang dapat membantu tubuh dan pikiran lebih rilaks (Johan, 2009). Intervensi WSE ini dilaksanakan secara bersama-sama (klasikal) di kelas. Penerapan model klasikal ini dapat menimbulkan motivasi dan akhirnya berdampak pada berkurangnya rasa bosan. Hal ini sesuai dengan pendapat Farozin (2012) bahwa metode klasikal dapat meningkatkan motivasi belajar. Menurut Ginting (2010) bahwa semakin tinggi kohesivitas kelompok maka akan semakinmeningkatkan motivasi. Responden dalam penelitian ini berasal dari 2 kelas pada 2 Jurusan, dimana setiap anggota kelompok telah berinteraksi selama satu tahun lebih, hal ini menunjukkan bahwa kohesivitas pada responden semakin tinggi. Hal ini ditandai dengan terjalinnya kerjasama, saling berkomunikasi, merasa bertanggung jawab terhadap tugas dan memiliki tujuan pembelajaran yang sama. Menurut Tarwaka (2011), bahwa kebosanan juga dapat dipicu oleh tuntutan tugas yang melampaui kapasitas individu, hal ini berarti rasa bosan yang dipicu oleh stress. Proses pembelajaran yang berkepanjangan sering memunculkan rasa bosan yang ditandai dengan rasa kesal, lelah, lemas, dan menurunnya konsentrasi serta ingin beralih dari aktivitas tersebut( Kroemer dan Grandjean ,2000). Hal ini sesuai dengan kondisi yang dialami oleh responden, dimana durasi pembelajaran dikampus seringkali lebih dari 8 jam setiap harinya, dan disamping pembelajaran dikampus, responden masih harus mengerjakan tugas mata kuliah di luar jam belajar di kampus. PEMBAHASAN Kebosanan Hal ini akan bertambah buruk jika suasana pembelajaran monoton, dan peserta didik cenderung pasif. g Responden dalam penelitian ini berasal dari 2 kelas pada 2 Jurusan, dimana setiap anggota kelompok telah berinteraksi selama satu tahun lebih, hal ini menunjukkan bahwa kohesivitas pada responden semakin tinggi. Hal ini ditandai dengan terjalinnya kerjasama, saling berkomunikasi, merasa bertanggung jawab terhadap tugas dan memiliki tujuan pembelajaran yang sama. Pada bagian akhir WSE terdapat gerakan pernafasan dengan menerapkan teknik Pursed lip breathing, dan WSE ditutup dengan gerakan ambil nafas (inspirasi) melalui hidung dan dikeluarkan (ekspirasi) melalui mulut disertai suara teriakan ‘huuu/haaa’. Teknik Pursed lip breathing yaitu menarik nafas melalui hidung, seperti membau bunga mawar (mulut tertutup), kemudian mengeluarkan nafas melalui mulut (seperti meniup lilin). Beberapa manfaat melakukan pursed lip breathing adalah mengurangi kecemasan, Pemberian intervensi WSE dapat berpengaruh terhadap penurunan kebosanan belajar mahasiswa, faktor pendukung hal ini antara lain adalah adanya musik WSE, intervensi dilaksaksanakan secara klasikal, dan gerakan akhir pada WSE. Musik instrumen yang dipergunakan untuk mengiringi WSE ini adalah musik Sojiro Jepang dengan tempo sedang. Tempo musik adalah kecepatan dalam ukuran langkah tertentu. Kriteria musik tempo Lis Sarwi Hastuti, Pengaruh Workplace Stretching Exercise 121 berakibat terjadi spasme yang berlebihan dan menimbulkan nyeri (Elnaggar et al.,1990). Nyeri terjadi karena vasokontriksi pembuluh darah di otot akibat kontraksi berlebihan yang terus menerus dan akhirnya menimbulkan ischemia jaringan. Mekanisme ischemia dan spasme otot yang berlebihan akan merangsang nociceptor yang berperan dalam menghantarkan impuls nyeri ke otak (Cameron, 1999). menjaga konsentrasi, mengurangi sesak nafas, menjadikan tubuh dan pikiran lebih relaks (Huntley, 2008). Dengan melakukan gerakan pernafasan ini akan membantu fisik dan pikiran responden lebih relaks. Teriakan pada gerakan pernafasan, bagian akhir WSE membantu responden untuk menyalurkan rasa bosan yang dirasakan. Berdasarkan hasil uji paired- sample t test menunjukkan bahwa WSE berpengaruh menurunkan kelelahan pada responden. Hal ini disebabkan oleh efek/manfaat stretching pada WSE. Stretching merupakan suatu usaha untuk memperpanjang otot istirahat (relaksasi), sehingga dapat mencegah ketegangan otot (spasme). Stretching yang dilakukan secara rutin dapat berdampak pada kelenturan (fleksibilitas), yaitu kemampuan sendi untuk bergerak dalam jangkauan penuh (Alter, 2003). Orang yang selalu aktif beraktifitas seperti responden penelitian ini memerlukan fleksibilitas yang cukup untuk dapat tetap melakukan pekerjaannya dengan nyaman. Pada otot yang mengalami spasme, akan terjadi pemendekan muscle fiber karena anyaman-anyaman myofilamen mengalami overlapping satu sama lain. PEMBAHASAN Kebosanan Pada saat dilakukan stretching dengan penahanan beberapa detik pada posisi otot memanjang, struktur muscle fiber terutama sarcomer akan mengalami peregangan karena anyaman-anyaman myofilamen yang overlapping akan berkurang dan secara otomatis menyebabkan struktur muscle fiber menjadi memanjang. Dengan pemanjangan struktur muscle fiber tersebut, maka spasme dapat berkurang (Appleton, 1998). Kontraksi dinamik pada otot-otot yang terjadi saat melaksanakan WSE menyebabkan lebih banyak darah mengalir ke otot, sehingga pasokan oksigen dan nutrisi yang dibutuhkan untuk proses metabolisme dapat dipenuhi secara efektif. Melakukan WSE berarti merubah posisi kerja statis menjadi lebih dinamis Hal ini dapat menstimulasi kelancaran proses metabolisme dalam tubuh, sehingga penimbunan hasil metabolisme otot (asam laktat dan CO2) dapat diminimalisir dan kelelahan dapat dicegah atau dikurangi (Tarwaka, 2011). Pemberian stretching dapat mengurangi spasme karena proprioceptor otot atau muscle spindle yang teraktivasi saat stretching terjadi. Muscle spindle bertugas untuk mengatur sinyal ke otak tentang perubahan panjang otot dan perubahan tonus yang mendadak dan berlebihan. Jika ada perubahan tonus otot yang mendadak dan berlebihan, maka muscle spindle akan mengirimkan sinyal ke otak untuk membuat otot tersebut berkontraksi sebagai bentuk pertahanan dan mencegah cedera. Oleh karena itu, saat melakukan stretching dilakukan penahanan beberapa saat dengan tujuan untuk memberikan adaptasi pada muscle spindle terhadap perubahan panjang otot Pada peserta didik dengan posisi belajar statis seperti responden penelitian ini, akan terjadi kontraksi isometrik otot- otot tertentu secara terus menerus dan 122 Jurnal Keterapian Fisik, Volume 2, No 2,November 2017, hlm 75-125 rendah terhadap kekuatan otot, fleksibilitas, dan keseimbangan terhadap lansia menunjukkan hasil bahwa pada kelompok perlakuan terjadi kenaikan lebih signifikan terhadap kekuatan otot, fleksibilitas, dan keseimbangan dibandingkan kelompok kontrol. Usia responden dalam penelitian ini rentangnya antara 18-24 tahun, hal ini kemungkinan turut berkontribusi terhadap manfaat WSE, karena ditinjau dari usia secara fisiologis belum banyak terjadi perubahan, sehingga kelompok usia ini lebih responsif terhadap efek exercise. yang kita berikan, sehingga sinyal dari otak untuk mengkontraksikan otot menjadi berkurang. Dengan kontraksi otot yang minimal pada saat stretching, akan memudahkan muscle fibers untuk memanjang dan spasme otot dapat berkurang (Appleton, 1998; Costa & Vieira, 2008). Menurut Cameron (1999), bahwa pemberian stretching juga dapat merangsang serabut saraf berpenampang tebal (A alpha dan A beta) sehingga mampu menutup gerbang kontrol nyeri. PEMBAHASAN Kebosanan Kondisi responden ini telah sesuai dengan ketentuan waktu istirahat, namun demikian jika dilihat data tentang kelelahan dan kebosanan responden sebelum intervensi cukup tinggi. Hal ini dimungknkan berkaitan dengan rendahnya angka kebiasaan rutin berolahraga pada responden . Terdapat 73,3% responden yang tidak melakukan kegiatan olahraga secara rutin. Dengan melakukan olahraga rutin, maka tubuh akan lebih bugar, otot dan sendi menjadi lebih terlatih dan siap dalam melakukan aktifitas (ACSM, 2010). Cone C., 2006. Ergonomic Exercise Break. Worksite wellness. Council of Volucia County Costa & Vieira., 2008. Stretching to reduce work related musculoskeletal disorders; journal rehabilitation med 2008, (Internet). Available from : http://www.occmed.oxfordjournals .org. (accessed 10 August 2012) Depkes R.I., 2007. Keselamatan dan Kesehatan Kerja di Laboratorium. Depkes R.I., Jakarta. Dharma, A & Lukmanto,P., 1993. Fisiologi Kedokteran. (A. G Terjemahan). Edisi 5. Penerbit Buku Kedokteran EGC, Jakarta Djohan., 2009. Psikologi Musik. Yogyakarta, Best Publisher. Djohan., 2009. Psikologi Musik. Y k B P bli h PEMBAHASAN Kebosanan Mekanisme stretching termasuk dalam kategori stimulasi mekanik yang dapat mengaktivasi fungsi serabut saraf berpenampang tebal non-nociceptif (A alpha dan A beta) dan menutup gerbang kontrol sehingga nyeri yang dibawa serabut saraf berpenampang tipis (A delta dan C) tidak dapat diteruskan ke otak. Karakteristik IMT turut berkontribusi terhadap manfaat WSE untuk menurun kan kebosanan dan kelelahan, karena rata-rata IMT responden adalah kategori normal . Ozguler at al. (2000) menuliskan bahwa kelelahan banyak dialami individu dengan Body Mass Index (BMI) > 24,92 kg/m2 yaitu individu yang masuk kategori overweight dan obese. Pendapat ini sejalan dengan Vessy at al (1990) yang dikutip oleh Tarwaka (2011) bahwa wanita gemuk memiliki resiko mengalami kelelahan 2x lipat dibandingkan wanita kurus. Hal ini disebabkan oleh kondisi keseimbangan struktur rangka dalam menerima beban, baik beban berat tubuh maupun beban tambahan lainnya. Salah satu indikator untuk mengetahui status gizi seseorang adalah dengan melihat Indeks Masa tubuh (IMT). Jika status gizi kurang (underweight) , maka beberapa akibat yang akan timbul antara lain daya tahan tubuh rendah, produksi energi rendah sehingga lebih mudah merasa lelah dan produktifitas belajar menurun. Jika status gizi berlebih (overweight dan obese), maka dikaitkan dengan resiko terjadinya beberapa penyakit degeneratif, misalnya p Kontraksi dinamik pada otot-otot yang terjadi saat melaksanakan WSE menyebabkan lebih banyak darah mengalir ke otot, sehingga pasokan oksigen dan nutrisi yang dibutuhkan untuk proses metabolisme dapat dipenuhi secara efektif. Melakukan WSE berarti merubah posisi kerja statis menjadi lebih dinamis Hal ini dapat menstimulasi kelancaran proses metabolisme dalam tubuh, sehingga penimbunan hasil metabolisme otot (asam laktat dan CO2) dapat diminimalisir dan akhirnya berdampak pada berkurangnya kelelahan belajar. Secara umum ketika individu berusia di atas 40 tahun terjadi beberapa penurunan kemampuan secara fisik maupun fisiologis, misalnya menurunnya kemampuan panca indera, berkurangnya elastisitas tonus jaringan (tissue elasticity and tone), dan menurunnya kapasitas VO2 max (Mills, 1991). Penelitian Mills (1991) tentang efek aerobik intensitas Lis Sarwi Hastuti, Pengaruh Workplace Stretching Exercise 123 stroke, jantung, diabetes melitus, dan lain- lain (Nurmianto, 2003). stroke, jantung, diabetes melitus, dan lain- lain (Nurmianto, 2003). Cameron., Michele.,1999. Physical Agents in Rehabilitation; W.B.Saunders company, Philadelphia. Rata-rata responden beristirahat/tidur selama 6,4 jam dalam periode satu hari (24 jam), hal ini sesuai dengan pendapat Grandjean (1993), bahwa tidur orang dewasa antara 6-8 jam, dan hutang tidur (sleep debt) dapat berdampak pada rasa kantuk, lelah, dan bosan dalam melakukan aktifitas. KESIMPULAN DAN SARAN Grandjean, E., 1995. Fitting the task to the man. A text book of occupational ergonomic 4 th edition. Tailor & Francis, New York. Berdasarkan hasil penelitian terhadap 86 orang responden di Kampus I Politeknik Kesehatan Surakarta, yang diberikan intervensi berupa Workplace Stretching-Exercise (WSE) selama 4 minggu, dan berdasarkan uji paired- sample t test diperolah hasil p value sebesar 0,001 (p < 0,05), dapat disimpulkan hal-hal sebagai berikut: Berdasarkan hasil penelitian terhadap 86 orang responden di Kampus I Politeknik Kesehatan Surakarta, yang diberikan intervensi berupa Workplace Stretching-Exercise (WSE) selama 4 minggu, dan berdasarkan uji paired- sample t test diperolah hasil p value sebesar 0,001 (p < 0,05), dapat disimpulkan hal-hal sebagai berikut: Huntley, N., 2008. Cardiac & Pulmonary Diseases. In M.V. Radomski & C.A. Trombly Latham (eds). Occupational Therapy for Physical Dysfunction 6th ed. Baltimore: Huntley, N., 2008. Cardiac & Pulmonary Huntley, N., 2008. Cardiac & Pulmonary Diseases. In M.V. Radomski & C.A. Trombly Latham (eds). Diseases. In M.V. Radomski & C.A. Trombly Latham (eds). Occupational Therapy for Physical Dysfunction 6th ed. Baltimore: Lippincott Williams & Wilkins 1. Pemberian WSE berpengaruh menurunkan kebosanan belajar pada responden Irwanti, N.K.D., 2010. Peregangan otot di sela pembelajaran mengurangi kebosanan, kelelahan, dan keluhan muskuloskeletal pada peserta didik kelas X SMK Pariwisata Triatma Jaya Bandung. (Tesis). Program Pascasarjana Universitas Udayana Denpasar, Bali 2. Pemberian WSE berpengaruh menurunkan kelelahan belajar responden Kroemer, K.H.E & Grandjean, E., 2000. Fitting the task to the Human. A 124 Jurnal Keterapian Fisik, Volume 2, No 2,November 2017, hlm 75-125 Textbook of Occupational Ergonomics. Fifth ed. Taylor and Francis. Back Pain according To Various Definition of Low Back Pain. J Epudemiol Community Health. Mukhmad., 2011. Pengertian Kelelahan Fisiologis (Internet), Teori Fisiologi Kelelahan. Available from : [http://www. rajauntung.blogspot.com/2011/12/f is-kel.html] (accessed 7 Nopember 2012) Paquet V. and Nirmale, R., 2004. Trunk Flexion and Musculoskeletal Stress During Light Assembly Work, Journal of Applied Biomechanics. Phoon, W.O., 1988., Practical Occupational health. PG Publishing.Singapore. pp. 281- 323. National Institute for Occupational Safety and Health., 1996. A Guide to Safety in Confined Space. U.S. Department of Health and Human Service, America. Robergs, R.A., Keteyian,S.J., (2003). Fundamentals of Exercise Physiology fo Fitness, Performance, and Health (2nd ed). New York: Mc Graw Hill Notoatmojo, S., 2002. Metodologi Penelitian Kesehatan. PT. Rineka Cipta, Jakarta. Sinaki M., Mokri B., 1996. Low Back Pain and Disorders of The Lumbar Spine . WB Sounders Company, Philadelphia. KESIMPULAN DAN SARAN Nunuk, S., 2005 Metode Penelitian (Pendidikan Teori dan Prosedur). Surakarta, Fakultas Keguruan dan Ilmu Pendidikan. Universitas Sebelas Maret, Surakarta Sugiyono., 2003. Statistik untuk Penelitian. Ed 7, CV Alfabeta, Bandung. Nurhikmah., 2011. Faktor-faktor yang berhubungan dengan musculosceletal disorder pada pekerja furniture di kecamatan Benda kota Tangerang. (skripsi). Program Studi Kesehatan Masyarakat Fakultas Kedokteran dan Ilmu kesehatan Universitas Islam Negeri Syarief Hidayatullah. Jakarta Sutajaya., IM. 2006. Pembelajaran Melalui Pendekatan Sistemik Holistik Interdisipliner dan Partisipatori (SHIP) Mengurangi Kelelahan Keluhan Muskuloskeletal dan Kebosanan serta Meningkatkan Luaran Proses Belajar 76 Mahasiswa Biologi IKIP Singaraja. (Disertasi). Program Pascasarjana Universitas Udayana, Denpasar. Nurmianto, E., 2003. Ergonomi, Konsep dan Aplikasinya. Edisi 1. PT.Guna Widya, Surabaya y p Tarwaka., 2011. Ergonomi Industri. Dasar-dasar pengetahuan ergonomi dan aplikasi di tempat kerja. Harapan Press, Solo. Occupational Safety and Health Administration., 2000. Sewing and Related Procedures Ergonomics. U.S. Department of Labor. California, www.osha.gov. Teja, A.R. 2009., Ergonomic Exercise pasca nerve mobilitation. Makalah ilmiah. Available from [http://ansharphysio.blogspot.com/ Ozguler A., Lecler C.A., Londre M.F., Pietri F., 2000. Individual and Occupational Determinant of Low Lis Sarwi Hastuti, Pengaruh Workplace Stretching Exercise 125 2009/03/senam-ergonomi.html] (accessed 12 Oktober 2012) Triaminah., 2010. Reflek-reflek Fisiologis (Internet), Laporan Praktikum Faal. Available from : [http://www.blokspot.com/2010/12 /lap-prak.html] (accessed 7 Nopember 2012) Departemen perindustrian., 1984. Undang-Undang No.5 Tentang Perindustrian. Jakarta Waikar,A.M.,Bradshaw,M.E., 1995 Exercises in the Workplace Employee Preferences. Emerald (Internet), October, 16 (9) pp. 16- 30. Available (accessed 7 November 2012) Wiradharma, N., 2012. Praktikum odontektomi berorientasi ergonomi meningkatkan kinerja praktikan di Jurusan Kedokteran Gigi Universitas Mahasaraswati Denpasar. (Tesis). Program Pascasarjana Universitas Udayana Denpasar, Bali Yogisutanti G., 2009. Pengaruh Pelatihan Patient Handling terhadap Penurunan Keluhan Muskuloskeletal pada Perawat. Program Studi Ilmu Kesehatan Kerja, Universitas Gadjah Mada, Yogyakarta. 2009/03/senam-ergonomi.html] (accessed 12 Oktober 2012) Triaminah., 2010. Reflek-reflek Fisiologis (Internet), Laporan Praktikum Faal. Available from : [http://www.blokspot.com/2010/12 /lap-prak.html] (accessed 7 Nopember 2012) Departemen perindustrian., 1984. Undang-Undang No.5 Tentang Perindustrian. Jakarta Waikar,A.M.,Bradshaw,M.E., 1995 Exercises in the Workplace Employee Preferences. Emerald (Internet), October, 16 (9) pp. 16- 30. Available (accessed 7 November 2012) Wiradharma, N., 2012. Praktikum odontektomi berorientasi ergonomi meningkatkan kinerja praktikan di Jurusan Kedokteran Gigi Universitas Mahasaraswati Denpasar. (Tesis). Program Pascasarjana Universitas Udayana Denpasar, Bali Yogisutanti G., 2009. Pengaruh Pelatihan Patient Handling terhadap Penurunan Keluhan Muskuloskeletal pada Perawat. Program Studi Ilmu Kesehatan Kerja, Universitas Gadjah Mada, Yogyakarta.
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Operative Zugangswege zur Hüfte beim älteren Menschen
˜Derœ Orthopäde
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M. Nogler1 · M. Thaler2 M. Nogler1 · M. Thaler2 1Experimentelle Orthopädie, Medizinische Universität Innsbruck, Innsbruck, Österreich 2Universitätsklinik für Orthopädie, Medizinische Universität Innsbruck, Innsbruck, Österreich Orthopäde 2017 · 46:18–24 DOI 10.1007/s00132-016-3366-2 Online publiziert:21. Dezember 2016 © Der/die Autor(en) 2016. Dieser Artikel ist eine Open-Access-Publikation. Orthopäde 2017 · 46:18–24 DOI 10.1007/s00132-016-3366-2 Online publiziert:21. Dezember 2016 © Der/die Autor(en) 2016. Dieser Artikel ist eine Open-Access-Publikation. Grundsätzliche Überlegung Anders als etwa in der Kniegelenkendo- prothetik stehen dem Operateur in der Hüftendoprothetik mehrere gleicherma- ßen gut belegte Zugänge zur Auswahl. Dies gilt unabhängig vom Alter des Pati- enten. Grundsätzlich werden die Zugän- genachihrerLokalisationunterschieden, wobei die Zugänge eng mit der Lagerung g g Jeder operative Eingriffführt zwangs- läufig zu einer zugangsbedingten Gewe- beschädigung, dem Zugangsschaden. Ziel der „Minimalinvasivität“ ist, diesen so klein wie möglich zu halten, ohne das Operationsergebnis negativ zu beeinflus- sen. Die Frage, was nun weniger invasiv ist, wird breit diskutiert und ist nicht vollständig entschieden, hängt sie doch, wie im Abschnitt über die Zugangswahl ausgeführt, auch von der Umsetzung ei- nes Zugang im Hinblick auf Erfahrung, operationstechnische Kompetenz sowie verwendete Implantate und Instrumente ab. Grundsätzlich ist das Ziel, den Scha- den, der durch die Operation und den gewählten Zugang besteht, möglichst gering zu halten, eines, das akzeptiert wird. Als Schaden im Bereich des Hüft- gelenkes sind v. a. Schädigungen der Muskulatur zu betrachten. Ein wesentliches Thema der aktuellen Diskussion ist jenes über die Bedeutung vonminimalinvasivenZugängenim Ver- gleich zu klassischen Zugangsvarianten. Eine entsprechende Suche in PubMed mit den Stichworten „THA minimally invasive“ (http://www.ncbi.nlm.nih.gov/ pubmed/?term=tha+minimally+invasive) ergab 220 Treffer mit Arbeiten zu diesem Thema; 14 Arbeiten waren im Jahr 2016 publiziert. Auch dieses Thema ist aktuell und wird breit diskutiert. des Patienten verbunden sind: des Patienten verbunden sind: 4 dorsaler Zugang: Seitenlage [1], 4 dorsaler Zugang: Seitenlage [1], 4 direkt lateraler, transglutealer Zugang (Bauer): Seitenlage/Rückenlage [2], 4 anterolateraler Zugang (Watson Jones): Seitenlage/Rückenlage [3], 4 direkt anteriorer Zugang (DAA): Rückenlage [4]. 4 direkt lateraler, transglutealer Zugang (Bauer): Seitenlage/Rückenlage [2], 4 anterolateraler Zugang (Watson Jones): Seitenlage/Rückenlage [3], 4 direkt anteriorer Zugang (DAA): Rückenlage [4]. 4 anterolateraler Zugang (Watson Jones): Seitenlage/Rückenlage [3], 4 direkt anteriorer Zugang (DAA): Rückenlage [4]. TechnischoperativsinddieUnterschiede zwischen diesen Zugängen klar ersicht- lich. Wesentlich schwieriger ist die Beur- teilung der Bedeutung der Zugangswahl für das Operationsergebnis, das Patien- ten-Outcome. Es gibt in der Literatur eine große Anzahl von Studien, die Un- terschiede zwischen den Zugängen un- tersuchen[5–7]. Esgibtallerdingskeinen Konsens darüber, dass einer dieser Zu- gänge der Goldstandard der Hüftendo- prothetik wäre. Alle beschriebenen Zu- gänge in zahlreichen Varianten finden weltweit in der Hüftendoprothetik Ver- wendung. durch ein Kunstgelenk, bei allen Zugän- gen gleich ist. Hüftendoprothetik breit diskutiert wird. Die Bandbreite der Artikel reicht da- bei von Vergleichen einzelner Zugänge miteinander im Hinblick auf Operati- onsergebnisse bis zu technischen oder anatomischen Details eines speziellen Zugangs. Orthopäde 2017 · 46:18–24 DOI 10.1007/s00132-016-3366-2 Online publiziert:21. Dezember 2016 © Der/die Autor(en) 2016. Dieser Artikel ist eine Open-Access-Publikation. Invasivität Neuere Zugangswege werden häufig als minimalinvasivbeschrieben. Gemeintist hier das Ausmaß des Muskeltraumas, da die Invasivität im Hinblick auf das Ge- lenk, also dessen Entfernung und Ersatz Aus anatomischen Gründen sind 2 Zugänge zum Hüftgelenk streng in- termuskulär. Der DAA und der antero- laterale Zugang verwenden jeweils ein Intervall zwischen Muskeln. Der dor- Abb. 1 9 Chirur- gische Portale zum Hüftgelenk.DAA di- rekt anteriorerZu- gang,TFLTensorfas- ciae latae,Glut.Med. Glutaeus medius Bei einer einfachen Literaturre- cherche im Juli 2016 ergab die Su- che nach „THA Approach“ auf Pub- Med (http://www.ncbi.nlm.nih.gov/ pubmed/?term=tha+approach)710Tref- fer; 99 Artikel stammen dabei aus dem Jahr 2016. Diese Zahlen zeigen deut- lich, dass die Diskussion um Vor- und Nachteile verschiedener Zugänge in der 18 Der Orthopäde 1 · 2017 Tab. 1 Hüftgelenkmuskeln undihre Funktion Flexion Extension Adduktion Abduktion Außenrotation Innenrotation Iliacus Glutaeus maximus Adduktoren Glutaeus medius Piriformis Tensor fasciae latae Psoas Glutaeus minimus Pectineus Glutaeus minimus Obturator ex- ternus Glutaeus medius Sartorius Glutaeus medius Gracilis Tensor fasciae latae Obturator inter- nus Glutaeus minimus Rectus femoris Hamstrings – Glutaeus maximus Quadratus femoris – Tensor fascia lata – – Piriformis Glutaeus medi- us et minimus – Weichteilgewebe dar. In . Tab. 2 sind die Zugänge den verschiedenen Mus- kelgruppen zugeordnet, die durch den Zugang tangiert werden können. Aus unserer Sicht ist der DAA besonders ge- eignet, die Abduktoren des Hüftgelenkes zu schonen. Gerade beim alten Menschen ist das Gehen eine der entscheidendsten Funktionen und Voraussetzung für de- ren Mobilität im Alltag. Der Flexion/ Extension und Abduktion kommt hier wohl die wichtigste Bedeutung zu. Mus- kuläre oder neurogene Störungen der Extension oder Flexion als Folgen von Hüftendoprothesenoperationen treten vergleichsweise selten auf. Iatrogene Störungen des Gangmechanismus be- treffen am häufigsten die Abduktion. Hauptmuskeln sind hier M. glutaeus medius und minimus. Eine Schädigung kann durch eine direkte Zerstörung von Muskelgewebe erfolgen. Ebenso kann die Funktion durch eine Zerstörung der Knochenansätze dieser Muskeln auftre- ten, wenn etwa größere Knochendefekte oder Frakturen an der Spitze (Glutaeus minimus [G-min]) und/oder am la- teralen Aspekt des Trochanter major (Glutaeus medius [G-med]) entstehen. Beide Muskeln werden vom N. glutaeus superior innerviert. Eine Schädigung dieses Nervs resultiert in einer Parese der Abduktoren. Tab. Invasivität 2 Potenziell vom operativen Zugangsweg betroffene Muskelgruppen Flexion Extension Adduktion Abduktion Außenrotation Innenrotation DAA Lateral Dorsal Lateral Dorsal Dorsal DAA Lateral Dorsal Iliacus Glutaeus maximus Adduktoren Glutaeus medius Piriformis Tensor fasciae latae Psoas Glutaeus minimus Pectineus Glutaeus minimus Obturator externus Glutaeus me- dius Sartorius Glutaeus medius Gracilis Tensor fasciae latae Obturator internus Glutaeus mini- mus Rectus femoris Hamstrings – Glutaeus maximus Quadratus femoris – Tensor fascia lata – – Piriformis Glutaeus me- dius und mini- mus – DAA direkt anteriorer Zugang, Lateral antero/lateraler Zugang, Dorsal Dorsaler Zugang Tab. 2 Potenziell vom operativen Zugangsweg betroffene Muskelgruppen jSpaltung und Ablösung des M. vas- tus lateralis als Bestandteil des M. quadriceps femoris; Tab. 3 Mittelwert derOperationsdauerin Minutenüber2Operateure:Vergleichdirekt anteriorerZugang (DAA)–Bauer-Zugang (2004/2005) DAA Bauer-Zugang Jahr 1 82 ± 20 85 ± 28 Jahr 2 64 ± 17 70 ± 11 Tab. 3 Mittelwert derOperationsdauerin Minutenüber2Operateure:Vergleichdirekt anteriorerZugang (DAA)–Bauer-Zugang (2004/2005) DAA Bauer-Zugang Jahr 1 82 ± 20 85 ± 28 Jahr 2 64 ± 17 70 ± 11 4 anterolateraler Zugang: Eine Schädigung der Abduktions- funktion führt zu einer glutealen Insuffi- zienz. Im Gangzyklus ist es Aufgabe der Abduktoren, das Becken in der Phase des Einbeinstandes stabil zu halten, um ein Vorschwingen des kontralateralen Beines zu ermöglichen. Ist diese Funkti- on gestört, ist der Patient nicht mehr in der Lage, das Becken im Einbeinstand zu stabilisieren, Das Becken kippt ab, der Patient zeigt ein positives Trendel- lenburg-Zeichen. jIntervall zwischen M. tensor fasciae latae und M. glutaeus medius; 4 direkt anteriorer Zugang: jIntervall zwischen M. sartorius und M. rectus femoris (medial) und M. tensor fasciae latae lateral. sale (D) und der laterale transgluteale ZugangerfordernjeweilsdasSpaltenbzw. Ablösen von Muskeln und Muskelgrup- pen. In . Abb. 1 sind die anatomischen Portale, über die das Hüftgelenk erreicht werden kann, dargestellt: Die . Tab. 1 zeigt die relevanten Muskeln mit ihrer Funktion. Diese Muskeln werden in unter- schiedlicher Weise bei den verschiede- nen Zugängen tangiert. Einerseits kann eine Muskelschädigung direkt durch den operativen Zugang selbst erfolgen, wenn dieser die Durchtrennung, Ablösung oder Spaltung des Muskels notwendig macht. Andererseits ist die Muskulatur auch durch die Manipulationen während derOperationgefährdet.RotierendeFrä- sen, Raspeln oder Retraktoren stellen eine mechanische Belastung für das 4 dorsaler Zugang: Eine Glutealinsuffizienz bedeutet für denälterenPatienteneinemassiveBeein- trächtigung der Gehfähigkeit und macht häufig ein freies Gehen ohne Gehhilfe unmöglich. Darüber hinaus ist die Geh- strecke massiv reduziert [8]. 4 dorsaler Zugang: jSpaltung des M. glutaeus maximus, jAblösen der kurzen Außenrota- toren (M. obturatorius internus, M. Abstract the probability of dislocations. The direct anterior approach has some advantages; however, minimally invasive approaches should be used with care. Surgical experience, experience with a specific approach and the availability of specialized instruments as well as sufficient training are mandatory for a successful performance. A surgeon should always choose the approach with which he has most experience. There are a large number of different approa- ches to the hip joint. Several conventional and minimally invasive portals exist and numerous technical variations with different instrument sets are available. All approaches can be regarded as standard and can be utilized lege artis. The main differences between approaches are their potential to preserve muscles and avoid damage which would slow down rehabilitation and mobilization. In the elderly the preservation of abductor function is, in the authors’ opinion, of utmost importance. A well- functioning gluteal system provides a limp- free gait, supports joint stability and reduces Keywords Minimally invasive · Gluteal insufficiency · Dislocation · Joint stability · Surgery Nicht direkt rekonstruierbar sind chronischeSchädigungendesN.glutaeus superior und eine Parese der Gluteal- muskulatur. Die denervierten Muskeln zeigen nach einiger Zeit eine vollständi- ge fettige Atrophie. Die fettige Atrophie der Muskulatur ist in der Magnetreso- nanztomographie (MRT) gut darstellbar (. Abb. 4). termajormassiveSchmerzenverbunden. Hier kann mit einer Lappenüberdeckung nach unserer Erfahrung eine erhebliche SchmerzreduktionodergarSchmerzfrei- heit beim älteren Patienten erzielt wer- den, wobei häufig ein TFL-Lappen dafür ausreicht. bei auch nur eine Teilfunktionsverbesse- rung erwartet werden kann, da die bei- den Muskellappen schon aufgrund ih- rer Faserrichtung, Ursprünge und An- sätze rein biomechanisch nur eine Teil- funktion des ausgefallenen Abduktions- systems übernehmen können (. Abb. 5). Allerdings ist die Wiedererlangung ei- ner Teilfunktion der Abduktion für äl- tere Patienten oft eine große Erleichte- rung,daeinfreiesGehendannzumindest über kürzere Strecken möglich ist und zu Hause auf eine Gehhilfe verzichtet wer- den kann. Häufig sind mit Schädigun- gen der Glutealmuskulatur am Trochan- bei auch nur eine Teilfunktionsverbesse- rung erwartet werden kann, da die bei- den Muskellappen schon aufgrund ih- rer Faserrichtung, Ursprünge und An- sätze rein biomechanisch nur eine Teil- funktion des ausgefallenen Abduktions- systems übernehmen können (. Abb. 5). Allerdings ist die Wiedererlangung ei- ner Teilfunktion der Abduktion für äl- tere Patienten oft eine große Erleichte- rung,daeinfreiesGehendannzumindest über kürzere Strecken möglich ist und zu Hause auf eine Gehhilfe verzichtet wer- den kann. Surgical access routes to the hip joint in the elderly Surgical access routes to the hip joint in the elderly Surgical access routes to the hip joint in the elderly Surgical access routes to the hip joint in the elderly Zusammenfassung · Abstract Zusammenfassung · Abstract Operative Zugangswege zur Hüfte beim älteren Menschen Operative Zugangswege zur Hüfte beim älteren Menschen Zusammenfassung Orthopäde 2017 · 46:18–24 DOI 10.1007/s00132-016-3366-2 © Der/die Autor(en) 2016. Dieser Artikel ist eine Open-Access-Publikation. Muskelansätze versucht werden. Im Hin- blick auf die Schwere der Beeinträch- tigung sollte dies so früh wie möglich versucht werden, wenn der Ansatz des G-medbetroffenist.DasSpektrumreicht hier von einer Muskel- bzw. Fasziennaht oder dem Versuch einer knöchernen Re- Insertion bis hin zur Osteosynthese bei Frakturen des Trochanter major (TM). Zu beachten ist hier, dass der relevantere G-medanderAußenseitedesTMansetzt und nur bei großen Trochanterdefekten betroffen ist. Bei den häufigeren, kleinen Trochanterspitzenfrakturen ist dieser in derRegelnichttangiert.Die. Abb.2zeigt ein Beispiel aus der Praxis, in dem es zu einerFrakturdesTMbeieinerRevisions- operation kam. Der TM wurde mit einer Krallenplatte refixiert und in situ gehal- ten. Der Verlauf und Ansatz des G-med sind farblich gekennzeichnet. Zusammenfassung · Abstract Orthopäde 2017 · 46:18–24 DOI 10.1007/s00132-016-3366-2 © Der/die Autor(en) 2016. Dieser Artikel ist eine Open-Access-Publikation. Zusammenfassung vermindert die Luxationswahrscheinlichkeit. Der direkt anteriore Zugang bringt hier einige wesentliche Vorteile. Allerdings sollte ein minimalinvasiver Zugang nicht kritiklos eingesetzt werden. Chirurgische Erfahrung, Erfahrung mit dem Zugang und die Verfügbarkeit von einem kompletten Instrumentenset sowie die Beherrschung desselben sind Grundvoraussetzung für einen erfolgreichen Einsatz. Grundsätzlich sollte ein Chirurg daher jenen Zugang wählen, mit dem er die größte Erfahrung hat. vermindert die Luxationswahrscheinlichkeit. Der direkt anteriore Zugang bringt hier einige wesentliche Vorteile. Allerdings sollte ein minimalinvasiver Zugang nicht kritiklos eingesetzt werden. Chirurgische Erfahrung, Erfahrung mit dem Zugang und die Verfügbarkeit von einem kompletten Instrumentenset sowie die Beherrschung desselben sind Grundvoraussetzung für einen erfolgreichen Einsatz. Grundsätzlich sollte ein Chirurg daher jenen Zugang wählen, mit dem er die größte Erfahrung hat. Die Anzahl der Zugänge zum Hüftgelenk ist vergleichsweise groß. Es gibt mehrere konventionelle und minimalinvasive Portale und zahlreiche technische Variationen mit unterschiedlichen Instrumentensets. Alle Zugänge sind als Standardzugänge zu bezeichnen und können lege artis eingesetzt werden. Die Unterschiede zwischen den Zugängen liegen in ihrem Potenzial, Muskeln zu schonen und Schädigungen sowie nachfolgende Rehabilitations- und Mobilisationsprobleme zu verhindern. Beim älteren Menschen steht aus Sicht der Autoren besonders die Erhaltung des Abduktionsap- parates im Vordergrund. Eine funktionsfähige Glutealmuskulatur erlaubt ein hinkfreies Gehen, unterstützt die Gelenkstabilität und Schlüsselwörter Minimalinvasiv · Gluetalinsuffizienz · Luxation · Gelenkstabilität· Chirurgie Schlüsselwörter Minimalinvasiv · Gluetalinsuffizienz · Luxation · Gelenkstabilität· Chirurgie Nicht unbedingt bei Primäroperatio- nen, aber in der Folge bei Revisionsope- rationen kann es zu einer völligen Zer- störung oder notwendigen Entfernung des proximalen Femurs kommen. In sol- chenFällenfehltnunjeglicherAnsatzder Glutealmuskulatur am Knochen. Solche Fälle, wie in . Abb. 3 dargestellt, führen unausweichlich zur glutealen Insuffizi- enz, da die Muskulatur nur an der late- ralen Narbenplatte des Weichteilgewebes inseriert werden kann. Da diese Situati- onauchmiteinemhohenLuxationsrisiko verbunden ist, wurde für diese Szenarien eine Dual-mobility-Pfanne mit mobilem PE-Inlay entwickelt und von uns erfolg- reich angewendet (z. B. Trident MDM®, STRYKER, Mahwah, NJ, USA). Invasivität gemellus superior/gemellus inferior); 4 lateraler, transglutealer Zugang: Eine orthetische Verbesserung der Si- tuation ist nicht möglich. Bei rein me- chanischen Schädigungen der Muskula- tur kann chirurgisch eine Refixation der 4 lateraler, transglutealer Zugang: jSpaltung und Ablösung des M. glutaeus medius, jSpaltung und Ablösung des M. glutaeus medius, 19 Der Orthopäde 1 · 2017 Der Orthopäde 1 · 2017 Erfahrung Wenn auch mehrere Zugänge zur glei- chen Operation zur Verfügung stehen, so sollte die Auswahl doch einigen ob- jektiven Kriterien unterliegen. Dies ist aus unserer Sicht in der Primärendopro- thetik zunächst nicht alleine eine anato- misch technische Überlegung, sondern die Frage nach der Erfahrung des Ope- rateurs. In Zeiten des Internets und gut vorinformierter Patienten ist dies nicht immer einfach zu vermitteln, da Patien- ten häufig mit dem dezidierten Wunsch nacheinembestimmten,besonderenZu- gang – vielleicht sogar besonders bewor- benen Zugang – kommen. Muskelscho- nende Zugänge sind bei Patienten aller Altersgruppenzubevorzugen.Allerdings ist eine Umstellung des eigenen Stan- dardzugangs eine Herausforderung, die nur gut vorbereitet und mit mit einer entsprechendenLernkurvedurchgeführt werden kann. Eine entsprechende Aus- bildung ist hier unbedingt erforderlich, um Patienten diese Verfahren mit der nötigen Sicherheit anbieten zu können. nitionsgemäß keinen direkten Zugangs- schaden im Glutealbereich. Allerdings befindet sich im Intervall des AL zwi- schen Tensor fasciae latae (TFL) und G-med etwa 4 cm proximal der Tro- chanterspitze ein kreuzender Ast des N. glutaeus superior, der direkt durch den Zugang gefährdet wird. Dies ist zwar nicht der Hauptast, eine Teilschädigung des Nervs kann jedoch zur Denervie- rung von Anteilen (anterior) des G-min führen. In dieser Hinsicht ist der DAA überlegen. In seinem Intervall liegen keine motorischen Nervenbahnen. Der Glutaeusmuskel ist durch den Tensor fasciae latae geschützt. Abstract Häufig sind mit Schädigun- gen der Glutealmuskulatur am Trochan- Der verbreiterte laterale Zugang schä- digt die Glutealmuskulatur zugangsbe- dingt direkt. Der dorsale Zugang ver- meidet eine direkte Schädigung von G-med/G-min, setzt aber zwangsläufig einen Schaden am G-max. Eine vollständige Rekonstruktion ist in diesen Fällen nicht möglich. Zur Ver- besserung der Stabilisierung ist eine Lap- penplastik eines Anteils des M. glutaeus maximus (G-max) [9] evtl. in Kombina- tion mit einem sog. Tensor-fasciae-latae (TFL)-Lappen möglich [10]. Die Opera- tion ist aufwendig und ausgedehnt, wo- Beide rein intermuskulären Zugänge (DAA und anterolateraler) setzen defi- 20 Der Orthopäde 1 · 2017 Abb. 2 8 Refixierung eines abgerissenen Tro- chantermajormittels einerKrallenplatte. Der AnsatzdesGlutaeusmediusliegtlateralamTro- chantermajor Abb. 3 8 Das Röntgenbilddes Hüftgelenkes zeigt ein völliges Fehlen des proximalenFemurs einschließlich des Trochantermajor » Bei älteren Patienten sollte die Luxationssicherheit höchste Priorität haben Gracia-Rey et al. [14] haben erst kürz- lich auf die Bedeutung der Abdukto- ren in der Luxationsprävention hinge- wiesen. Der DAA bietet hier eine be- sonders sichere Alternative und hat ge- genüber dem luxationsanfälligen dorsa- len Zugang massive Vorteile. Luxationen sindbeikonsequenterSchonungderkur- zen Außenrotatoren, der hinteren Kap- sel und der Glutealmuskulatur durch den DAA eine seltene Komplikation. Auswahl des Zugangs Wie in den vorangegangenen Abschnit- ten dargestellt, gibt es mehrere grund- sätzliche Zugangswege zum Hüftgelenk. Diese Zugänge sind in zahlreichen Vari- anten und technischen Variationen be- schrieben. Ebenso gibt es konventionelle und minimalinvasive Konzepte für die- se Zugänge. Es liegt also am Chirurgen, den „richtigen“ Zugang für eine Endo- prothesenversorgung auszuwählen. Im- mer dann, wenn es eine große Auswahl gibt, sind die Auswahlkriterien schwie- rig zu definieren, da Vor- und Nachteile, Schwierigkeiten und Herausforderungen unterschiedlich sind, sich aber teilweise auch überschneiden. Wenn auch keine eindeutigen Vorgaben aus dem derzeiti- gen Stand des Wissens zu machen sind, so glauben wir doch, dass es sinnvoll ist, einige grundsätzliche Überlegungen an- zustellen. Abb. 3 8 Das Röntgenbilddes Hüftgelenkes zeigt ein völliges Fehlen des proximalenFemurs einschließlich des Trochantermajor Abb. 2 8 Refixierung eines abgerissenen Tro- chantermajormittels einerKrallenplatte. Der AnsatzdesGlutaeusmediusliegtlateralamTro- chantermajor offen oder geschlossen in Narkose ein relevantes Risiko dar. Daher sollte in dieser Patientengruppe die Luxations- sicherheit des verwendeten Verfahrens höchste Priorität haben. Neben Maß- nahmen in der Auswahl des verwen- deten Prothesenkonzeptes (Großkopf, „dual mobility“) [11, 12] und Implan- tatpositionierung (Pfanneninklination/ Anteversion, Schafttorsion) sind die Zugänge mit unterschiedlichen Luxati- onswahrscheinlichkeiten assoziiert. Mit dem DAA ist die Erhaltung der Ab- duktorfunktion gut möglich und die Luxationsrate reduziert [13]. Luxationen Postoperative Luxationen der Hüftendo- prothese sind bei älteren Menschen eine wichtige Komplikation, die es zu ver- meiden gilt. Neben den mit der Luxation verbundenen Schmerzen ist die aus dem Luxationserlebnis resultierende Unsi- cherheit besonders belastend und für die Mobilisierung des alten Menschen einschränkend. Beim älteren Patienten stellt auch die Reposition des Gelenkes Muskelschonung Es gibt heute keinen Grund mehr, aus- gedehnte Zugänge zum Hüftgelenk an- zuwenden. Minimalinvasive oder – bes- ser–,„muskelschonende“Verfahrensind mittlerweile grundsätzlich etabliert. Die- se Zugänge werden gelehrt und in vielen Zentren erfolgreich angewandt. Gerade ältere Patienten profitieren von der Re- Implantatwahl 4 Platten am Femur, die weiter distal liegen und über einen lateralen Zugang entfernt werden müssen, stimmten, speziellen Instrumenten. Dies inkludiert in aller Regel ein komplexes Set aus allgemeinen und prothesenspe- zifischen Instrumenten. Hinzu kommen Retraktoren, die üblicherweise auf das Verfahren abgestimmt sind. Erfahrungs- gemäß funktionieren Operationstechni- kennurdannreibungslos,wennalleabge- stimmtenInstrumente korrekteingesetzt werden. Dies bedeutet, dass für ein spe- zielles Zugangsverfahren auch die geeig- neten Instrumente vollständig zur Ver- fügung stehen müssen. Allerdings nicht nur für den Standardfall, sondern auch für intraoperativ eintretende Variationen oder Komplikationen. So ist etwa zum Setzen einer Bohrung in der Pfanne bei den minimalinvasiven Zugängen ein ge- winkeltes Instrumentarium Grundvor- aussetzung und sollte auch dann zur Ver- fügung stehen, wenn nicht grundsätzlich eine Verschraubung der Pfanne geplant ist, sondern dies nur in speziellen Fällen durchgeführt wird. Die Wahl des Implantates richtet sich in einer idealen Welt nach der anatomisch- biomechanischen Konfiguration des Ge- lenkes des Patienten. In der Realität ha- ben wir ein Set von vorausgewählten Im- plantatsystemen zur Verfügung. Aus die- sem wird das für den vorliegenden Fall am besten geeignete System für den Pati- enten ausgewählt. Auch hier scheint uns die Erfahrung des Operateurs mit dem System ein wesentlicher Parameter der Auswahl zu sein. Im Hinblick auf den Zugang ist die Implantatwahl wichtig. Einerseits bestimmen Form und Größe desImplantatesdenbenötigtenZugangs- undPräparationsweg,andererseitsgehen mit der Wahl des Implantates auch Ein- schränkungen aufseiten der Instrumen- tarien einher. 4 dorsale Verplattungen des Azeta- bulums, die nicht belassen werden können, da etwa die Schrauben weit in das Azetabulum hineinragen, 4 intramedulläre Verknöcherungen nach Umstellungsosteotomien, die das Auffräßen mit langen, geraden Instrumentarien erfordern, 4 Hautläsionen, die im Zugangsgebiet liegen, 4 Tattoos, die der Patient erhalten möchte und die im Zugangsgebiet liegen. » Der Operateur sollte den Zugang wählen, mit dem er die größte Erfahrung hat Abb. 5 8 Kreuzlappenplastik zurBehandlung einerglutealen Insuffizienz beiAbduktoren- atrophie.GM Gluteus maximus In der Primärendoprothetik sind auch in der Literatur alle Zugänge zum Hüftge- lenk gut belegt. Insofern ist jeder Zugang anwendbar.AllerdingsgibtesspezielleSi- tuationen, welche die Zugangswahl be- einflussen können: kommt den Herstellern eine besondere Verantwortung zu. Es sollte auch hier Erfahrung mit dem verwendeten System bestehen. Abb. 4 8 Bei Zustandnach Hüfttotalendopro- these zeigen sich M. glutaeus medius undmini- mus komplett atrophiert 4 Marknägel, die entfernt werden müssen und lateral eingebracht wurden, Instrumentarium Zugänge, v. a. moderne minimalinvasive Zugänge, sind häufig abhängig von be- 21 Der Orthopäde 1 · 2017 Leitthema sind uns bewusst, dass man auch argu- mentieren könnte, dass die Konfigurati- on des Patienten und die Besonderheiten den Zugang diktieren sollten. Aus eige- ner Erfahrung und aus der Erfahrung des Lehrens von Zugängen sind wir je- doch überzeugt, dass es aufwendig ist, verschiedene Zugänge gleichermaßen zu beherrschen,undlangeLernkurvenbeim Erlernen eines neuen Verfahrens beste- hen. Insofern, sind wir der Auffassung, dass ein Operateur im Rahmen seiner größten Erfahrung agieren sollte, wann immer es möglich ist. Abb. 4 8 Bei Zustandnach Hüfttotalendopro- these zeigen sich M. glutaeus medius undmini- mus komplett atrophiert Abb. 5 8 Kreuzlappenplastik zurBehandlung einerglutealen Insuffizienz beiAbduktoren- atrophie.GM Gluteus maximus » Der Operateur sollte den Zugang wählen, mit dem er die größte Erfahrung hat Schlussfolgerung Es gibt eine große Anzahl von Zu- gangswegen und -techniken für die Prothesenversorgung des Hüftgelenkes. Der ältere Mensch benötigt eine sichere Operation mit minimalem Muskeltrau- ma, eine rasche Rehabilitation und ein geringes Luxationsrisiko. Minimalinva- sive Zugänge, insbesondere der DAA, sind neben der Auswahl des Implan- tatsystems wesentliche Faktoren für das Umsetzen dieser Forderungen. Open access funding provided by University of Innsbruck and Medical University of Innsbruck. Einhaltung ethischer Richtlinien Interessenkonflikt. M.Noglergibtan,dasserals BeraterfürSTRYKERundDJO tätigist.M.Thalergibt an,dasskeinInteressenkonfliktbesteht. DieserBeitragbeinhaltetkeinevondenAutoren durchgeführtenStudienanMenschenoderTieren. Open AccessDieserArtikelwirdunterderCreative CommonsNamensnennung4.0InternationalLizenz (http://creativecommons.org/licenses/by/4.0/deed. de)veröffentlicht,welchedieNutzung,Vervielfäl- tigung,Bearbeitung,VerbreitungundWiedergabe injeglichemMediumundFormaterlaubt,sofern Sieden/dieursprünglichenAutor(en)unddieQuelle ordnungsgemäßnennen,einenLinkzurCreativeCom- monsLizenz beifügenundangeben,obÄnderungen vorgenommenwurden. Patientenspezifische Überlegungen Es mag erstaunen, dass wir Patienten- spezifisches nicht an erster Stelle in Be- zug auf die Zugangswahl anführen. Wir Über die Verfügbarkeit hinaus soll- te die Anwendung des speziellen Instru- mentariums auch eingeübt sein. Hierbei 22 Der Orthopäde 1 · 2017 4 Minimalinvasive Zugänge sind gut etabliert. Der DAA erlaubt es, die Glutealmuskulatur zu schonen und Luxationen zu vermeiden. Operationszeit Die Operationszeitangaben in der Lite- ratur für eine primäre Hüftendoprothese schwanken in Abhängigkeit von Kom- plexität des Falles, Verfahren und ope- rativer Erfahrung. Die Operationsdauer ist ein relevanter Parameter im Hinblick auf perioperative Parameter wie Blutver- lust und Infektionsgefahr – Parameter, diebeimälterenMenschenbesondersbe- deutsam sind. Im Rahmen einer eigenen Studie aus dem Jahr 2005, am Beginn unserer Erfahrung mit dem DAA, zeig- te sich einerseits, dass es bei gleichen Operateuren zwar keine Unterschiede in der mittleren Operationsdauer zwischen einzelnen Verfahren gab, es konnte aber nachgewiesen werden, dass die Operati- onsdauer während des Studienzeitraums beidengleichenOperateurendurchfort- gesetztes Üben reduziert werden konnte (. Tab. 3). Fazit für die Praxis 4 Schonung der Weichteilstrukturen ist für den älteren Patienten besonders wichtig. 4 SchädigungenderGlutealmuskulatur führen zu massiven Problemen in der Rehabilitation, schränken die Gehfähigkeit bis hin zu ihrem Verlust massiv ein. Die oben genannten Daten sind über 10Jahrealt.DerDAAwurdebeiuns2002 eingeführt, diese Studie wurde 2 Jahre nach Einführung des direkten anterio- ren Zugangs bei uns begonnen. Aktu- ell lag die durchschnittliche Operations- dauer der letzten 200 konsekutiven pri- mären Hüftendoprothesen eines der Au- toren (MN) bei 55 min (Min 22, Max 133 – mithin eine Stabilisierung der Opera- tionsdauer deutlich unter 1 h). Gerade beim älteren Menschen ist es wichtig, die BelastungdurchdieOperation–undhier ist die Dauer der Narkose ein wesentli- cher Aspekt – gering zu halten. Korrespondenzadresse Korrespondenzadresse duktion des Zugangsschadens und der mittlerweile gut belegten rascheren Re- habilitation. Aus unserer Sicht ist der DAA ein besonders schonender Zugang. Univ. Prof. Mag. Dr. M. Nogler, MAS, MSc. Experimentelle Orthopädie, Medizinische Universität Innsbruck Innrain 36, 6020 Innsbruck, Österreich Michael.nogler@i-med.ac.at Open access funding provided by University of Innsbruck and Medical University of Innsbruck. Univ. Prof. Mag. Dr. M. Nogler, MAS, MSc. Experimentelle Orthopädie, Medizinische Universität Innsbruck Innrain 36, 6020 Innsbruck, Österreich Mi h l l @i d t 2. Bauer R, Kerschbaumer F, Poisel S, Oberthaler W (1979) The transgluteal approach to the hip joint. ArchOrthopTraumaSurg95(1–2):47 1. BauerGJ,SarkarMR(1997)Injuryclassificationand surgicalapproachinhipdislocationsandfractures. Orthopäde26(4):304 Infobox Operationsvideos zum Thema Eine wesentliche Frage in der Wahl ei- nes Zugangs ist die nach der Erweiter- barkeit. Grundsätzlich sind alle Zugänge erweiterbar – sowohl proximal hin zum Becken als auch distal entlang des Ober- schenkels. Wir haben diese kürzlich auch imHinblickaufdenDAAgezeigt,derwie die anderen Zugänge auch für komple- xe Erweiterungen in beide Richtungen eingesetzt werden kann [15]. Eine we- sentliche Einschränkung ergibt sich je- doch aus der Lagerung des Patienten. In Rückenlage sind Becken und Hüfte von dorsalnichterreichbar.EineErweiterung des Zugangs ist ohne Umlagerung nicht möglich. (Registrierung erforderlich!) Allgemein: https://www.vumedi.com Direkt anteriorer Zugang (DAA): https://www. vumedi.com/video/direct-anterior-approach- total-hip-arthroplasty/ Crossflap: https://www.vumedi.com/video/ cross-flap-technique-for-the-treatment-of- gluteal-insufficiency/ (Registrierung erforderlich!) Allgemein: https://www.vumedi.com Direkt anteriorer Zugang (DAA): https://www. vumedi.com/video/direct-anterior-approach- total-hip-arthroplasty/ Crossflap: https://www.vumedi.com/video/ cross-flap-technique-for-the-treatment-of- gluteal-insufficiency/ 4 Minimalinvasive Zugänge benötigen spezialisiertes Instrumentarium und erfordern Training. 4 Bei der Wahl des Zugangs sollte die eigene Erfahrung eine wesentliche Rolle spielen. 3. Bertin KC, Rottinger H (2004) Anterolateral mini- incision hip replacement surgery: a modified Watson-Jones approach. Clin Orthop Relat Res 429:248. doi:10.1097/01.blo.0000150294.81825. 8c Ausschreibung Oskar-Medizinpreis 2017 withlowerdislocationriskwithouthigherrevision risk.ClinOrthopRelatRes473(11):3409 withlowerdislocationriskwithouthigherrevision risk.ClinOrthopRelatRes473(11):3409 withlowerdislocationriskwithouthigherrevision risk.ClinOrthopRelatRes473(11):3409 6. Dienstknecht T, Luring C, Tingart M, Grifka J, Sendtner E (2014) Total hip arthroplasty through the mini-incision (Micro-hip) approach versus the standard transgluteal (Bauer) approach: a prospective, randomised study. J Orthop Surg (HongKong)22(2):168 Die Stiftung Oskar-Helene-Heim för- dert die Wissenschaft und Forschung auf dem Gebiet der Medizin. Als be- sonderes Förderprojekt verleiht die Stiftung jährlich den mit 50.000 € dotierten Oskar Medizin-Preis. Mit diesem Medizinpreis werden hervor- ragende Leistungen gewürdigt und die Weiterführung von Forschungen unterstützt. 7. Sendtner E, Borowiak K, Schuster T, Woerner M, Grifka J, Renkawitz T (2011) Tackling the learning curve: comparison between the anterior, minimally invasive (Micro-hip(R)) and the lateral, transgluteal(Bauer)approachforprimarytotalhip replacement.ArchOrthopTraumaSurg131(5):597 Der prämierte Erkenntnisgewinn soll für eine breite Öffentlichkeit vermittelbarsein. 4 Dem Antrag sollen der Lebenslauf, die wichtigsten Arbeiten aus den letzten drei Jahren zum Thema „Prävention durch Bewegung“ (Arbeiten, die bereits eine anderweitige Prämierung erhalten haben, sind ausgeschlossen) sowie eine inhaltliche Zusammenfassung der Forschungsergebnisse ergänzt um die weitere Forschungsplanung beigefügt werden. 4 Dem Antrag sollen der Lebenslauf, die wichtigsten Arbeiten aus den letzten drei Jahren zum Thema „Prävention durch Bewegung“ (Arbeiten, die bereits eine anderweitige Prämierung erhalten haben, sind ausgeschlossen) sowie eine inhaltliche Zusammenfassung der Forschungsergebnisse ergänzt um die weitere Forschungsplanung beigefügt werden. 8. HarrasserN,BankeI,GollwitzerH,WilkenF,Toepfer A, von Eisenhart-Rothe R, Hauschild M (2016) Glutealinsufficiency:pathogenesis,diagnosisand therapy.ZOrthopUnfall154(2):140 9. Whiteside LA (2012) Surgical technique: transfer of the anterior portion of the gluteus maximus muscle for abductor deficiency of the hip. Clin OrthopRelatRes470(2):503 Literatur 1. BauerGJ,SarkarMR(1997)Injuryclassificationand surgicalapproachinhipdislocationsandfractures. Orthopäde26(4):304 4 Wenn die Glutealinsuffizienz auf Abrissen der Knochenansätze der Muskulatur beruht, sollte eine chirur- gische Revision rasch erfolgen. 4 Ein Ersatz der atrophierten Gluteal- muskulatur ist teilweise durch eine Kreuzlappenplastik aus TFL und dem anterioren Anteil des G-max möglich. 3. Bertin KC, Rottinger H (2004) Anterolateral mini- incision hip replacement surgery: a modified Watson-Jones approach. Clin Orthop Relat Res 429:248. doi:10.1097/01.blo.0000150294.81825. 8c 4 Besonderes Augenmerk sollte auf die Vermeidung von Luxationen gerichtet sein. Die Wahl des Zugangs kann hier einen wesentlichen Beitrag leisten. 4. Rachbauer F (2005) Minimally invasive total hip arthroplasty via direct anterior approach. Orthopäde34(11):1103 5. Clyburn TA (2015) CORR insights®: anterior and anterolateral approaches for THA Are associated 23 Der Orthopäde 1 · 2017 Fachnachrichten Im Jahr 2017 wird dieser Medizinpreis auf dem Gebiet der Orthopädie und Unfallchirurgie ausgeschrieben. Im Jahr 2017 wird dieser Medizinpreis auf dem Gebiet der Orthopädie und Unfallchirurgie ausgeschrieben. 10. Nogler M, Schwabegger A (2012) Crossed muscular flap technique for the treatment gluteal insufficiency.AAOSAnnualMeeting,SanFrancisco. Regelmäßige körperliche Aktivität ist nach- weislich gesundheitsfördernd und wird als integraler Bestandteil eines gesunden Le- bensstils angesehen. Die Arbeit im Büro, die bevorzugte Freizeitgestaltung – all das findet überwiegend im Sitzen statt. Ein briti- scher Gesundheitsexperte bezeichnet dieses Verhalten als „Gehen Mangel-Syndrom“. ZEIT online 2012 nennt für das Sitzen einen Durchschnittswert von 11,5 Stunden pro Tag. Dieses „Vielsitzen“ verkürzt die Rücken- muskulatur, schwächt Bauch-, Bein- und Ge- säßmuskeln. und verursacht Rückenleiden, Muskel- und Gelenkerkrankungen. Bewe- gung erhalten und verlorene Bewegung wie- derherstellen wird als eine Kernaufgabe der Orthopädie und Unfallchirurgie angesehen. Dieser Anforderung stellt sich der Deutsche Kongress für Orthopädie und Unfallchirurgie 2017 mit dem Motto „Bewegung ist Leben“. 11. HailerNP,WeissRJ,StarkA,KarrholmJ(2012)Dual- mobility cups for revision due to instability are associated with a low rate of re-revisions due to dislocation: 228 patients from the Swedish Hip ArthroplastyRegister.ActaOrthop83(6):566 4 Das Preisgeld ist für Forschungszwecke nach der freien Entscheidung des Preisträgers zu verwenden. Der Preisträger hat der Stiftung diese Verwendung in geeigneter Weise zu belegen. 12. JacobsJJ(2012)Large(36or40-mm)femoralheads decreasedtherateofdislocationafterrevisiontotal hiparthroplasty.JBoneJointSurgAm94(22):2095 13. Tsukada S, Wakui M (2015) Lower dislocation rate following total hip arthroplasty via direct anterior approach than via posterior approach: five-year- averagefollow-upresults.OpenOrthopJ9:157 4 Der Antrag ist bis zum 31. Mai 2017 bei der Stiftung Oskar-Helene-Heim, Walterhöferstr. 11, 14165 Berlin einzureichen (info@stiftung-ohh.de). 14. Garcia-Rey E, Garcia-Cimbrelo E (2016) Abductor Biomechanics Clinically Impact the Total Hip ArthroplastyDislocationRate:AProspectiveLong- TermStudy.JArthroplasty31(2):484 Weitere Informationen erteilt der Geschäfts- führer der Stiftung, Werner Ukas (wer- ner.ukas@stiftung.de, Tell. 030/8102-1100). 15. Nogler M, Thaler M (2016) The Direct Anterior Approach for Hip Revision: Accessing the entire femoraldiaphysiswithoutendangeringthenerve supply.JArthroplasty.doi:10.1016/j.arth.2016.07. 044 ZweckderStiftungOskar-Helene-Heimistdie Förderung von Wissenschaft und Forschung in der Medizin,insbesondere der Orthopädie, der Lungenheilkunde,Gastroenterologie und Viszeralchirurgie sowie der Orthopädietech- nik. Zu diesem Zweck unterstütztdie Stiftung Forschungsprojekte oder sonstige gemein- nützige gesundheitsfördernde Vorhaben, verleiht Stipendien und vergibt jährlich den Oskar-Medizinpreis und die Helene-Medaille. Zur Verbesserung von Diagnostik und The- rapie soll mit dem Oskar Medizinpreis 2017 ein/e Mediziner/in oder Wissenschaftler/in ausgezeichnet werden, die/der zum Thema „Prävention durch Bewegung“ besonders relevante Ergebnisse der Grund- lagen- und/oder klinischen Forschung in Deutschland erzielt hat. Fachübergreifende Arbeitsgruppen sind ebenfalls zur Bewer- bung zugelassen. Quelle: Stiftung Oskar-Helene-Heim, www.stiftung-ohh.de Quelle: Stiftung Oskar-Helene-Heim, www.stiftung-ohh.de 24 Der Orthopäde 1 · 2017 24 24 Der Orthopäde 1 · 2017
https://openalex.org/W3025680686
https://www.cambridge.org/core/services/aop-cambridge-core/content/view/C9730961E7C7C37AA5E204A3EDD84965/S0008423920000517a.pdf/div-class-title-do-covid-19-conspiracy-theory-beliefs-form-a-monological-belief-system-div.pdf
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Do COVID-19 Conspiracy Theory Beliefs Form a Monological Belief System?
Canadian journal of political science
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© Canadian Political Science Association (l’Association canadienne de science politique) and/et la Société québécoise de science politique 2020. 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. Do COVID-19 Conspiracy Theory Beliefs Form a Monological Belief System? Joanne M. Miller* Political Science and International Relations, University of Delaware, 347 Smith Hall, 18 Amstel Ave., Newark, Delaware 19716, USA *Corresponding author. Email: jomiller@udel.edu Political Science and International Relations, University of Delaware, 347 Smith Hall, 18 Amstel Ave., Newark, Delaware 19716, USA *Corresponding author. Email: jomiller@udel.edu Political Science and International Relations, University of Delaware, 347 Smith Hall, 18 Amstel Ave., Newark, Delaware 19716, USA *Corresponding author. Email: jomiller@udel.edu Canadian Journal of Political Science (2020), 53, 319–326 doi:10.1017/S0008423920000517 Introduction Along with criticisms of the U.S. government’s response to the COVID-19 pandemic, the disruptions to home, work, and school life resulting from social dis- tancing orders recommended by public health experts, as well as the uncertainty about how long the disruptions will be necessary and when (if ever) we will have a vaccine, have come COVID-19 conspiracy theories (CTs). CTs are beliefs that powerful actors are engaging in wide-ranging secret (often illegal or ambiguously legal) activities for their own personal gain (Miller et al., 2016). Uscinski et al. (2020) find that 29 per cent of respondents in a recent, nation- ally representative sample agree that the threat of COVID-19 has been exaggerated to damage President Trump and 31 per cent agree that the virus was purposefully created and spread. A recent YouGov/The Economist poll found that 13 per cent believe it is a hoax. Such beliefs can have negative health effects; for example, Motta et al.’s (2020) data suggest that individuals who endorse conspiracy theories about COVID-19 are less likely to accept public health experts’ warnings about the severity of the crisis. The motives that give rise to CT beliefs—conspiratorial thinking (the tendency to view events as the product of a conspiracy; Uscinski and Parent, 2014), denialism (the tendency to deny official/authoritative accounts of events; Uscinski et al., 2020), partisan-motivated reasoning (the desire to bolster/protect one’s worldview; Miller et al., 2016; Pennycook et al., 2020), and uncertainty/powerlessness (van Prooijen and Acker, 2015)—are all certainly activated during the current pandemic. As such, it is not surprising that specific COVID-19 CTs are predicted by these motives (Uscinski et al., 2020). To gain a better understanding of how to counteract COVID-19 CTs, it is important not only to examine the predictors of individual CT beliefs, but also to explore their underlying structure. A robust finding in the study of CTs is that belief in one predicts belief in others (Goertzel, 1994). In essence, CTs form a https://doi.org/10.1017/S0008423920000517 Published online by Cambridge University Press Joanne M. Miller 320 “unitary, closed-off worldview in which beliefs come together in a mutually suppor- tive network known as a monological belief system” (Wood et al., 2012: 767). When CTs fit a similar theme, this makes sense: belief in one acts as “evidence” for another, and vice versa. However, Wood et al. Introduction (2012) demonstrate that people are quite willing to believe contradictory CTs (for example, that Osama Bin Laden is still alive and that he was already dead when the U.S. raid took place), and also that a belief system of even incompatible CTs is “held together” by the higher-order belief that government officials are often deceptive. To the extent that COVID-19 CTs—even contradictory ones—form a monolog- ical belief system, attempts at debunking them individually will likely fail. A more effective approach may be to tackle the higher-order needs/beliefs that give rise to the belief system in the first place. Higher order beliefs, such as conspiratorial thinking, denialism, and distrust of government, are certainly likely culprits. But in the context of a pandemic, uncertainty looms large. According to a Pew survey conducted March 19–24, 2020, 43 per cent of Americans reported feeling anxious most of the time or occasionally over the previous week. And as of May 16, 2020, the Civqs rolling cross-section survey indicates that 61 per cent of Americans are extremely or moderately worried about a coronavirus outbreak in their local area. Such situationally induced uncertainty may lead to the type of explanation- seeking that would cause people to accept multiple, even contradictory, CTs. This study has two goals: (1) to assess whether COVID-19 CTs that have become part of the pandemic discourse form a monological belief system and (2) to exam- ine whether personal uncertainty binds CT beliefs together. https://doi.org/10.1017/S0008423920000517 Published online by Cambridge University Press https://doi.org/10.1017/S0008423920000517 Published online by Cambridge University Press Material and Methods U.S. adults (n = 3,019) recruited via Lucid Theorem completed an online survey between April 24 and 28, 2020. Lucid provides a quota sample (using U.S. Census benchmarks) of individuals who have agreed to participate in scientific research. Data were also weighted based on the 2018 Current Population Survey (CPS) benchmarks for education, income, sex, race, and ethnicity to more accu- rately represent the U.S. population (see Appendix A, available online in the Supplementary Materials, for a comparison of sample characteristics with the CPS). pp y p p The survey assessed belief in the CTs described in Table 1 that have been recently circulating (Lynas, 2020). The order of the CTs was randomized across respondents. Responses were coded from 1–4 (definitely not; probably not; proba- bly; definitely); higher numbers equal greater belief. To examine the psychological/political correlates of COVID-19 CT beliefs, the survey included measures of partisanship, conspiratorial thinking, and uncertainty. Partisanship was coded into two dummy variables, one for Republicans/leaners vs. everyone else and one for pure Independents vs. everyone else. Democrats/leaners are the comparison group. Conspiratorial thinking was measured via a four-item scale (Uscinski et al., 2016). Personal uncertainty was measured via a three-item scale that assessed how uncertain people felt about themselves, their place in the world, and their future. See Appendix B, available online, for complete question wordings. All independent and control variables were recoded to range from 0–1. Canadian Journal of Political Science 321 Table 1 COVID-19 conspiracy theories Table 1 COVID-19 conspiracy theories Variable label Question wording BioChina The virus is a biological weapon intentionally released by China. AccChina The virus was accidentally released by China. AccUS The virus was accidentally released by the U.S. Scientists Scientists are exaggerating the seriousness to make President Trump look bad. Media The media are exaggerating the seriousness to make President Trump look bad. Hoard Democratic governors are hoarding ventilators to make President Trump look bad. Tests Democratic governors are not distributing coronavirus tests to make President Trump look bad. 5GTech 5 G technology is causing the coronavirus to spread faster. NotReal The coronavirus isn’t real. Gates Former Microsoft CEO Bill Gates is creating a tracking device to be injected with the coronavirus vaccine. WorldPop The coronavirus was intentionally created to reduce the world’s population. Question wording Figure 1 Distribution of COVID-19 CT beliefs. Results and Discussion COVID-19 CT Beliefs Are Widespread Figure 1 displays the distribution of responses to the 11 CT questions. Consistent with Uscinski et al. (2020), a striking percentage (between 3% and 19%) of respon- dents believe that each of these conspiracies is definitely occurring. Whereas only 8 per cent believe that the virus is probably (5%) or definitely (3%) not real, 49 per cent and 52 per cent report that they believe the virus was either probably or def- initely intentionally created (as a biological weapon) or accidentally released by China. Fewer people believe it was accidentally released by the U.S. or that the virus is not real. Figure 2 graphs the total number of CTs respondents endorsed. Approximately 85 per cent believe more than one; 60 per cent believe three or more, and 30 per cent believe at least six. Figure 2 The unweighted percentages of people who endorse multiple CTs, combining the probably and definitely response options. 322 Joanne M. Miller 322 Joanne M. Miller Figure 2 The unweighted percentages of people who endorse multiple CTs, combining the probably and definitely response options. p y BioChina AccChina AccUS Scientists Media Hoard Tests 5GTech Gates WorldPop NotReal BioChina * AccChina .43 * AccUS .27 .26 * Scientists .39 .27 .33 * Media .37 .30 .25 .63 * Hoard .46 .30 .34 .54 .58 * Tests .34 .22 .22 .42 .41 .52 * 5GTech .38 .24 .47 .43 .33 .41 .24 * Gates .40 .22 .31 .32 .30 .36 .25 .42 * WorldPop .56 .29 .39 .41 .36 .46 .33 .48 .39 * NotReal .14 .11 .27 .36 .28 .25 .26 .31 .18 .27 * BioChina AccChina AccUS Scientists Media Hoard Tests 5GTech Gates WorldPop NotReal https://doi.org/10.1017/S0008423920000517 Published online by Cambridge University Press COVID-19 CT Beliefs Form a Monological Belief System Not only are these beliefs widespread, but they are also interrelated. As Table 2 shows, the CTs are moderately to strongly positively correlated (all are statistically significant at p < .001). At the high end, the Scientists and Media CTs are correlated at .63. At the low end, the NotReal and AccChina CTs are correlated at .11. Given the floor effect in belief in the NotReal CT, it is not surprising that the correlations between it and the others are among the weakest. g To formally test whether the 11 COVID-19 CT beliefs form a monological belief system, a principal factor analysis (PFA) was performed on the correlation matrix. The PFA yielded one factor with an Eigenvalue greater than 1 (3.96); the proportion of variance explained by the factor is .96 (the Eigenvalue for the sec- ond factor is .50). All 11 CTs positively load on this single factor, with factor load- ings ranging from .40 to .73. Both the correlation matrix and the factor analysis provide strong support for the notion that the COVID-19 CTs are a monological belief system. https://doi.org/10.1017/S0008423920000517 Published online by Cambridge University Press Canadian Journal of Political Science 323 Figure 3 The unstandardized regression coefficients from ordinary least squares models that include controls for age, education, income, gender, race, and ethnicity. The dots represent the coefficients and the lines represent the 95 per cent confidence intervals. See Appendix C, available online, for the full models. Figure 3 The unstandardized regression coefficients from ordinary least squares models that include controls for age, education, income, gender, race, and ethnicity. The dots represent the coefficients and the lines represent the 95 per cent confidence intervals. See Appendix C, available online, for the full models. https://doi.org/10.1017/S0008423920000517 Published online by Cambridge University Press https://doi.org/10.1017/S0008423920000517 Published online by Cambridge University Press Personal Uncertainty Moderates the Effect of a CT Belief on a Contradictory CT Belief To examine whether the COVID-19 belief system is driven by personal uncer- tainty, uncertainty was interacted with AccChina, BioChina, and AccUS predict- ing one another (in separate models). If the belief system is held to together by the situational induction of personal uncertainty surrounding the pandemic, then the relationship between mutually contradictory CTs should be stronger for those higher in uncertainty than their counterparts. To display the shape of the interactions, Figure 4 show the effects AccChina and BioChina for people in the bottom and top quartiles of uncertainty. Consistent with expectations, this is exactly the case (results for the AccChina and BioChina dependent variables are similar; see Appendix D, available online). In sum, the COVID-19 belief system is a more tightly knit structure (that is, it is more constrained) for those who are currently experiencing a great deal of uncertainty, likely due to the pandemic itself. Contradictory CTs Are Positively Related, Even When Controlling for Standard Predictors On one hand, such a monological belief system could arise because the individual beliefs within it are mutually reinforcing. In other words, one CT belief becomes “evidence” for others (Goertzel, 1994). However, even the most obviously contra- dictory conspiracy theories are positively correlated, such as BioChina and AccUS (.27), AccChina and AccUS (.26), and BioChina and AccChina (.43). Fifteen per cent of respondents simultaneously believe that the BioChina and AccUS CTs are probably/definitely true; 15 per cent believe that both the AccChina and AccUS CTs are probably/definitely true, and 34 per cent believe that both the BioChina and AccChina CTs are probably/definitely true. These results hold in multivariate models controlling for standard predictors (see Figure 3; NotReal was not included in these models due to lack of variance; see Figure 1); mutually incompatible CTs are statistically significant, positive pre- dictors of one another. Specifically, endorsement of the theories that the virus was accidentally released by China, the virus was accidentally released by the U.S., and that it is a biological weapon intentionally released by China are statisti- cally significant, positive predictors of one another. People are quite willing and able to endorse contradictory COVID-19 CTs. Consistent with past research, conspiratorial thinking and uncertainty are statis- tically significant, positive predictors of all three CTs. Republicans are more likely pp BioChina at the lowest quartile of uncertainty is not sign on AccUS. Predicted values for the left panel are derived from Model 1 in online es of uncertainty are all statistically significant (b = .45, p < .001; b = .08, p = .05; a Model 2 in online Appendix C. The interaction and the effect of BioChina at th < .001). The effect of BioChina at the lowest quartile of uncertainty is not sign values for the left panel are derived from Model 1 in online all statistically significant (b = .45, p < .001; b = .08, p = .05; a pendix C The interaction and the effect of BioChina at t on AccUS. Predicte s of uncertainty a odel 2 in online A .001). The effect AccUS. Predict f uncertainty a el 2 in online 1). The effect Canadian Journal of Political Science 325 than Democrats to believe that the virus was accidentally released by China and that it is a Chinese biological weapon, and Independents are more likely to believe all three CTs than Democrats. Supplementary material. To view supplementary material for this article, please visit https://doi.org/10. 1017/S0008423920000517. Supplementary material. To view supplementary material for this article, please visit https://doi.org/10. 1017/S0008423920000517. Acknowledgements. The author thanks Erin Cassese and Matt Motta for feedback on an earlier draft of this manuscript. Acknowledgements. The author thanks Erin Cassese and Matt Motta for feedback on an earlier draft of this manuscript. Cite this article: Miller JM (2020). Do COVID-19 Conspiracy Theory Beliefs Form a Monological Belief System? Canadian Journal of Political Science 53, 319–326. https://doi.org/10.1017/S0008423920000517 References Goertzel, Ted. 1994. “Belief in Conspiracy Theories.” Political Psychology 15 (4): 731–42 Lynas, Mark. 2020. “COVID: Top 10 Current Conspiracy Theories.” Cornell Alliance for Science, Ap https://allianceforscience cornell edu/blog/2020/04/covid-top-10-current-conspiracy-theories/ (May 28 Lynas, Mark. 2020. “COVID: Top 10 Current Conspiracy Theories.” Cornell Alliance for Science, April 24. https://allianceforscience.cornell.edu/blog/2020/04/covid-top-10-current-conspiracy-theories/ (May 28, 2020). y , p p y f , p https://allianceforscience.cornell.edu/blog/2020/04/covid-top-10-current-conspiracy-theories/ (May 28, 2020 Miller, Joanne M., Kyle L. Saunders, and Christina E. Farhart. 2016. “Conspiracy Endorsement as Motivated Reasoning: The Moderating Roles of Political Knowledge and Trust.” American Journal of Political Science 60 (4): 824–44. Motta, Matt, Dominik Stecula, and Christina Farhart. 2020. “How Right-Leaning Media Coverage of COVID-19 Facilitated the Spread of Misinformation in the Early Stages of the Pandemic in the US.” Canadian Journal of Political Science/Revue canadienne de science politique. doi: 10.1017/ S0008423920000396. Pennycook, Gordon, Jonathon McPhetres, Bence Bago and David G. Rand. 2020. “Predictors of Attitudes and Misperceptions about COVID-19 in Canada, the U.K., and the U.S.A.” PsyArXiv. April 14. doi: 10.31234/osf.io/zhjkp. Wood, Michael J., Karen M. Douglas, and Robbie M. Sutton. 2012. “Dead and Alive: Beliefs in Contradictory Conspiracy Theories.” Social Psychological and Personality Science 3 (6): 767–73. Conclusion Taken together, these findings provide strong support for the theory that COVID-19 CTs form a monological belief system. Not only are the CTs highly cor- related, but a large majority believe more than one. Moreover, mutually contradic- tory CTs are positively related to one another, even when standard predictors of CT beliefs are taken into account. Finally, this belief system is more constrained for people who are currently experiencing a great deal of uncertainty. Two caveats to these conclusions are in order. First, a full accounting of the variety of COVID-19 CTs that are currently circulating was beyond the scope of this manu- script. It is possible that some of the ones out there that were not included in this study do not fit within this monological belief system. Second, it is possible that some of the CT responses represent expressive responding rather than true beliefs (Schaffner and Luks, 2018). As such, the distribution of responses to the CT questions may be biased upward. However, widespread expressive responding would likely attenuate relationships between CT beliefs and standard predictors evi- denced here. The breadth, and, for all intents and purposes, interchangeability of these beliefs may make attempts to debunk them by tackling each one individually akin to a futile game of whack-a-mole. Such attempts may be effective in the short term, but if the psychological needs that give rise to the beliefs in the first place remain intact, then a debunked CT will likely be replaced by another. A more effective, long-term strategy may be to address the political/psychological needs that give rise to the belief system in the first place. Alleviating situationally induced uncer- tainty, powerlessness, and concomitant anxiety (for example, van Prooijen, 2018) may be a promising antidote. 326 Joanne M. Miller 10.31234/osf.io/zhjkp. van Prooijen, Jan-Willem. 2018. “Empowerment as a Tool to Reduce Belief in Conspiracy Theories.” In Conspiracy Theories & the People Who Believe Them, ed. Joseph E. Uscinski. New York: Oxford University Press. van Prooijen, Jan-Willem and Michelle Acker. 2015. “The Influence of Control on Belief in Conspiracy Theories: Conceptual and Applied Extensions.” Applied Cognitive Psychology 29 (5): 753–761. Schaffner, Brian F., and Samantha Luks. 2018. “Misinformation or Expressive Responding? What an Inauguration Crowd Can Tell Us about the Source of Political Misinformation in Surveys.” Public Opinion Quarterly 82 (1): 135–47. Uscinski, Joseph E., Adam M. Enders, Casey M. Klofstad, Michelle Seelig, John Funchion, Caleb Everett, Stephan Wuchty, Kamal Premaratne and Manohar Murthi. 2020. "Why Do People Believe COVID- 19 Conspiracy Theories?" The Harvard Kennedy School (HKS) Misinformation Review. doi: 10.37016/ mr-2020-015. Uscinski, Joseph E., Casey Klofstad, and Matthew D. Atkinson. 2016. “What Drives Conspiratorial Beliefs? The Role of Informational Cues and Predispositions.” Political Research Quarterly 69 (1): 57–71. Uscinski, Joseph E., and Joseph M. Parent. 2014. American Conspiracy Theories. Oxford: Oxford University Uscinski, Joseph E., Casey Klofstad, and Matthew D. Atkinson. 2016. “What Drives Conspiratorial Beliefs? The Role of Informational Cues and Predispositions.” Political Research Quarterly 69 (1): 57–71. Uscinski, Joseph E., and Joseph M. Parent. 2014. American Conspiracy Theories. Oxford: Oxford University Press. Wood, Michael J., Karen M. Douglas, and Robbie M. Sutton. 2012. “Dead and Alive: Beliefs in Contradictory Conspiracy Theories.” Social Psychological and Personality Science 3 (6): 767–73. https://doi.org/10.1017/S0008423920000517 Published online by Cambridge University Press
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https://figshare.com/articles/journal_contribution/Supplementary_Table_4_from_Methylated_RASSF1a_Is_the_First_Specific_DNA_Marker_for_Minimal_Residual_Disease_Testing_in_Neuroblastoma/22443308/1/files/39894269.pdf
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Supplementary Table 2 from Methylated RASSF1a Is the First Specific DNA Marker for Minimal Residual Disease Testing in Neuroblastoma
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Supplementary table S4. RASSF1a methylation in control bone marrow samples (n=50) and clinical bone marrow samples (n=127) no. of triplicates amplified 0/3+ 1/3† 2/3§ 3/3‡ total BM Control 49 1* 0 0 50 stage 1-3 BM diagnosis 19 6** 1 0 26 stage 4s BM diagnosis 1 1** 2 2 6 stage 4 BM diagnosis 2 1** 3 39 45 BM follow up 25 4** 4 17 50 RASSF1aM + none of triplicate amplified; † one of triplicate amplified; § two of triplicate amplified; ‡ three of triplicate amplified; * Ct value 40.9; ** Ct values below arbitrary threshold of 39.9 Ct Supplementary table S4. RASSF1a methylation in control bone marrow samples (n=50) and clinical bone marrow samples (n=127) no. of triplicates amplified 0/3+ 1/3† 2/3§ 3/3‡ total BM Control 49 1* 0 0 50 stage 1-3 BM diagnosis 19 6** 1 0 26 stage 4s BM diagnosis 1 1** 2 2 6 stage 4 BM diagnosis 2 1** 3 39 45 BM follow up 25 4** 4 17 50 RASSF1aM + none of triplicate amplified; † one of triplicate amplified; § two of triplicate amplified; ‡ three of triplicate amplified; * Ct value 40.9; ** Ct values below arbitrary threshold of 39.9 Ct Supplementary table S4. RASSF1a methylation in control bone marrow samples (n=50) and clinical bone marrow samples (n=127) + none of triplicate amplified; † one of triplicate amplified; § two of triplicate amplified; ‡ three of triplicate amplified; * Ct value 40.9; ** Ct values below arbitrary threshold of 39.9 Ct
https://openalex.org/W3120283736
https://zenodo.org/records/1888193/files/article.pdf
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A Boat on Wheels
Scientific American
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public-domain
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SOME INTERESTING FEATURES OF THE KRUPP WORKS AT ESSEN. BY, BENRY L. GEIS8EL. One of the most attractive features at the Columbian Expo­ sition in Chicago was that of the great Krupp works. Since that time this firm has made no sim­ ilar €·xhibition, but it is just an­ nounced that a display, which will cost over a million dollars, is being arranged for the indus­ trial exposition to be held in Diisseldorf. There is no indus­ trial plant in the world the story of which is more interesting. This vehicle was built by Charles S. and Carl H. Hoyt, brothers, of Cleveland, 0., eight months ago, and has been con­ stantly in use since, running thousands of miles. Her own­ ers have a gold mine in the buttes near the station of Rosa­ mond, on the western border of the desert, and owing to lack of a suitable site they established their camp nine miles away. Between this place and the mine' is a remarkable dry lake. Its surface is as hard as concrete, and swept as smooth as a tennis court by the sands forever driven over it by the fierce winds rushing down through the Tehachepi Pass. While trudg­ ing wearily over this level tract, before a gale that almost blew them off their feet, one of the Hoyts suggested that if they had a wagon with sails they might make the trip easier and quicker. This idea was followed out and with surprising suc­ cess. Within a few German miles of the Rhine, north of the Ruhr, in fertile, undulating, yet not attractive country, surrounded by the most important coal mines in Germany, lies the town of Essen. With the exception of a time-honored cathedral, Es­ sen, despite its age, can boast of no historical relic of ancient days. The square before the town hall is embellished by a bronze monument from the mas­ ter hand of the sculptor Schaper, representing not a king or hero, but a man clad in a simple citizen's coat, whose right hand rests on an anvil, and whose pene­ trating eyes are overhung by a thinker's brow. The granite pedestal bears in golden letters the words: "ALFRED KRuPP " YACHTING ON THE GREAT DESERT. "ALFRED KRuPP." You tie a string to y to your suspender; your handke your neck and goes sailing and IN THE GUN SHOPS -away out of sight almost befo gone. The wind here is differ blew in any other part of the fairly fiying now and but a lit is filled with sand and pebble The town of Essen has erected this life-like statue in grateful commemoration for generations now and to come of her most distinguished son_ For this man, sprung from an' old and honorable family in Essen, within the time of half a generation, raised the small unknown country town to its present importance and celebrity. He did not sit in the Council of Aldermen, but in the small steel foundry, inherited from his fa­ ther, which employed hardly a dozen workmen. This steel foundry, however, rose to world-wide fame, and grew beyond all limits. At the time of Alfred Krupp's death, in 1887, the number' of employes and workmen of his works was 25,000. These are familiar scenes, and at first you notice them. Then the wind grows stronger and the pace madder. You tie a string to your hat and anchor it to your suspender; your handkerchief is whipped from your neck and goes sailing and writhing up and away On April 1, 1901, the number of hands employed in all the Krupp works, including 3,823 engineers and office men, amounted to 46.077. A few figures, for which the writer is indebted to the courtesy of the administration of the Krupp works (who also, by the way, sup­ plied the photographs reproduced with this. article), show the im­ mense extent of Krupp's establish ments. IN THE GUN SHOPS AT ESSEN. During the year 1900 the works at Essen con· sumed 937,172. tons of coal, or an average of 3,123 tons per day, rep­ resenting four railroad trains of 40 cars, each of a capacity of 20 tons. The other Krupp works consumed 655,125 tons, making a total of 1,592,- 296 tons, or 5,307 tons per day. The consump­ tion of water at the Es­ sen works, during the same year, amounted to 660,000,000 cubic feet, or equaled t h e annual water consumption of the city of Amsterdam. The gas consumption at the Essen works in 1900 was 665,000,000 cubic feEo1t, as compared with 660,000,000 cubic feet consumed in 1900 by the city of Breslau. SOME INTERESTING FEATURES OF THE KRUPP WORKS AT ESSEN. BY, BENRY L. GEIS8EL. arms outstretched to reach you; horned toad s scurry away over the hot sands, and lizards dart, looking like blue streaks, for the shelter, but not always quickly enough, for the "Queen's" wheels have crushed many before they could move; jackrabbits go skittering through the brush; and little ash-colored desert chip­ munks scatter the sand about in their frenzied haste to get into their retreats; now and then a coyote, long and gray and lean-the picture of starved want-rises upon his scraggy hind legs and sniffs; occasionally you will run over a deadly "sidewinder'" (rattlesnake) and hear the whirring of the rattles, or pass the bleaching bones of some poor creature that suffered the horrors of starvation and probably sucked the blood from its own parched tongue before the end came. With only saw and ax and hammer and knives for tools, the young men began the work of construction. The material available consisted of the odds and ends to be found around a mining camp. The first requisite was a support for the machine, and for this the axle of an ordinary worn-out buggy was used, two iron wheels 30 inches in diameter, which had done service on a farming implement, being attached. Other parts were improvised with similar skill and ingenuity, and after a month of diligent application the work­ men turned out a stanch "boat," 14 feet long, 8 feet across in front and tapering to the rear, with a mast 15. feet high, mainsail 10 feet on the boom and 10 feet on the mast, jib and jibboom to match. A steering contrivance like those on hook-and-ladder trucks was devised, and "Desert Queen" stood ready for her trial trip. The initial run was doomed to end in disaster. While tear­ ing along b e f o r e a strong wind at a ter­ rific rate the machine got beyond control and a sudden gust brought her to grief with a crash. Bruised men, bro k e n timbers and wrecked sails littered the ground. Neither of the Hoyts will ever for­ get· the experience, for they will always bear the marks of the cas­ ualty as a reminder. A BOAT ON WHEELS. BY JOHN L. VON BLON. The queerest ship that ever sailed is a yacht on wheels, a graceful land-going clipper, that glides over the pathless stretches of sun-blistered plain, and car­ ries her plucky navigators to and from their gold mine in the desert. Solitary gold hunters who have seen her white sails silhouetted against the bleak brown background in their aimless wanderings have brought to the outer world strange and ludicrous tales of a phantom ship that sped by them like a bird on the wing. The spectacle of a trim-built craft such as ordinarily belongs to the sea skimming over that barren expanse where not a drop of water ever falls might well alarm less superstitious persons. A fast ride on "Desert Queen," amid surroundings more desolate than the lonely sea itself, is a thrilling and exciting experience. You go dodging between the dots of greasewood and cacti as you leave the camp for the solitude when the wind rises. Here and there grotesque yucca trees stand like sentinels, with gaunt cling to! YACHTING ON THE GREAT DESERT. Scientific Alnerican forty miles in eighty minutes. She answers her helm perfectly and sails about as "close" to the wind as the ordinary water craft of her size. forty miles in eighty minutes. She answers her helm perfectly and sails about as "close" to the wind as the ordinary water craft of her size. talns to Death Valley. Wilder becomes the speed, and you hang on frantically with both hands and find it hard to catch your breath. The man at the helm and the man hauling in canvas are too busy to see you g.asp and shiver, but at last the sails are all lowered and the wonderful voyage is ended. But then it has not begun to blow yet on the Mojave Desert! Thirty minutes later you could not stand anywhere on the ground over which you have passed without a .post to cling to! SOME INTERESTING FEATURES OF THE KRUPP WORKS AT ESSEN. BY, BENRY L. GEIS8EL. Nothing daunted, they set to work rebuilding, and after many days re­ paired the damage and m a d e necessary im­ provements, and now she carries her owners and their tools and sup­ plies to and from the mine daily, and often on Sundays and holi­ days they take out ex­ cursion parties of half a dozen people, usually admiring visitors who have gone many miles to see the sight. Hundreds have been attracted to Rosa­ mond from all directions for a look at "Desert Queen." S d i th t i hi lit f th ft d "ALFRED KRuPP." With only saw and ax and hammer and knives for tools, the young men began the work of construction. The material available consisted of the odds and ends to be found around a mining camp. The first requisite was a support for the machine, and for this the axle of an ordinary worn-out buggy was used, two iron wheels 30 inches in diameter, which had done service on a farming implement, being attached. Other parts were improvised with similar skill and ingenuity, and after a month of diligent application the work­ men turned out a stanch "boat," 14 feet long, 8 feet across in front and tapering to the rear, with a mast 15. feet high, mainsail 10 feet on the boom and 10 feet on the mast, jib and jibboom to match. A steering contrivance like those on hook-and-ladder trucks was devised, and "Desert Queen" stood ready for her trial trip. The initial run was doomed to end in disaster. While tear­ ing along b e f o r e a strong wind at a ter­ rific rate the machine got beyond control and a sudden gust brought her to grief with a crash. Bruised men, bro k e n timbers and wrecked sails littered the ground. Neither of the Hoyts will ever for­ get· the experience, for they will always bear the marks of the cas­ ualty as a reminder. Nothing daunted, they set to work rebuilding, and after many days re­ paired the damage and m a d e necessary im­ provements, and now she carries her owners and their tools and sup­ plies to and from the mine daily, and often on Sundays and holi­ days they take out ex­ cursion parties of half a dozen people, usually admiring visitors who have gone many miles to see the sight. Hundreds have been attracted to Rosa­ mond from all directions for a look at "Desert Queen." Speed is the astonishing quality of the craft and through the brush; and little a munks scatter the sand about to get into their retreats; now a and gray and lean-the picture upon his scraggy hind legs an you will run over a deadly "sid and hear the whirring of the bleaching bones of some poor the horrors of starvation and blood from its own parched t came. These are familiar scenes, a them. Then the wind grows madder. "ALFRED KRuPP." The number of gas-lights outside of the workshops was 2,658, and that within the shops 43,012. The gas works of the Krupp works is the sixth largest in the German Empire. IN THE GUN SHOPS AT ESSEN. -away out of sight almost before you realize that it is gone. The wind here is different from any that ever blew in any other part of the world. The "Queen" is fairly fiying now and but a little sail is up. The air is filled with sand and pebbles as large as buckshot, and they pelt you h;u-d; all around towering spirals of dust-small end of the spiral down-go springing across the plain, whirling up sand to feed the terrible storm that is sweeping fro/ the Sierra Madre Moun- -away out of sight almost before you realize that it is gone. The wind here is different from any that ever blew in any other part of the world. The "Queen" is fairly fiying now and but a little sail is up. The air is filled with sand and pebbles as large as buckshot, and they pelt you h;u-d; all around towering spirals of dust-small end of the spiral down-go springing across the plain, whirling up sand to feed the terrible storm that is sweeping fro/ the Sierra Madre Moun- Speed is the astonishing quality of the craft, and almost beyond belief. Time and again she has sailed fifty miles an hour on the dry lake in favorable winds. On the open desert she has been speeded up consider­ ably, and once is said to have made a straight run of The electric plant of the steel works includes seven distributing stations, 125 /nes o! calM, 1,062 arc
https://openalex.org/W4307268609
https://www.mdpi.com/1422-0067/23/21/12811/pdf?version=1666617201
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Host–Pathogen Interaction 3.0
International journal of molecular sciences
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International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences Editorial Host–Pathogen Interaction 3.0 Andreas Burkovski Microbiology Division, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany; andreas.burkovski@fau.de; Tel.: +49-9131-85-28086 Microbiology Division, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany; andreas.burkovski@fau.de; Tel.: +49-9131-85-28086 Microorganisms can interact with plants, animals and humans in many different ways, e.g., beneficially as symbionts, indifferently as commensals or harmfully as pathogens. Today, a wide variety of molecular and cell biology tools, including advanced microscopy and -omics techniques, allow us to study these interactions at a molecular level. It is the ded- icated aim of this serial of Special Issues, i.e., “Host–Pathogen Interaction”, Host–Pathogen Interaction 2.0” and “Host–Pathogen-Interaction 3.0”, to provide an overview about various aspects of pathogenic microorganisms (viruses, bacteria, fungi and protozoa) and their interaction with their host organisms. In fact, contributions to “Host–Pathogen Interaction 3.0” and the preceding Special Issues on this subject may be divided into different topics: (i) characterization of model systems, (ii) analysis of bacteria–host interactions, (iii) analysis of viral compounds, and (iv) characterization of fungal and protozoal pathogenicity. p g p p g y Invertebrate model systems provide a cost effective and ethically unproblematic alternative to animal trials, especially in the beginning of a research project to gain first insights in the respective pathosystem or in high-throughput studies. A prerequisite is a proper characterization of the model. One of the best investigated model systems in this respect is the nematode Caenorhabditis elegans due to advantageous properties such as short life span, transparency, genetic tractability and ease of culture. Interestingly, Guo et al. [1] showed that plasmids of its standard bacterial diet Escherichia coli can interfere with nematode metabolism, an unexpected observation, which may also have an impact on studies of pathogenic bacteria. Other established model systems are protozoa such as Acanthamoeba castellanii, which was used to study Salmonella enterica sv. Typhimurium [2], the yellow mealworm Tenebrio molitor, which was analyzed in respect to antimicrobial peptide production in several of the submitted manuscripts [3–5], and larvae of the greater wax moth Galleria mellonella [6]. Citation: Burkovski, A. Host–Pathogen Interaction 3.0. Int. J. Mol. Sci. 2022, 23, 12811. https:// doi.org/10.3390/ijms232112811 Received: 17 October 2022 Accepted: 20 October 2022 Published: 24 October 2022 Citation: Burkovski, A. Host–Pathogen Interaction 3.0. Int. J. Mol. Sci. 2022, 23, 12811. https:// doi.org/10.3390/ijms232112811 Invertebrate model systems are especially valuable for the analysis of bacterial pathogens. In fact, studies describing bacterium-host interaction were the majority of submissions to the abovementioned Special Issues. Reviews submitted summarize our current knowledge on important pathogens such as Corynebacterium diphtheriae [7], Staphy- lococcus aureus [8,9], Legionella species [10] and Neisseria meningitidis [11], while research articles highlight specific aspects of host–pathogen interaction. Different mycobacterial pathogens [12,13], Corynebacterium species [14,15], Helicobacter pylori [16], Mycoplasma fer- mentans [17], rickettsiae [18] and S. enterica [19] were studied in respect to their interaction with human cell lines. In addition to other human pathogens [20–24], animal- [25,26] and plant-pathogenic bacteria [27–30] were investigated. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. In addition to pathogenic bacteria, fungi are important pathogens, especially in respect to agricultural plant diseases. Papers dealing with Fusarium infection of wheat [31] and flax [32], crop infection by Rhizoctonia solani [33], grapevine infection by Lasiodiplodia theobromae [34] and the role of fatty acid synthesis in the infection process by Magnaporthe oryzae [35] were submitted. Copyright: © 2022 by the author. 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/). y In addition to bacterial and fungal pathogens, plants are affected by viral diseases. Pathogenicity factors of Tobacco Curly Shoot Virus and Tobacco Mosaic Virus were studied in manuscripts submitted to the abovementioned Special Issues [36,37]. Of course, humans https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 12811. https://doi.org/10.3390/ijms232112811 Int. J. Mol. Sci. 2022, 23, 12811 2 of 4 are also infected by viruses. Prominent examples are SARS-CoV-2 [38,39], influenza [40,41], Ebola virus [42] and human papillomavirus [43]. Other papers submitted to this Special are also infected by viruses. Prominent examples are SARS-CoV-2 [38,39], influenza [40,41], Ebola virus [42] and human papillomavirus [43]. Other papers submitted to this Special Issue serial are focusing on viruses infecting animals such as pigs [44] and ducks [45]. Issue serial are focusing on viruses infecting animals such as pigs [44] and ducks [45]. Taken together, this serial of Special Issues provides an excellent overview about various aspects of viral, bacterial and eukaryotic pathogens and their interactions with their respective human, animal and plant hosts. The fact that several Special Issues were already successfully launched in the International Journal of Molecular Sciences emphasizes the scientific interest and importance of this research field at the interface of molecular biology and medicine. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Conflicts of Interest: The author declares no conflict of interest. Conflicts of Interest: The author declares no conflict of interest. References 1. Guo, R.; Li, G.; Lu, L.; Sun, S.; Liu, T.; Li, M.; Zheng, Y.; Walhout, A.J.M.; Wu, J.; Li, H. The Plasmid pEX18Gm Indirectly Increases Caenorhabditis elegans Fecundity by Accelerating Bacterial Methionine Synthesis. Int. J. Mol. Sci. 2022, 23, 5003. [CrossRef] [PubMed] [ ] 2. Balkin, A.S.; Plotnikov, A.O.; Gogoleva, N.E.; Gogolev, Y.V.; Demchenko, K.N.; Cherkasov, S.V. Cappable-Seq Reveals Specific Patterns of Metabolism and Virulence for Salmonella Typhimurium Intracellular Survival within Acanthamoeba castellanii. Int. J. Mol. Sci. 2021, 22, 9077. [CrossRef] [PubMed] 3. Ko, H.J.; Jo, Y.H.; Patnaik, B.B.; Park, K.B.; Kim, C.E.; Keshavarz, M.; Jang, H.A.; Lee, Y.S.; Han, Y.S. IKKγ/NEMO Is Required to Confer Antimicrobial Innate Immune Responses in the Yellow Mealworm, Tenebrio molitor. Int. J. Mol. Sci. 2020, 21, 6734. [CrossRef] 4. 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Palusi´nska-Szysz, M.; Zdybicka-Barabas, A.; Luchowski, R.; Reszczy´nska, E.; ´Smiałek, J.; Mak, P.; Gruszecki, W.I.; Cytry´nska, M. Choline Supplementation Sensitizes Legionella dumoffii to Galleria mellonella Apolipophorin III. Int. J. Mol. Sci. 2020, 21, 5818. [CrossRef] [ ] 7. Ott, L.; Möller, J.; Burkovski, A. Interactions between the Re-Emerging Pathogen Corynebacterium diphtheriae and Host Cells. Int. J. Mol. Sci. 2022, 23, 3298. [CrossRef] 8. Wójcik-Bojek, U.; Ró˙zalska, B.; Sadowska, B. Staphylococcus aureus—A Known Opponent against Host Defense Mechanisms and Vaccine Development—Do We Still Have a Chance to Win? Int. J. Mol. Sci. 2022, 23, 948. [CrossRef] 9. Mrochen, D.M.; de Oliveira, L.M.F.; Raafat, D.; Holtfreter, S. Staphylococcus aureus Host Tropism and Its Infection Models. Int. J. Mol. Sci. 2020, 21, 7061. References C on the Pathogenicity and Lifestyle of Rhizoctonia solani. Int. J. Mol. Sci. 2021, 22, 2183. 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Attitudes towards Research among Bachelor of Pharmacy and Doctor of Pharmacy Students: A Study from Tertiary Medical Institute
International journal of Indian psychology
2,016
cc-by
2,705
© 2016 S Pasupula, L Obulesu, K Reddy; licensee IJIP. This is an Open Access Research 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. 1 M.D, Assistant Professor, Department of Psychiatry, RVS medical college, Chittoor, India 2 M.D., Prof & Dean, Department of Forensic medicine, RVS medical college ,Chittoor, India 3 Graduate, SVCP, Rvs Nagar, Chittoor, India 4 M.Pharm, Ph.D, SVCP, Rvs Nagar, Chittoor, India *Responding Author ABSTRACT Background: Research plays a key role in day to day life and research became a part of study curriculum. Attitude towards research plays a key role in knowledge among medical as well as Para medical students. Aim: To assess the attitude towards research among pharmacy internees. Methodology: This study was carried out at department of psychiatry, RVS medical college & hospital..Study sample includes the pharmaceutical students (Bachelor of Pharmacy and Doctor of Pharmacy) doing their internship at Sri venakteswara college of pharmacy are included in the study. Attitudes towards the research was measured using Attitude towards research scale (ATR Scale). Conclusion: Attitude towards the research among pharmacy internees is variable which indicates the need to increase awareness of research among their graduate period. The International Journal of Indian Psychology ISSN 2348-5396 (e) | ISSN: 2349-3429 (p) Volume 4, Issue 1, No. 77, DIP: 18.01.077/20160401 ISBN: 978-1-365-51571-2 http://www.ijip.in | October-December, 2016 http://www.ijip.in | October-December, 2016 Dr. Sai kiran Pasupula1*, Dr. L. C. Obulesu2, Ammajan3, Dr. K. Bhaskar Reddy4 Dr. Sai kiran Pasupula1*, Dr. L. C. Obulesu2, Ammajan3, Dr. K. Bhaskar Reddy4 Dr. Sai kiran Pasupula1*, Dr. L. C. Obulesu2, Ammajan3, Dr. K. Bhask Attitudes towards Research among Bachelor of Pharmacy and Doctor of Pharmacy Students: A Study from Tertiary Medical Institute Attitudes towards Research among Bachelor of Pharmacy and Doctor of Pharmacy Students: A Study from Tertiary Medical Institute [4,5,6] and future career achievements in academic activities .Limited data is available on research attitude among Pharmacy graduates and current study focuses on the study of attitude towards research among Bachelor of pharmacy and Doctor of Pharmacy internees. METHODOLOGY This study was carried out at department of psychiatry, RVS medical college and hospital. Study sample includes the pharmaceutical students (B-Pharmacy and Pharm-D) studying at Sri venakteswara college of pharmacy. Attitudes towards the research was measured using Attitude towards research scale (ATR Scale). Statistical analysis was done by using EPI INFO and M.S. Excel software's. Attitude towards research questionnaire: It is a 30-item, self-rated, Likert scale which measures research attitude on the basis of five factors: The five factors included in the questionnaire were: usefulness of research in a career, research anxiety, positive attitudes toward the research, relevance of the research to life, and research difficulty. Attitudinal statements were scored on a 7-point Likert scale varying from 1.Strongly disagrees to 7. Strongly agree. More the mean value of the response, more is the positive attitude and vice versa. Keywords: Research, Attitude, Pharmacy internees. Research plays a prominent role in day to day life and research is one of the most important intellectual possessions for every human being to change his way life in accordance to the needs and demands of the society. Research is a key ingredient in shaping up the world that man lives in and the new experiences they see and encounter in their surroundings.[1] Students at the undergraduate level usually tend to view research methods courses negatively [2]. Many records could show evidences of the students’ negative attitudes towards research in relation to courses in, statistics and mathematics[3]. Attitude towards research is the significant indicator for academic performance and achievement and more positive one’s attitude towards an academic subject, the higher the possibility for an individual to perform well academically. Attitude towards research among the medical and paramedical professionals is variable and research experience during graduate period is strongly associated with postgraduate research initiatives Aims and Objectives 1. To assess the attitude towards research among Bachelor of pharmacy and Doctor of Pharmacy internees. 2. To compare the attitude towards research between Bachelor of pharmacy and Doctor of Pharmacy internees. 2. To compare the attitude towards research between Bachelor of pharmacy and Doctor of Pharmacy internees. Table 2 Table 2 Questionnaire Mean value of Pharm D students n=50 Std Dev of Pharm D students Mean value of B-Pharm students n = 50 SD of Pharm B- Pharm Total mean value n = 50 Total Std Dev 1. Research makes me anxious. 4.6667 1.0328 4.6585 1.6675 4.5833 1.661 2 Research should be taught to all students. 5.5 1.3784 5.7805 1.525 5.6667 1.5755 3. I enjoy research. 5.1667 1.472 5.3659 1.3371 5.2917 1.3677 4. Research is interesting. 5.6667 1.0328 5.6098 1.3762 5.5833 1.3342 5. I like research. 5.8333 0.9832 5.3171 1.5722 5.375 1.4964 6. Research scares me. 3 1.0954 3.122 1.9519 3.1667 1.8831 7. Research is useful for my career. 5 1.5492 4.9512 1.7168 5 1.6885 8. I find it difficult to understand the concepts of research. 5.1667 1.6021 3.7317 1.5973 3.9583 1.6626 9. I make many mistakes in research. 4 1.7889 4.0244 1.6806 4.0417 1.6626 10. I love research. 6 1.2649 5.0976 1.4284 5.1875 1.4241 11. I am interested in research. 5.1667 1.8348 5.3171 1.3498 5.25 1.4217 12. Research is connected to my field of study. 5.8333 0.9832 5.6585 1.4596 5.6042 1.4838 13. Most students benefit from research. 5.6667 1.0328 5.4146 1.5326 5.3542 1.5911 14. Research is stressful. 4.8333 2.2286 4.1951 1.8871 4.2292 1.9267 15. Research is very valuable. 6 1.2649 5.7561 1.3744 5.7292 1.3951 16. Research makes me nervous. 4.1667 1.9408 3.9268 1.4559 3.9583 1.4869 17. I use research in my daily life. 4.6667 2.2509 3.8537 1.7544 3.9792 1.8041 18. The skills I have acquired in research will be helpful to me in the future 4.8333 1.169 5.3415 1.4935 5.2917 1.4434 19. Research is useful to every professional. 5.5 1.2247 6.1707 1.2228 6.1042 1.2246 20. Knowledge from research is as useful as writing. 4.6667 1.633 5.0488 1.7882 5.0208 1.7442 21. Research is irrelevant to my life. 3.5 2.0736 2.6829 1.6192 2.8333 1.6927 22. Research should be indispensable in my professional training 4.8333 1.8348 4.2195 1.5734 4.2917 1.5839 23. Research is complicated 5.8333 0.9832 4.8537 1.7112 4.9375 1.6683 24. Research thinking does not apply to my personal life. 5 2.1909 3.4878 1.9381 3.6667 1.9929 25. I will employ research approaches in my profession. 5.5 1.0488 4.9512 1.7882 4.9792 1.7196 26. Research is difficult. 5.8333 1.472 4.5366 1.8721 4.6667 1.8604 27. I am inclined to study the details of research procedures carefully. 5.1667 0.7528 4.6585 1.1093 4.7083 1.0711 28. Research is pleasant 4.5 2.0736 3.9512 1.8296 4.0417 1.833 29. Socio demographic profile As shown in Table 1, total sample consists of hundred house surgeons which constitutes by fifty B-Pharm and Pharm D individuals respectively. Males constitute forty eight and a female constitutes fifty two in number. Mean age of participants is 23.5 years. Table 1: Variable Value Age : Mean Age of all the sample 23.5 Sex : Male Female 48 52 Table 1: Variable Value Age : Mean Age of all the sample 23.5 Sex : Male Female 48 52 © The International Journal of Indian Psychology, ISSN 2348-5396 (e)| ISSN: 2349-3429 (p) | 171 © The International Journal of Indian Psychology, ISSN 2348-5396 (e)| ISSN: 2349-3429 (p) | 171 © The International Journal of Indian Psychology, ISSN 2348-5396 (e)| ISSN: 2349-3429 (p) | 171 Attitudes towards Research among Bachelor of Pharmacy and Doctor of Pharmacy Students: A Study from Tertiary Medical Institute Attitudes towards Research among Bachelor of Pharmacy and Doctor of Pharmacy Students: A Study from Tertiary Medical Institute Attitudes towards Research among Bachelor of Pharmacy and Doctor of Pharmacy Students: A Study from Tertiary Medical Institute Among total population, most of the individuals had variable and uncertain responses. People shared favourable responses (Mean value = 5.5 to 7.0 ) towards eleven items -2,4,12,15,19, . Uncertain attitude (Mean value = 3.0 to 5.5 ) was observed in seven items - 1,3,5,6,7,8,9,10,11,13,14,16,17,18,20, ,22,23,24,25,26,27,28,29,30. Unfavourable response (1.0 - 3.0) is observed for the item 21. DISCUSSION AND CONCLUSION Attitude towards research is variable and proper education with training after assessing the attitude helps the individuals to change the individuals attitude more favourable and helps in better ongoing of research .The study revealed that there was a positive significant relationship between students’ research self-efficacy and some of their personal and professional characteristics, including age and numbers of published papers. Earlier studies on research attitude found that several factors such as personal and professional characteristics, including age and numbers of published papers. [7,8] In our study we found that among the total population, individuals have more uncertain responses than favourable or unfavourable responses. More number of uncertain responses can be explained by the experience of research as a tough and difficult course and want to avoiding research during the undergraduate training. It also indicates the need of education and research training during their undergraduate level. Gender plays a key role on research attitudes and earlier studies shows that males experienced lower levels of anxiety compared to females. Graduating students whether male and female from the Faculties of Education, Arts and Social Sciences had negative attitude towards research [9] . In our study female individuals showed favorable responses than male individuals which differs from earlier studies. The current study found that individuals from B-pharm and Pharm D have different responses and most of the Pharm D individuals are strongly agreeing that they love research and research is very valuable ( 6.0 + 1.264 ) and they are strongly disagreeing for the question Research scares me (3.0 + 1.9). B-pharm individuals are strongly agreeing that research is useful to every professional (6.1707+1.2228) and at the same time they are disagreeing that Research is irrelevant to my life. (2.6829 + 1.6192). Much varied responses and much variation is seen among the items I will employ research approaches in my profession, Research is difficult, I am inclined to study the details of research procedures carefully, Research is pleasant , Research- orientated thinking plays an important role in my daily life . The variation among two groups can be explained by the fact of early exposure of research to the individuals of Pharm D than B- pharm individuals and moreover inclusion of Research and methodology subject in the educational curriculum of Pharm D course. Table 2 Research-orientated thinking plays an important role in my daily life 5.3333 1.0328 4.7805 1.8099 4.875 1.7213 30. Research is a complex subject 4.8333 1.169 5.1707 1.7015 5.1667 1.6417 © The International Journal of Indian Psychology, ISSN 2348-5396 (e)| ISSN: 2349-3429 (p) | 172 © The International Journal of Indian Psychology, ISSN 2348-5396 (e)| ISSN: 2349-3429 (p) | 172 Attitudes towards Research among Bachelor of Pharmacy and Doctor of Pharmacy Students: A Study from Tertiary Medical Institute Attitudes towards Research among Bachelor of Pharmacy and Doctor of Pharmacy Students: A Study from Tertiary Medical Institute DISCUSSION AND CONCLUSION Attitude towards research is variable and proper education with training after assessing the attitude helps the individuals to change the individuals attitude more favourable and helps in better ongoing of research .The study revealed that there was a positive significant relationship between students’ research self-efficacy and some of their personal and professional characteristics, including age and numbers of published papers. Earlier studies on research attitude found that several factors such as personal and professional characteristics, including age and numbers of published papers. [7,8] In our study we found that among the total population, individuals have more uncertain responses than favourable or unfavourable responses. More number of uncertain responses can be explained by the experience of research as a tough and difficult course and want to avoiding research during the undergraduate training. It also indicates the need of education and research training during their undergraduate level. Attitudes towards Research among Bachelor of Pharmacy and Doctor of Pharmacy Students: A Study from Tertiary Medical Institute Acknowledgments The author appreciates all those who participated in the study and helped to facilitate the research process. Attitudes towards Research among Bachelor of Pharmacy and Doctor of Pharmacy Students: A Study from Tertiary Medical Institute Attitudes towards Research among Bachelor of Pharmacy and Doctor of Pharmacy Students: A Study from Tertiary Medical Institute towards research to personals at undergraduate level and inclusion of research in undergraduate curriculum is required for more favourable attitude which helps in better professional outcomes. DISCUSSION AND CONCLUSION [10] As the sample recruited for the study is from single institution results cannot be generalised to the whole population is one of the limitation in our study. Future research is needed with large sample size at clinical and community level .The results are concluding that attitude towards research among Pharmacy internees is variable and Pharm D individuals are having more favourable responses than B-Pharm internees. This results shows the need of research oriented programmes to improve the awareness and knowledge regarding the research. Proper awareness ternational Journal of Indian Psychology, ISSN 2348-5396 (e)| ISSN: 2349-3429 (p) | 173 Conflict of Interests The author declared no conflict of interests. REFERENCES 1. Faustino e. oguan jr et al., Attitude and anxiety towards research, its influence on the students’ achievement in the course. Asian journal of management sciences & education vol. 3(4) October 2014. 2. Papanastasiou, e. c. (2005). Factor structure of the attitudes towards research scale. statistics education research journal, 4(1), 16-26. www.stat.auckland.ac.nz/serj 3. Adams, n. a., & Holcomb, w. r. (1986). Analysis of the relationship between anxiety about mathematics and performance. psychological reports, 59, 943-948. 3. Adams, n. a., & Holcomb, w. r. (1986). Analysis of the relationship between anxiety about mathematics and performance. psychological reports, 59, 943-948. 4. Houlden, R.L 2004. Medical students’ perceptions of an undergraduate research elective. med. teach. 26 (7), 659–661. 4. Houlden, R.L 2004. Medical students’ perceptions of an undergraduate research elective. med. teach. 26 (7), 659–661. 5. Solomon, s.s., tom, s.c., Pichert, j., Wasserman, d., powers, a.c.,2003. Impact of medical student research in the development of physician-scientists. j. invest. med. 51 (3), 149–156. 5. Solomon, s.s., tom, s.c., Pichert, j., Wasserman, d., powers, a.c.,2003. Impact of medical student research in the development of physician-scientists. j. invest. med. 51 (3), 149–156. 6. Brancati, f.l., mead, l.a., levine, d.m., martin, d., margolis, s., klag, m.j., 1992. Early predictors of career achievement in academic medicine. jama 267 (10), 1372–1376. 6. Brancati, f.l., mead, l.a., levine, d.m., martin, d., margolis, s., klag, m.j., 1992. Early predictors of career achievement in academic medicine. jama 267 (10), 1372–1376. 7. Dr. naser zamani-miandashti et al., The relationship between research self-efficacy, research anxiety and attitude toward research: a study of agricultural graduate students ., journal of educational and instructional studies in the world November 2013, volume: 3 issue: 4 article: 09 issn: 2146-7463 . 7. Dr. naser zamani-miandashti et al., The relationship between research self-efficacy, research anxiety and attitude toward research: a study of agricultural graduate students ., journal of educational and instructional studies in the world November 2013, volume: 3 issue: 4 article: 09 issn: 2146-7463 . 8. Sadia shaukat et al ., Postgraduate students’ attitudes towards research .,bulletin of education and research june 2014, vol. 36, no. 1 pp. 111-122) . 8. Sadia shaukat et al ., Postgraduate students’ attitudes towards research .,bulletin of education and research june 2014, vol. 36, no. 1 pp. 111-122) . 9. Bandele s.o. © The International Journal of Indian Psychology, ISSN 2348-5396 (e)| ISSN: 2349-3429 (p) | 174 REFERENCES et al., Patterns of university graduating students’ attitude to research work et al ., international journal of educational research and technology ., ijert: volume 4 [3] September 2013: 98-103. 9. Bandele s.o. et al., Patterns of university graduating students’ attitude to research work et al ., international journal of educational research and technology ., ijert: volume 4 [3] September 2013: 98-103. 10. Educational curriculum of B-Pharm and Pharm D individuals. How to cite this article: S Pasupula, L Obulesu, K Reddy (2016), Attitudes towards Research among Bachelor of Pharmacy and Doctor of Pharmacy Students: A Study from Tertiary Medical Institute, International Journal of Indian Psychology, Volume 3, Issue 4, No. 77, ISSN:2348- 5396 (e), ISSN:2349-3429 (p), DIP:18.01.077/20160304, ISBN: 978-1-365-51571-2 ternational Journal of Indian Psychology, ISSN 2348-5396 (e)| ISSN: 2349-3429 (p) | 174
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Transformações do capitalismo e formação do indivíduo: contribuições da Escola de Frankfurt na análise das eleições presidenciais nos EUA e Brasil
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Capitalism’s transformations and individual’s formation: contributions of the Frankfurt School in the analysis of the presidential elections in the USA and Brazil Abstract Abstract This paper has as theoretical reference the contributions of the authors of the first generation of the Frankfurt School. It aims to present the changes occurred in capitalism in the XIX and XX centuries and their impact in individual’s formation – still noticed nowadays. Therefore, characteristics of the liberal and monopoly capitalism are presented, as well as the ideology produced by such configurations of the production system. From this, it is highlighted as these elements affect the formation of the individual and how there are changes in its social function: opposition to adhesion. In order to do so, it has been analyzed some psychosocial phenomena that occurred in the last presidential elections in the USA and Brazil. If some propositions of the first generation of the Frankfurt School become anachronistic, as to the means, the contributions regarding mass manipulation become extremely actual. The reduction of individuals to algorithms demonstrates the bankruptcy of the model of bourgeois individuality. Far from being a nostalgic reference to the liberal model of individual, the article aims to demonstrate its contradictions in the contemporary world. Keywords: liberal capitalism; monopoly capitalism; ideology; individual’s formation; Frankfurt School. O presente artigo visa destacar alguns elementos acer­ ca das transformações do capitalismo em seu processo histórico. São utilizadas as reflexões da primeira geração da Escola de Frankfurt, dando ênfase, especialmente à formação do indivíduo e alterações na ideologia a respeito dele. Vale destacar que a obra Capitalismo monopolista: ensaio sobre a ordem social americana, de Baran e Swee­ zy (1966), é utilizada como suporte para tal discussão. Os autores não fazem parte da primeira geração da Escola de Frankfurt. Optou-se por tal procedimento com a finalida­ de de facilitar a compreensão dos leitores a respeito dos processos sociais analisados pelos frankfurtianos. ricos que possibilitaram as transformações no capitalismo, nem o longo processo de ascensão, conquista e dominação dos ideais burgueses. Entretanto, apresentar-se-ão caracte­ rísticas gerais das duas formas do capitalismo, quando em funcionamento pleno na sociedade ocidental. A subida da burguesia ao poder provocou profundas alterações nas esferas econômica, política, cultural e for­ mas de socialização da sociedade. H Pesquisa financiada pela CAPES. HH Endereço para correspondência: Prefeitura Municipal de Sumaré, CRAS Área Cura. Rua Deusdete Alves, 206, Parque Santo Antônio – Sumaré, SP – Brasil. CEP: 13173343. E-mail: psycogus@hotmail.com Os dados completos do autor encontram-se ao final do artigo. Fractal: Revista de Psicologia, v. 31, n. 2, p. 83-90, maio-ago. 2019. doi: https://doi.org/10.22409/1984-0292/v31i2/5619 Fractal: Revista de Psicologia, v. 31, n. 2, p. 83-90, maio-ago. 2019. doi: https://doi.org/10.22409/1984-0292/v31i2/5619 Artigos Transformações do capitalismo e formação do indivíduo: contribuições da Escola de Frankfurt na análise das eleições presidenciais nos EUA e BrasilH Gustavo Henrique Carretero    HH Pontifícia Universidade Católica de São Paulo, São Paulo, SP, Brasil Resumo O artigo teve como referencial teórico as contribuições dos autores da primeira geração da Escola de Frankfurt. Ele visa apre­ sentar, em termos gerais, as alterações que ocorreram no capitalismo nos séculos XIX e XX e os impactos delas na formação do indivíduo – ainda percebidos na contemporaneidade. Para tanto, são apresentadas características do capitalismo liberal e mono­ polista, assim como da ideologia produzida por tais configurações desse sistema produtivo. A partir disso, é destacado como tais elementos incidem na formação do indivíduo e como há alterações em sua função social: da oposição para a adesão. Para tanto, são analisados alguns fenômenos psicossociais ocorridos nas últimas eleições presidenciais nos EUA e no Brasil. Se algumas proposições da primeira geração da Escola de Frankfurt se tornam anacrônicas, quanto aos meios, as contribuições a respeito da manipulação da massa se tornam extremamente atuais. A redução de indivíduos a algoritmos, passíveis de manipulação, demons­ tra a falência do modelo de individualidade burguês. Longe de ser uma referência nostálgica ao modelo liberal de indivíduo, o artigo visa demonstrar as contradições da formação do indivíduo na contemporaneidade. lavras-chave: capitalismo liberal; capitalismo monopolista; ideologia; formação do indivíduo; Escola de Frankfurt. Recebido em: 18 de março de 2016 1ª avaliação: 8 de outubro de 2018 2ª avaliação: 28 de novembro de 2018 Aceito em: 7 de fevereiro de 2019 Este trabalho está licenciado com uma Licença Creative Commons Attribution 4.0 International License. A. Capitalismo Liberal Baran e Sweezy (1966) destacam que a burguesia, nos seus primórdios, era formada por centenas de empre­ endedores individuais que usavam de sua riqueza para fazer fortunas. Inicialmente, tal processo se dava pelo comércio, venda de produtos raros na Europa comprados por valores irrisórios ou trocas altamente vantajosas. No­ vas forma de obtenção de lucro se abriram com a Revolu­ ção Industrial, que possibilitou que o excedente, ou mais valia, fosse obtido por meio da produção de mercadorias. Os custos de produção abriam possibilidade de venda a valores maiores do que a importação. Pode-se falar, nesse momento, de uma nova confi­ guração do sistema capitalista que assume diversas no­ menclaturas, tais como: capitalismo tardio, sociedade industrial avançada, capitalismo monopolista, sociedade administrada. Tais transformações do sistema produtivo designam alterações profundas na organização social, política, econômica. Tais transformações minam as bases de uma das mais ilustres “invenções” possibilitadas pela ideologia burguesa: o indivíduo como portador de razão e autonomia e capaz de opor-se à sociedade na defesa de seus interesses racionais. Nesse contexto surgem milhares de pequenas empre­ sas que produzem para um mercado em expansão. O pre­ ço do produto é imposto pela concorrência com diversas outras pequenas empresas que produzem no mesmo se­ tor. A tentativa de inovação tecnológica é buscada como forma de solapar toda concorrência e se constitui como meta na disputa entre pequenas firmas que produzem para um mercado anônimo. A mecanização e racionalização da produção levaram o competidor mais fraco a submeter-se ao domínio de grandes empresas da indústria mecanizada. Isso abole o sujeito econômico livre. Com o surgimento do capitalis­ mo monopolista, a ideia de indivíduo torna-se ambígua: de um lado está a insistência no desempenho social li­ vre e eficiência produtiva e do outro a massificação da produção, do trabalho, dos produtos e da subjetividade (MARCUSE, 1998a). Os direitos e liberdades individu­ ais passam a ser interpretados no âmbito da privacidade e afastamento da sociedade, perdendo assim seu caráter social e tornando-se particular. Os autores destacam que o excedente, nessa modali­ dade do capitalismo, é absorvido por meio do consumo e da acumulação. Tal excedente seria obtido pela explo­ ração do trabalho de milhares de homens, mulheres e crianças expulsos dos campos e que migraram para as cidades com a derrocada da ordem feudal. Tais popula­ ções se constituíram como mão de obra desqualificada e, portanto, barata. Capitalism’s transformations and individual’s formation: contributions of the Frankfurt School in the analysis of the presidential elections in the USA and Brazil Abstract Os autores da primeira geração da Escola de Frankfurt afirmam que a concepção de subjetividade como conhecida na atualidade é fruto do mundo burguês (ADORNO, 1991; HORKHEIMER, 2010), podendo ser encontrados elementos do protótipo do indiví­ duo burguês já no período clássico grego, como na Odisseia de Homero (HORKHEIMER, 2010; ADORNO, 1985). O Capitalismo não se manteve homogêneo e inalterado desde seu aparecimento e implantação pela burguesia. Ob­ servam-se, durante a história, diversas fases, concepções e ideologias que sustentaram a implantação e manutenção deste sistema. Todavia, para o artigo é interessante fazer a contraposição entre o capitalismo liberal e o capitalismo tardio ou monopolista. Nesta sucinta reflexão não serão aprofundados, dentre outros temas, nem os aspectos histó­ As revoluções burguesas culminaram na tomada do poder por tal classe social pelo solapamento da antiga ordem. Não se deve pensar que tal processo iniciou-se economicamente. Percebe-se que, com a chegada de tal classe ao poder, a esfera da produção torna-se o principal fundamento da existência humana. O surgimento, ascen­ são e conquista do poder pela burguesia ocorreu durante vários séculos. Os Renascimentos Cultural e Comercial da Europa podem ser datados como o surgimento da clas­ se e início da derrocada da ordem feudal. Vários even­ Este trabalho está licenciado com uma Licença Creative Commons Attribution 4.0 International License. Gustavo Henrique Carretero tos históricos colaboraram e resultaram do esforço de tal classe para o estabelecimento de sua ordem social; dentre outros podem ser destacados: a Reforma Protestante no século XVI, o surgimento do método científico e do Ilu­ minismo nos séculos XVII e XVIII; a Revolução Glorio­ sa no século XVII; a Revolução Francesa e a Revolução Industrial no século XVIII. Dois aspectos fundamentais da ideologia liberal são: 1. liberdade política, econômica e intelectual (MAR­ CUSE, 1973); 2. igualitarismo ideológico de oportuni­ dades, nas quais as desigualdades não seriam frutos de instituições injustas, mas dotes pessoais superiores e in­ feriores (BARAN; SWEEZY, 1966). Para Marcuse (1973) a liberdade de pensamento, de palavra e de consciência ajudou a promover uma das ca­ racterísticas mais marcantes do período liberal: o livre empreendimento. Este foi um dos baluartes do sistema, possibilitando o surgimento da figura do empresário in­ dividual (BARAN; SWEEZY, 1966), que, por meio da acumulação de riquezas granjeadas no mercado, pela livre iniciativa e enfrentamento de riscos, procurava os maiores lucros possíveis. Capitalism’s transformations and individual’s formation: contributions of the Frankfurt School in the analysis of the presidential elections in the USA and Brazil Abstract Para refletir sobre as profundas alterações sociais promovidas pela burguesia é necessário destacar os as­ pectos ideológicos que perpassam tal processo. Assim, apresentar características do sistema político e econô­ mico imposto por essa classe sem destacar os aspectos ideológicos seria ingênuo, senão incorreto. No século XIX houve o apogeu do liberalismo, sendo que em suas últimas décadas acentuaram-se as transformações no sis­ tema capitalista, que culminaram com o surgimento de novas configurações do sistema produtivo. Marcuse (1998a) afirma que (na passagem do capi­ talismo liberal ao monopolista) o processo de produção de mercadorias solapou a base econômica sobre a qual a racionalidade individualista se constituiu e se assentava. Além disso, a formação de indivíduos no modelo liberal se torna anacrônica no modo de produção monopolista. Se as mercadorias são produzidas em massa e para as massas não há a necessidade social de indivíduos pensantes, autô­ nomos e críticos. Crochik (1998) ressalta que cada socie­ dade “produz” indivíduos necessários à sua reprodução. A. Capitalismo Liberal Marcuse (1973) destaca que a liberdade de empre­ endimento não foi vantajosa para toda a sociedade. Ela significou, para os trabalhadores, a “liberdade” de tra­ balhar ou morrer à míngua. A liberdade de empreendi­ mento tornou-se, portanto, labuta (trabalho sem sentido), insegurança e temor para a maioria da população. Os tra­ balhadores explorados eram obrigados a assumir a con­ dição de sujeitos econômicos livres, apesar de não terem condições para tal. Eles foram submetidos à concorrência em contraste com as formações “coletivistas” feudais. Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 B. Capitalismo Monopolista Baran e Sweezy (1966) destacam que o capitalismo monopolista se caracteriza pelo predomínio de grandes em­ presas que produzem em grande escala. A essas cabe uma parcela significativa da produção de uma indústria (ramo de produção) ou mesmo várias. Tais empresas são capazes de controlar os preços, tipos e volumes de investimento. O termo monopolista não designa exclusividade, mas oligopólio, ou seja, poucos produtores dominando o mer­ cado e produtos que se substituem um ao outro de forma Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 84 Transformações do capitalismo e formação do indivíduo: contribuições da Escola de Frankfurt na análise das eleições presidenciais nos EUA e Brasil mais ou menos satisfatória. Tal elemento levou à derroca­ da da concorrência e estabilização dos preços em certos patamares. Uma das consequências desse processo é a perda da importância da pequena empresa, que continua existindo, mas com menor relevância e sem potencial de exercer iniciativas próprias e independentes. nizações da indústria em sua situação social dominante) que dita em larga escala a quantidade, a forma e o tipo de mercadorias a serem produzidas. Assim, segundo Mar­ cuse (1973), a tecnologia serve à fusão da economia, po­ lítica, cultura e racionalidade. A tecnologia acaba por se tornar totalitária por determinar não apenas as oscilações, habilidades e atitudes socialmente necessárias, mas tam­ bém necessidades e aspirações individuais. A existência privada dos homens é invadida pela pública, invalidando tal contraposição e transformando necessidades sociais em individuais. Dessa maneira, a tecnologia serve para produzir novas e mais eficazes formas de controle e coe­ são social, ao mesmo tempo em que fica exposta a falácia da noção de “neutralidade” da tecnologia. Para Baran e Sweezy (1966), no capitalismo mono­ polista o tipo sociológico representado pelo empresário individualista (preocupado exclusivamente com seus interesses e os de sua família) é substituído pelos admi­ nistradores (que defendem os interesses da empresa). Em outras palavras, do dominador da instituição para o dominado pela instituição. O caráter pessoal no controle das empresas é substituído pelo da administração e/ou da Sociedade Anônima (S. A.), que possui três característi­ cas: 1. controle nas mãos de uma junta de diretores e prin­ cipais funcionários executivos (o poder real não está nas mãos de quem administra a empresa); 2. a administração se dá por um grupo que se autoperpetua; 3. B. Capitalismo Monopolista a empresa bus­ ca atingir independência financeira pela criação interna de fundos que permanecem à disposição da administração. Baran e Sweezy (1966) defendem que o capitalismo monopolista é uma resposta ao alto grau de excedente eco­ nômico produzido pela sociedade e que não seria passível de investimento. Eles defendem que os mercados não tinham mais a possibilidade de absorção do excedente produzido. Dessa forma, o excedente obtido no capitalismo liberal pela exploração de mão de obra, consumo e acumulação teve que encontrar novas formas de ser produzido na nova con­ figuração do sistema. Os autores destacam três estratégias para produção de excedente: campanhas de venda, Estado ou Governo e militarismo com imperialismo. Os oligopólios conseguiram uma crescente indepen­ dência tanto em relação aos banqueiros como aos acionis­ tas dominantes. Suas políticas ligaram-se, em proporções cada vez maiores, aos seus próprios interesses, ao invés de subordinar-se às conveniências de um grupo. A tecnologia, como já referido, tem papel fundamen­ tal no investimento e aumento do excedente, pelas cam­ panhas de vendas. Novos métodos de produção e novos produtos aumentam o lucro, acentuando o caráter da bus­ ca dirigida, lucrativa e institucionalizada pela novidade. A procura da invenção de novos produtos é constante, mas com limitações. Assim, as campanhas de vendas são uma das estratégias criadas para a manutenção do con­ sumo em certa escala. Outra estratégia é a obsolescên­ cia planejada, ou seja, alterações mínimas de produtos, variação de aspectos e embalagem, modificação de mo­ delo. A obsolescência planejada visa à criação de novas necessidades de consumo a fim de serem sanadas por “novos” produtos. Tal processo segue infinitamente, con­ tendo em si uma lógica perversa: escondendo a ausência de novidades no mecanismo. Esse processo também gera o alheamento dos indivíduos de suas necessidades pela imposição de novas, relacionadas ao consumo. Com isso, na figura dos administradores se dá um pro­ cesso de mediação entre os interesses individuais e os da empresa: na medida em que a empresa avança, o indivíduo seria recompensado, confundindo-se o progresso da insti­ tuição com o pessoal. Tais elementos acabam por se tornar as finalidades e valores subjetivos do mundo dos negócios, porque são as exigências do sistema. O caráter deste de­ termina a psicologia dos seus membros, e não o inverso. O verdadeiro capitalista no sistema monopolista não é o empresário individual, mas as empresas. Estas são uma versão ampliada e institucionalizada do capitalista pessoal. Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 B. Capitalismo Monopolista Essa mediação histórica ocorreu na consciência e na ação política das duas grandes classes que se defrontavam na sociedade: a burguesia e o proletariado. No mundo capita­ lista, ainda são as classes básicas. Contudo, o desenvolvi­ mento capitalista alterou a estrutura e a função dessas duas classes de tal modo que elas não mais parecem ser agentes de transformação histórica. Um interesse predominante na preservação e no melhoramento do status quo institucional une os antigos antagonistas nos setores mais avançados da sociedade contemporânea (MARCUSE, 1973, p. 16). produzido. Dessa maneira, os Estados nacionais transfor­ mam-se em geradores de renda. Além disso, a necessida­ de de dinheiro para a manutenção política faz com que as grandes empresas sejam os grandes agentes do capitalismo monopolista. O dinheiro se concentra em grandes volumes nas empresas e torna-se fonte de poder político. Quando as grandes oligarquias veem seu poder ou suas fontes de lucros ameaçados (pelo investimento em educação, moradia, assistência), poderosas instâncias de resistência são mobilizadas. Uma delas é a substituição do governo democrático pelo autoritário. As classes pri­ vilegiadas dominam os instrumentos de coação e lançam mão de suas artimanhas. Vale lembrar que o poder políti­ co é financiado por elas. Em países com histórico demo­ crático há preferência por pequenas concessões que não ferem a ordem estabelecida e reduz a fúria dos oposito­ res. Já em países com histórico autoritário há a ascensão (por vias democráticas ou não) de regimes fascistas. Dessa maneira, a produtividade serve como elemento de coerção e coesão social, impondo-se acima de inte­ resses individuais e grupais. O governo, no capitalismo monopolista, reafirma seu poder e se desenvolve a par­ tir da mobilização, organização e exploração com êxi­ to da produtividade técnica e mecânica à disposição na sociedade. Assim, é inútil falar de liberdade (política, econômica e intelectual) mediante o totalitarismo do sis­ tema. Esse deixa à mostra suas diversas irracionalidades, como a capacidade de conter a transformação social. Isso conduz a uma das grandes contradições do capitalismo monopolista que é a intensificação dos preconceitos e, consequentemente, o aumento da discriminação e segre­ gação. O nazismo é uma dessas manifestações. Baran e Sweezy (1966) destacam que uma das for­ mas para que investimentos públicos não alterem os pri­ vilégios das elites é direcionando-os para fontes que não apresentem riscos e que fortalecem o controle e a coesão social. A. Características da formação do indivíduo no capitalismo liberal Segundo Marcuse (1998a), a ideologia liberal, que deu sustentação à configuração do capitalismo liberal, preconizava a transformação do modelo burguês de in­ divíduo e dos valores atribuídos a este como unidade fundamental e fim da sociedade. A autonomia seria a ca­ racterística genuína do indivíduo de acordo com tendên­ cias políticas, econômicas e religiosas dos séculos XVI e XVII. O indivíduo deveria ser portador de certos padrões e valores fundamentais que nenhuma autoridade externa deveria desrespeitar. Tais padrões e valores referiam-se a formas de vida, tanto social como pessoal, que são a verdade da existência individual. Cada indivíduo seria portador de razão e capaz de encontrar suas verdades por meio da liberdade de pensamento, capacidade de ação e transformação da realidade. Aos indivíduos a sociedade deveria liberdade e a eliminação todas as restrições à sua linha de ação racional. Tal concepção é denominada de individualismo (que apregoa a busca do interesse próprio guiado pela razão individual ou pensamento autônomo). O individualismo é considerado a base do capitalismo li­ beral dos séculos XVIII e XIX. Baran e Sweezy (1966) destacam que o capitalismo monopolista gera a tendência de ciclos de estagnação e depressão. A constante racionalização e aumento da pro­ dução por empresas gigantes não é acompanhada de al­ terações na organização social e eliminação da miséria. Uma das consequências da irracionalidade do todo é o deserto ideológico, característico desse sistema. Em relação ao trabalho, Marcuse (1973) destaca que ele se torna labuta e estupefaciente. Isso ocorre quanto mais real se demonstra a sua não necessidade. Os mo­ dos de descanso mitigam e prolongam a estupefação. As liberdades acabam por se mostrar decepcionantes, tais como: consumo do inútil, livre competição a preços ad­ ministrados, uma imprensa livre que se autocensura. Baran e Sweezy (1966) ressaltam que é ideologia acreditar na livre iniciativa e na democracia do capitalis­ mo monopolista. Ele preconiza uma pequena oligarquia com enorme poder econômico que controla a tomada de decisões pela manipulação do aparato político e cultural. Marcuse (1998a) destaca, ainda, que o interesse pró­ prio racional da sociedade não coincidia com o interesse próprio imediato do indivíduo. O indivíduo, para alcan­ çar os seus interesses, dependeria dos padrões e deman­ das da ordem social dominante. Tais padrões e demandas seriam instituídos por autoridades externas e não pelo pensamento autônomo ou a consciência. B. Capitalismo Monopolista Os gastos militares e o imperialismo oferecem uma saída para tal situação. Entretanto, a militarização estimula forças reacionárias e irracionais na sociedade, inibindo e matando o que é progressista e humano. A militarização da sociedade leva ao respeito cego à auto­ ridade, atitudes de docilidade e conformismo. Elas são pregadas e impostas. A discordância é tratada como falta de patriotismo ou traição (ADORNO, 2006). A militari­ zação traz à tona os elementos fascistas e totalitários da sociedade capitalista. Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 B. Capitalismo Monopolista Elas possuem um horizonte temporal mais am­ plo e são calculadores mais racionais, pela racionalização da administração. As empresas transformam-se em tec­ nocracias, assumindo características próprias nas mãos da administração interna. Elas visam, portanto, à redu­ ção de custos, expansão da receita e aumento dos lucros para a instituição. Tais transformações têm impacto nos processos de subjetivação, pois há a substituição de in­ divíduos com racionalidade e interesse próprio por ins­ tituições com tais características, devendo os homens se submeter a tais pressupostos (BARAN; SWEEZY, 1966; HORKHEIMER, 2010). Os autores defendem que a publicidade é produto do capitalismo monopolista e procura envolver os pro­ dutos de certas características. Ela é, portanto, resultado da decadência da concorrência e leva os consumidores a fazerem escolhas por motivos irracionais. A publicidade mantém o sistema pela expansão do consumo, tendo em vista a tendência deste à estagnação. Nessa nova organização do capitalismo, a transfor­ mação tecnológica assume papel crucial. Segundo Mar­ cuse (1998a), a eficiência produtiva torna-se fundamental e favorece empresas com o equipamento industrial mais altamente mecanizado, racionalizado e avançado. Esse processo de acumulação do poder tecnológico favorece a concentração do poder econômico, com a formação de enormes empresas associadas. Tal conglomerado empre­ sarial forma um aparato (instituições, dispositivos e orga­ O Estado, segundo Baran e Sweezy (1966), tem a fi­ nalidade de servir aos interesses do capital monopolista, por meio da absorção de grande parte do excedente, direta ou indiretamente. Os usos que o governo faz do excedente está determinado por interesses do sistema monopolista e, com o passar do tempo, tal procedimento se torna cada vez mais irracional e destrutivo. Destaca-se que, na forma como o capitalismo monopolista se organiza, os meios pri­ vados não têm a capacidade de absorver todo o excedente Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 85 Gustavo Henrique Carretero Essa mediação histórica ocorreu na consciência e na ação política das duas grandes classes que se defrontavam na sociedade: a burguesia e o proletariado. No mundo capita­ lista, ainda são as classes básicas. Contudo, o desenvolvi­ mento capitalista alterou a estrutura e a função dessas duas classes de tal modo que elas não mais parecem ser agentes de transformação histórica. Um interesse predominante na preservação e no melhoramento do status quo institucional une os antigos antagonistas nos setores mais avançados da sociedade contemporânea (MARCUSE, 1973, p. 16). A. Características da formação do indivíduo no capitalismo liberal Marcuse (1973) ressalta que nesse contexto há a su­ focação da necessidade de libertação; ele mantém a so­ ciedade sob suas bases irracionais. O fim das lutas entre operários e elites ocorre mediante a assimilação dos pri­ meiros ao mundo do consumo. Tal elemento é um dos sintomas de tal sufocação: Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 86 Transformações do capitalismo e formação do indivíduo: contribuições da Escola de Frankfurt na análise das eleições presidenciais nos EUA e Brasil Transformações do capitalismo e formação do indivíduo: contribuições da Escola de Frankfurt na análise das eleições presidenciais nos EUA e Brasil No período liberal do capitalismo, o indivíduo era colocado contra a sociedade, buscando superar todas as imposições e impor os próprios interesses racionais. Isso levava a um estado de constante vigilância, apreen­ são e crítica, que rejeitava o que não fosse verdadeiro nem justificado pela livre razão. Em uma sociedade com grandes contradições, a possibilidade da crítica e análise racional dos próprios interesses constituía a possibilidade de inquietação e oposição. Portanto, a liberdade estava associada à crítica aos padrões vigentes, procura de pa­ drões verdadeiros e tentativa de realização (mesmo que confrontando toda a sociedade). a falta de relacionamento e interesse emocional entre as pessoas (como as redes sociais na contemporaneidade). Adorno (1991, 2006) propõe que a nova organização do capitalismo leva os indivíduos a estados regredidos de consciência, atrelando tal concepção ao narcisismo, uma das descobertas de Freud. Baran e Sweezy (1966) sugerem que a crescente ra­ cionalização das esferas econômicas e humanas levou ao desaparecimento de qualquer finalidade significativa em relação à vida. Isso ocorre tanto socialmente como in­ dividualmente. O trabalho perde o seu sentido, transfor­ mando-se em labuta, e o tempo livre causa aborrecimento devido ao ar de inutilidade de que a vida se reveste. Para que o indivíduo realizasse sua racionalidade se­ ria necessário um ambiente social e econômico adequa­ do, que atraísse indivíduos cujas condutas e realizações sociais fossem seu próprio trabalho. A sociedade liberal era ideal à racionalidade individualista, pois, pela livre concorrência, o indivíduo transformava (pelos seus feitos e ação) o produto de seu trabalho em parte das neces­ sidades da sociedade. Assim, o produto do trabalho de cada indivíduo se tornaria marca de sua individualidade. B. Características da formação do indivíduo no capitalismo monopolista No capitalismo monopolista, a contraposição entre in­ divíduo e sociedade (que visava a uma reforma militan­ te da segunda com base nos interesses do indivíduo) se desfaz pela forma como o sistema passa a se organizar. A oposição é substituída pela adesão. Tal processo prepara e justifica o afastamento do indivíduo da sociedade. A auto­ nomia, tema essencial da ideologia liberal, passa a ser vista como assunto privado em vez de público, um elemento de retirada em vez de agressão. A contenção e a resignação tornam-se cada vez mais presentes na filosofia individu­ alista, ilustrando o ocaso do modelo de indivíduo liberal. Para Baran e Sweezy (1966) um dos sintomas da “extinção” do indivíduo é o aborrecimento causado pe­ las horas livres, que podem ser mais opressivas do que o trabalho, de tal maneira que o tempo é preenchido com diversas trivialidades ou encontros sociais vazios. A vida e o tempo livre tornam-se ociosidade ou trivialidade. As conversas reduzem-se aos bate-papos entre amigos. Os elementos expostos têm relação direta com a for­ mação do indivíduo, ao ponto de os autores da primei­ ra geração da Escola de Frankfurt anunciarem o ocaso deste a partir do capitalismo monopolista e das altera­ ções políticas, sociais, econômicas e culturais geradas (HORKHEIMER, 2010; ADORNO, 1971, 1991; MAR­ CUSE, 1998b, 1999). Horkheimer e Adorno (1985) “criam” o conceito de Indústria Cultural para denotar como até mesmo nas ho­ ras livres o trabalho alienado e o totalitarismo da cultura invadem a vida. Tal processo destitui de sentido a divisão entre as esferas pública e privada e prepara os indivíduos para a adesão, consumo e labuta. Baran e Sweezy (1966), mesmo não pertencendo a tal referencial teórico, percebem os sintomas da derro­ cada da individualidade. Entre outros elementos, eles apontam para a desintegração da família burguesa, tendo como decorrência a crescente solidão e miséria no capi­ talismo monopolista. A solidão não pode ser superada, transformando cada um em uma partícula na multidão pela necessidade de adesão. A miséria de estar só e o horror da companhia de terceiros produzem uma atitude de ambivalência entre o envolvimento e o alheamento. Dessa maneira, são criados mecanismos para disfarçar Marcuse (1973) destaca que um dos aspectos mais per­ turbadores do capitalismo monopolista é o caráter racional de sua irracionalidade. A. Características da formação do indivíduo no capitalismo liberal Adorno (2006) destaca algumas qualidades do indivíduo em seu molde liberal: este é filho de uma sociedade libe­ ral, competitiva, individualista e está condicionado a se manter como unidade independente e autossustentável, sendo continuamente advertido de que deve ser duro e prevenido contra rendição. A sexualidade, a partir do fracasso do prazer no tra­ balho e em outras esferas da vida, no capitalismo mono­ polista torna-se uma das principais formas de satisfação, mas cuja finalidade não pode ser atingida em uma so­ ciedade em que os padrões morais humanos e os meios de satisfação encontram-se aquém do que poderiam e até mais regredidos do que em outros momentos da história. O indivíduo torna-se um mero apêndice da sociedade, um átomo social do qual a totalidade enlouquecida pode abrir mão (HORKHEIMER, 2010; ADORNO, 1991, 2006; MARCUSE, 1973, 1998b). Devido à crescente especialização e racionalização dos processos sociais, o indivíduo pode ser comprimido em esquemas redutores impostos de fora. O resultado de tal processo não pode ser chamado de individualidade ou subjetividade, mas um produto humano padronizado, racionalizado e sistematica­ mente controlado por meio de levantamentos estatísticos. As reações ditas individuais se tornam cada vez mais auto­ máticas e previsíveis. Horkheimer (2010) chega ao ponto de afirmar que toda defesa da individualidade e unicidade dos homens cai por terra diante de questionários ou testes que englobam a existência da maioria dos homens. Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 B. Características da formação do indivíduo no capitalismo monopolista A produtividade, eficiência, capaci­ dade para aumentar e disseminar comodidades, transfor­ mar resíduo em necessidade e a destruição em construção fazem com que tal sistema transforme o mundo objetivo em uma extensão da mente e do corpo humanos. Isso torna questionável até a própria noção de alienação, pois os in­ divíduos conseguem se reconhecer em suas mercadorias. O controle social do indivíduo se dá na produção de novas Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 87 Gustavo Henrique Carretero necessidades e altera o mecanismo que ata o indivíduo à sua sociedade: no liberalismo a oposição e no capitalismo monopolista a assimilação e adesão. A sociedade atende às necessidades do indivíduo pela forma como está organiza­ da e reduz a possibilidade de oposição: ilustração da estrutura da racionalidade tecnológica. Com isso, a razão se transforma: de força crítica para elemento de ajuste e submissão. Assim, a razão perde autonomia ao mesmo tempo em que os pensamentos, sentimentos e ações da humanidade são moldados pelas exigências téc­ nicas do aparato que ela mesma criou. O túmulo da razão é o sistema de controle, produção e consumo padronizado que acaba por padronizar os homens e a racionalidade. Essa repressão, tão diferente daquela que caracterizou as etapas anteriores, menos desenvolvidas, de nossa so­ ciedade, não opera, hoje, de uma posição de imaturidade natural e técnica, mas de força. As aptidões (intelectuais e materiais) da sociedade contemporânea são incomensura­ velmente maiores do que nunca dantes – significando que o alcance da dominação da sociedade sobre o indivíduo é incomensuravelmente maior do que nunca dantes. A nossa sociedade se distingue por conquistar forças sociais centrí­ fugas mais pela Tecnologia do que pelo Terror, com dúpli­ ce base numa eficiência esmagadora e num padrão de vida crescente (MARCUSE, 1973, p. 14). Para Marcuse (1998a), no capitalismo monopolista os padrões (de consumo, racionalidade, atitudes e individu­ alidade) passam a ser ditados de fora aos indivíduos, que devem se adequar ao aparato. A liberdade destes reduz-se à seleção dos meios mais adequados para alcançar uma meta determinada exteriormente. Os desafios lançados pelas alterações na formação do indivíduo nas transformações do capitalismo são temas extremamente importantes à Psicologia que se diz crítica. Adorno (1991) destaca a desproporção no campo so­ cial entre as grandes empresas, o governo, o exército e as instituições com o indivíduo. 3 - Formação do indivíduo na contemporaneidade O modelo de indivíduo das revoluções burguesas (pautado no modelo liberal) torna-se obsoleto no capi­ talismo monopolista. Tudo contribuiu para transformar os instintos, desejos e pensamentos humanos em canais que alimentam o aparato industrial. O sistema produtivo impõe suas exigências econômicas e políticas à defesa e à expansão do tempo de trabalho no tempo livre. A sociedade contemporânea é totalitária pela forma como organizou sua base tecnológica, pois esta invade todas as esferas da vida humana (MARCUSE, 1973). Destaca-se que as reflexões dos frankfurtianos não apontam a nostalgia pelo modelo liberal de indivíduo. Elas indicam possibilidades negadas pelo “desenvolvimento” e que só podem ser realizadas pela superação do capitalismo e de sua racionalidade/irracionalidade. Apenas a subversão da própria ordem objetiva poderia “libertar” as forças negadas no desenvolvimento de tal sistema (MARCUSE, 1999). Um dos elementos destacados por Horkheimer (2010) para o totalitarismo da sociedade sobre o indivíduo é a al­ teração da racionalidade. A razão se tornou independente do indivíduo. Ela não se pauta por elementos humanos, mas relacionados ao funcionamento do aparato e manu­ tenção da ordem. A explosão do nazismo na Alemanha é um dos exemplos mais contundentes de tal manifestação. As reflexões de Adorno, Marcuse e Horkheimer, ape­ sar de terem sido escritas em meados do século passado, permanecem extremamente atuais na contemporaneida­ de. Algumas críticas de tais autores se tornam fundamen­ tais à compreensão do atual estágio de desenvolvimento do capitalismo (financeiro), tais como: anacronismo do modelo burguês de individualidade, enfraquecimento de capacidades críticas e “fusão” entre interesses individu­ ais e coletivos. Tais elementos podem ser considerados sintomas da pertinência de tais contribuições. Ainda mais no momento do “reaparecimento” das massas tota­ litárias e fascistas por meio de seu controle tecnológico contemporâneo. Vale destacar que o fascismo não pode ser superado no capitalismo por ser decorrente da própria racionalidade de tal sistema produtivo. No mesmo sentido, Marcuse (1998a) destaca que os indivíduos, na atual sociedade, são despidos de sua indi­ vidualidade. Isso não ocorre pela coerção externa, mas pela própria racionalidade sob a qual vivem. O aparato ao qual o indivíduo deve adaptar-se é tão racional que o protesto e a libertação individual parecem, além de inú­ teis, absolutamente irracionais. A racionalidade individualista do período liberal trans­ forma-se em racionalidade tecnológica. Segundo o autor, esta estabelece padrões de julgamento e fomenta atitudes que predispõem os homens a aceitar e introjetar os dita­ mes do sistema. B. Características da formação do indivíduo no capitalismo monopolista Perante a grandeza das insti­ tuições são necessárias forças sobre-humanas para se opor. Até mesmo a sobrevivência se torna impossível fora do sistema. A vida reduz-se à submissão para a autoconserva­ ção. Para Marcuse (1998a), ao manipular a máquina o ho­ mem aprende que a obediência é o único meio de se obter resultados desejados. Portanto, a adaptação ao aparato é a única saída, e ela se impõe sobre a racionalidade individu­ alista, preconizada no período liberal do capitalismo. Os autores da primeira geração da Escola de Frank­ furt sugerem, a partir de suas reflexões, que a liberdade do sujeito econômico no capitalismo liberal foi submersa na eficiência, e a racionalidade se tornou poder social. Ao indivíduo não resta outra possibilidade senão a de adap­ tação sem ressalvas para a manutenção de sua existência, que se reduz à autoconservação. No capitalismo mono­ polista as condições que formam a individualidade se rendem às condições que tornam a individualidade des­ necessária (ADORNO, 1991, 2006). A padronização em todas as esferas demonstra que a criatividade e a origina­ lidade individuais tornam-se desnecessárias. Com o fim da era liberal, estas foram desaparecendo do domínio da produção material e tornando-se cada vez mais proprie­ dade exclusiva das elevadas atividades intelectuais, ten­ dendo a desaparecer destas devido à cultura de massas. Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 3 - Formação do indivíduo na contemporaneidade A eficiência submissa é a mais perfeita O desenvolvimento tecnológico das últimas déca­ das potencializou processos que sempre permaneceram latentes na sociedade capitalista. As últimas eleições Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 88 Transformações do capitalismo e formação do indivíduo: contribuições da Escola de Frankfurt na análise das eleições presidenciais nos EUA e Brasil o indivíduo: contribuições da Escola de Frankfurt na análise das eleições presidenciais nos EUA e Brasil a de Frankfurt na análise das eleições presidenciais nos EUA e Bras presidenciais nos EUA e no Brasil demonstram como algoritmos de computadores podem prever facilmente o comportamento dos “indivíduos”. A novidade de tal processo é a utilização política eleitoral de tecnologia anteriormente utilizada para campanhas publicitárias. Tal processo ocorre com o uso maciço de tecnologias pu­ blicitárias utilizando-se de conhecimentos sociológicos e psicológicos. Soma-se a isso, na contemporaneidade, tecnologias de controle da navegação na internet. Atualmente, a mensagem é encaminhada de modo pri­ vado, atingindo inconscientemente e de forma “personali­ zada” o indivíduo, de acordo com seu perfil. Tal fato só se tornou possível devido aos avanços tecnológicos usados contra a humanidade e na defesa de interesses escusos. Acrescenta-se que tais mensagens, por serem transmitidas por aplicativos de celulares e/ou computadores, apresen­ tam certa dificuldade de controle social e político. A partir do exposto, pode-se inferir que as contribui­ ções dos frankfurtianos tornaram-se anacrônicas quanto aos meios (tecnológicos) pelos quais são transmitidas, mas são extremamente atuais quanto aos seus efeitos psi­ cossociais. Além disso, processos de produção industrial e massificada de indivíduos e subjetividades, descritos pelos autores, ganham contornos jamais imaginados de­ vido ao desenvolvimento e controle tecnológico. Nas campanhas publicitárias é feita a segmentação da população em determinados perfis (de acordo com sites visitados, comportamento em redes sociais, etc.) e envio de mensagens eletrônicas “personalizadas” de acordo com “interesses” pessoais. Pode-se dizer que o mesmo procedimento foi utilizado para eleições presidenciais, tanto no Brasil como nos EUA. A tecnologia moderna re­ finou as estratégias dos agitadores fascistas transmitidas por rádio e TV e descritas por Adorno (2006). O docu­ mentário recente denominado “Driblando a democracia: como Trump venceu” (online) demonstra como foi utili­ zado controle tecnológico das redes sociais e algoritmos de computadores para vencer as últimas eleições presi­ denciais americanas. Ele aponta como o comportamen­ to político dos eleitores foi meticulosamente estudado e compilado por meio das redes sociais. APA Carretero, G. H. (2019). Transformações do capitalismo e formação do indivíduo: contribuições da Escola de Frankfurt na análise das eleições presidenciais nos EUA e Brasil. Fractal: Revista de Psicologia, 31(2), 83-90. doi:10.22409/1984-0292/v31i2/5619 As reflexões da primeira geração da Escola de Frank­ furt estão voltadas para os meios de comunicação de massa do período de seus escritos. Em tal período as mensagens dos agitadores fascistas eram transmitidas pelas ondas do rádio e da TV. Tais mensagens podiam ser alvo de controle e crítica social. Ao mesmo tempo, as mensagens deveriam possuir determinado grau de gene­ ralidade para atingir o maior número de indivíduos. Além disso, corriam o risco “de se perderem no ar”. 3 - Formação do indivíduo na contemporaneidade Pelo uso de algo­ ritmos, tais informações foram transformadas em perfis padronizados de eleitores. Cada segmento estava relacio­ nado à sensibilidade sobre determinadas temáticas, tais como: porte de armas, defesa da propriedade privada, liberdades civis de minorias, corrupção, etc. A possibilidade da defesa de certa autonomia frente ao todo não deve ser colocada em segundo plano. Mes­ mo que tal mote possua elementos ideológicos, ele deve superar seu viés liberal ainda mais em tempos do reapa­ recimento de movimentos totalitários. Tais movimentos, apesar das diferenças em relação aos do século passado, ainda visam à integração total dos indivíduos e tendem a desqualificar toda diferença. Portanto, o debate e a luta pelo indivíduo (único em meio ao total) em uma socie­ dade totalitária é também uma política pela superação do capitalismo, o futuro da humanidade, e pela própria supe­ ração da concepção burguesa de individualidade. Gustavo Henrique Carretero https://orcid.org/0000-0002-9633-2581   http://lattes.cnpq.br/6373778457510094 Possui graduação em Psicologia pela Pontifícia Universidade Católica de Campinas (2006), mestrado em Psicologia (Psicologia Social) pela Pontifícia Universidade Católica de São Paulo (2011) e doutorado em Psicologia (Psicologia Social) pela Pontifícia Universidade Católica de São Paulo (2018). Possui graduação em Psicologia pela Pontifícia Universidade Católica de Campinas (2006), mestrado em Psicologia (Psicologia Social) pela Pontifícia Universidade Católica de São Paulo (2011) e doutorado em Psicologia (Psicologia Social) pela Pontifícia Universidade Católica de São Paulo (2018). Como citar este artigo: Além da segmentação de perfis, se percebe também a produção industrial e massificada de notícias falsas. Es­ sas foram distribuídas de acordo com perfis de sensibili­ dade a temáticas específicas. Tal processo fez com que defensores da irracionalidade se travestissem de defen­ sores de interesses coletivos. Ao mesmo tempo, questio­ na-se a possibilidade de escolhas livres pelos indivíduos. Não se devem considerar as pessoas que foram manipu­ ladas como agentes meramente passivos. Havia, mesmo que inconscientemente, em cada indivíduo algo que os fazia aderir a tais mensagens (ADORNO, 1991, 2006). ABNT CARRETERO, Gustavo Henrique. Transformações do capitalismo e formação do indivíduo: contribuições da Escola de Frankfurt na análise das eleições presidenciais nos EUA e Brasil. Fractal: Revista de Psicologia, Niterói, v. 31, n. 2, p. 83-90, maio/ago. 2019. https://doi.org/10.22409/1984-0292/v31i2/5619 Informações sobre o autor: Mensagens “personalizadas” que apelavam para medos inconscientes de determinados segmentos eram encaminha­ das de forma privada no “Facebook”. E se nos EUA tal estra­ tégia foi arquitetada pelo uso do Facebook, no Brasil houve a preferência pela criação de grupos temáticos no WhatsA­ pp. Tal elemento é de conhecimento popular por meio de reportagem no jornal “Folha de São Paulo” (MELLO, 2018, online). Todavia, a reportagem não traz maiores informa­ ções sobre a segmentação dos perfis de mensagens. Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 Referências ADORNO, Theodor Ludwig Wiesengrund. La revision del Psicanalisis (1942). In: HORKHEIMER, Max; ADORNO, Theodor Ludwig Wiesengrund. Sociologica II. Madrid: Taurus, 1971. p. 133-156. ADORNO, Theodor Ludwig Wiesengrund. De la relación entre Sociología y Psicología (1955). In: ______. Actualidad de la Filosofia. Barcelona: Paidós, 1991. p. 135-205. Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 89 Gustavo Henrique Carretero ADORNO, Theodor Ludwig Wiesengrund. A teoria freudiana e o padrão de propaganda fascista (1951). Margem esquerda: ensaios marxistas, v. 7, p. 164-189, 2006. BARAN, Paul A.; SWEEZY, Paul. M. Capitalismo monopolista: ensaio sobre a ordem econômica e social americana. Rio de Janeiro: J. Zahar, 1966. CROCHIK, José Leon. Os desafios atuais do estudo da subjetividade na Psicologia. Psicologia USP, São Paulo, v. 9, n. 2, p. 69-85, 1998. http://dx.doi.org/10.1590/S0103- 65641998000200003 DRIBLANDO a democracia: como Trump venceu [documentário]. Direção de Thomas Huchon. Paris: Spicee, 2018. (52 min.), son., color. Disponível em: http://canalcurta. tv.br/filme/?name=driblando_a_democracia_como_trump_ venceu. Acesso em: 22 dez. 2018. HORKHEIMER, Max; ADORNO, THEODOR LUDWIG WIESENGRUND. Dialética do Esclarecimento (1944). Rio de Janeiro: J. Zahar, 1985. HORKHEIMER, Max. Eclipse da Razão (1955). São Paulo: Centauro, 2010. MARCUSE, HERBERT. Ideologia da sociedade industrial: o homem unidimensional (1964). Rio de Janeiro: J. Zahar, 1973. MARCUSE, HERBERT. Algumas implicações sociais da tecnologia moderna (1941). In: ______. Tecnologia, guerra e fascismo. São Paulo: UNESP, 1998a. p. 73-104. MARCUSE, HERBERT. A Obsolescência da Psicanálise (1963). In: ______. Cultura e Sociedade. Rio de Janeiro: Paz e Terra, 1998b. v. 2, p. 91-111. MARCUSE, HERBERT. Eros e civilização: uma interpretação filosófica do pensamento de Freud (1959). 8. ed. Rio de Janeiro: Livros Técnicos e Científicos, 1999. MELLO, Patrícia Campos. Empresários bancam campanha contra o PT pelo WhatsApp. Folha de São Paulo, São Paulo, 18 out. 2018. Disponível em: https://www1.folha.uol.com.br/ poder/2018/10/empresarios-bancam-campanha-contra-o-pt- pelo-whatsapp.shtml. Acesso em: 4 dez. 2018. Fractal, Rev. Psicol., v. 31 – n. 2, p. 83-90, 2019 90
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https://eprints.qut.edu.au/220844/1/Development.pdf
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Development of hybrid materials based on sponge supported reduced graphene oxide and transition metal hydroxides for hybrid energy storage devices
Scientific reports
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This may be the author’s version of a work that was submitted/accepted for publication in the following source: ( ) Development of hybrid materials based on sponge supported reduced graphene oxide and transition metal hydroxides for hybrid energy storage devices. Scientific Reports, 4, Article number: 7349 1-10. This file was downloaded from: https://eprints.qut.edu.au/220844/ OPEN Received 11 September 2014 Accepted 11 November 2014 Published 8 December 2014 Deepak P. Dubal1,2, Rudolf Holze2 & Pedro Gomez-Romero1,3 1Catalan Institute of Nanoscience and Nanotechnology, CIN2, ICN2 (CSIC-ICN), Campus UAB, E-08193 Bellaterra, Barcelona, Spain, 2Technische Universita¨t Chemnitz, Institut fu¨r Chemie, AG Elektrochemie, D-09107 Chemnitz, Germany, 3Consejo Superior de Investigaciones Cientı´ficas (CSIC), Spain. Earnest efforts have been taken to design hybrid energy storage devices using hybrid electrodes based on capacitive (rGO) and pseudocapacitive (Ni(OH)2 and Co(OH)2) materials deposited on the skeleton of 3D macroporous (indicate sponge material) sponge support. Conducting framework was formed by coating rGO on macroporous sponge on which subsequent deposition of Ni(OH)2 and Co(OH)2 was carried out. The synergetic combination of rGO and Ni(OH)2 or Co(OH)2) provides dual charge-storing mechanisms whereas 3D framework of sponge allows excellent accessibility of electrolyte to hybrid electrodes. Moreover, to further increase the energy density, hybrid devices have been fabricated with SP@rGO@Ni or SP@rGO@ Co and SP@rGO as positive and negative electrodes, respectively. These hybrid devices operate with extended operating voltage windows and achieve remarkable electrochemical supercapacitive properties which make them truly promising energy storage devices for commercial production. Correspondence and requests for materials should be addressed to D.P.D. (dubaldeepak2@ gmail.com) or P.G.-R. (pedro.gomez@cin2. es) N N owadays, energy-related science and technology are benefiting from a big tsunami of hybridization, starting from hybrid materials to hybrid devices1. In hybrid materials, there are synergistic effects of materials properties whereas hybrid devices combine the different mechanisms of devices in a single one. The field of hybrid energy storage devices is no exception. Among different energy storage systems playing on the chessboard of Ragone plot, supercapacitors are getting increasing attention from scientists and industrialists alike due to their excellent performance at high power rates2,3. Indeed, supercapacitors are intrinsically well suited to provide high power density by storing charge non-faradically at the electrode/electrolyte interface (electrochem- ical double layer capacitor, EDLC) or through redox mechanism at the electrode surface (pseudocapacitor) hence allowing for high power, fast charge-discharge rates and excellent cycling life4. As we look back, supercapacitors are highly effective and are integrated in many of our daily-live applications from portable devices such as mobile- phones, laptops, cameras to hybrid electric vehicles etc. Despite this success these systems still have boundaries such as low energy density in EDLC, or short cycle life and limited power capability in pseudocapacitors5. © Consult author(s) regarding copyright matters This work is covered by copyright. Unless the document is being made available under a Creative Commons Licence, you must assume that re-use is limited to personal use and that permission from the copyright owner must be obtained for all other uses. If the docu- ment is available under a Creative Commons License (or other specified license) then refer to the Licence for details of permitted re-use. It is a condition of access that users recog- nise and abide by the legal requirements associated with these rights. If you believe that this work infringes copyright please provide details by email to qut.copyright@qut.edu.au License: Creative Commons: Attribution 2.5 Notice: Please note that this document may not be the Version of Record (i.e. published version) of the work. Author manuscript versions (as Sub- mitted for peer review or as Accepted for publication after peer review) can be identified by an absence of publisher branding and/or typeset appear- ance. If there is any doubt, please refer to the published source. https://doi.org/10.1038/srep07349 Development of hybrid materials based on sponge supported reduced graphene oxide and transition metal hydroxides for hybrid energy storage devices Development of hybrid materials based on sponge supported reduced graphene oxide and transition metal hydroxides for hybrid energy storage devices Correspondence and requests for materials should be addressed to D.P.D. (dubaldeepak2@ gmail.com) or P.G.-R. (pedro.gomez@cin2. es) Results h 2(a, b) reveal, the 3D open porous nature of the sponge substrate is maintained even after the coating of rGO and deposition of Ni(OH)2 and Co(OH)2 nanostructures. Fig. 2(c and d) shows hierarchically porous well networked structure of thin b-Ni(OH)2 nanosheets and b-Co(OH)2 nanoflakes. These nanostructures are interconnected with each other, providing open porous structure which enables the full exposure of the arrays to the electrolyte. The open geometry between the arrays allows easier elec- trolyte penetration into the inner region of the electrode, increasing the utilization of the active materials. The skeleton of the sponge is fully covered by both Ni(OH)2 and Co(OH)2 nanostructures so that no rGO is exposed to the surface (Supporting information S3). The thicknesses of the Ni(OH)2 and Co(OH)2 coatings were found to be ,1.2 and ,1.3 mm, respectively which can be measured by the cross- section SEM images in Fig. 2(e–f). A possible growth process will be as follows: Initially, dissolved ammonia forms a complex with metal ions (Ni21 and Co21), which decreases the free metal ion concentra- tion and reduces the rate of crystal growth. Formation of metal ammine complex avoids homogeneous nucleation in the solution due to the binding of metal ions and provides priority to heterogen- eous nucleation on the substrate. Further, as the reaction time increases (at a constant temperature), hydrolysis of HMT molecules takes place. This increases the concentration of OH2 in the solution which encourages the formation of thin Ni(OH)2 nanosheets and Co(OH)2 nanoflakes15. Here, HMT provides controlled supply of ammonia via the hydrolysis reaction, while unreacted HMT selec- tively adsorbs on the metal hydroxide nanoparticles due to its high binding capacity16, resulting in the formation of nanosheets/nano- In this work we have successfully used both of those possible optimization pathways (CBD on foam substrates) to improve the performance of supercapacitors in aqueous system. According to the equation for energy storage i.e. E 5 0.5 CV2, the performance of the supercapacitors can be enhanced by increasing capacitance and extending the operational voltage window using different con- figurations. Results h graphene frameworks10–13. To this list of possible substrates for hybrid supercapacitive electrodes, a new very practical contender has been recently added, namely, commercially available sponges with 3D porous structure coated with carbon. The sponge substrate provides smooth inner and outer sides of the skeleton for the depos- ition of materials and its soft, lightweight, flexible and cost-effective nature makes it more attractive for flexible devices. Moreover, macroporosity and high surface area of sponge provide an easy path to the electrolyte to reach the entire surface of the electrodes where redox reactions can take place. graphene frameworks10–13. To this list of possible substrates for hybrid supercapacitive electrodes, a new very practical contender has been recently added, namely, commercially available sponges with 3D porous structure coated with carbon. The sponge substrate provides smooth inner and outer sides of the skeleton for the depos- ition of materials and its soft, lightweight, flexible and cost-effective nature makes it more attractive for flexible devices. Moreover, macroporosity and high surface area of sponge provide an easy path to the electrolyte to reach the entire surface of the electrodes where redox reactions can take place. Synthesis of SP@rGO@Ni and SP@rGO@Co hybrid electrodes involves two steps: first, coating of rGO onto the sponge substrate by ‘‘dip and dry’’ method and subsequently, depositions of nanos- tructured Ni(OH)2 and Co(OH)2 carried out by CBD method (see Fig. 1). The bare sponge was first coated with rGO nanosheets via dipping into rGO ink in aqueous solution and subsequent drying in an oven. By this way, 3D macroporous conducting framework with high surface area was formed which provides enough space for the heavy and uniform deposition of nanostructured Ni(OH)2 and Co(OH)2 onto the skeleton of the sponge easily. Sponge surface was uniformly covered with aggregates of rGO sheets (see Supporting information S2) with almost completely transparent and crumpled to a curly, wavy shape. In the long list of possible chemical methods, the chemical bath deposition (CBD) method is best suited to prepare large-scale thin coatings of different materials with diverse and controllable morphologies at low temperature and low cost on any type of sub- strate. Moreover, direct growth of material by CBD method enhances the interfacial contacts and avoids the use of unnecessary binders and additives. SEM images as shown in Fig. OPEN Looking at both systems, the demerits of one type are the strengths of the other and vice-versa. Thus, the hybridization of EDLC and pseudocapacitor materials is an appealing path to solve the problems related to both systems6. p p pp g p p y There are two kinds of hybrids; one is the ‘‘hybrid electrode’’ in which pseudocapacitive/faradic and non- faradic materials are combined in single electrode in order to take the advantage of both charge storing mechan- isms7, whereas the second is the ‘‘hybrid device or hybrid combination’’ in which a pseudocapacitive/faradic electrode and non-faradic one are combined in a single cell8. The most promising approach to develop hybrid electrodes is to integrate non-faradaic (carbon based) and pseudocapacitive/faradic materials (transition metal oxides/hydroxides, polyoxometalates, conducting polymers and Li-intercalates). Simple mixing of two different materials will not be an effective mode to fabricate hybrid electrodes due to difficulties in interfacial contacts between them. The proper hybridization of components through rational design with control over morphology, size and uniformity of active material, as well as facile processing are key parameters to obtain inexpensive yet high-performance hybrid electrodes9. An emerging new concept to fabricate hybrid electrodes is designing carbon supported substrates with pseudocapacitive materials under the control of their morphologies and sizes such as carbon nanofiber textile, carbon nanotube/graphene coated textile, and three dimensional macroporous SCIENTIFIC REPORTS | 4 : 7349 | DOI: 10.1038/srep07349 1 www.nature.com/scientificreports Figure 1 | Schematic representation with digital photographs of fabrication of hybrid materials based on rGO and transition metal hydroxides (Ni(OH)2 and Co(OH)2) onto skeleton of 3D macroporous sponge. Figure 1 | Schematic representation with digital photographs of fabrication of hybrid materials based on rGO and transition metal hydroxides (Ni(OH)2 and Co(OH)2) onto skeleton of 3D macroporous sponge. Results h The work we present here deals with: 1) Construction of unique hybrid electrodes to improve capacit- ance: Using macroporous 3D sponge (SP) substrate coated with reduced graphene oxide (rGO), we have deposited two different materials Ni(OH)2 and Co(OH)2 by inexpensive CBD method to fabricate open porous, high surface area hybrid electrodes to achieve synergistic effects of EDLC and pseudocapacitive mate- rials at low cost. 2) Fabrication of hybrid device to extend working potential win- dow: The hybrid device is configured by using hybrid electrode (above mentioned) as positive with rGO coated sponge as nega- tive electrodes in single cell to extend working potential window. Thus, we have presented an integrated approach from hybrid materials to hybrid devices. These hybrid materials effectively increase the capacitance and hybrid configurations extend the working potential windows of device in aqueous system which con- sequently enhances the supercapacitive properties of the devices. SCIENTIFIC REPORTS | 4 : 7349 | DOI: 10.1038/srep07349 2 Figure 2 | SEM images of (a), (c) and (e) SP@rGO@Ni and (b), (d) and (f) SP@rGO@Co hybrid at two different magnifications with corresponding cross-sectional images showing a thick layer of Ni(OH)2 and Co(OH)2 coated on SP@rGO support. Figure 2 | SEM images of (a), (c) and (e) SP@rGO@Ni and (b), (d) and (f) SP@rGO@Co hybrid at two different magnifications with corresponding cross-sectional images showing a thick layer of Ni(OH)2 and Co(OH)2 coated on SP@rGO support. flakes. According to thermodynamics, the surface energy of an indi- vidual nanosheet or nanoflake is high therefore they aggregate per- pendicularly to the surface planes in order to decrease the surface energy17. Therefore, as the reaction proceeds, the thin sheets and flakes would self-aggregate to form interconnected Ni(OH)2 nanosheets and Co(OH)2 nanoflakes clusters for minimizing the overall surface energy. line in nature. Due to the small content of rGO coated on the sponge substrate, the corresponding peak for rGO was not detected in XRD. Further, to investigate the oxidation states of elements, XPS analysis was carried out. The XPS Ni2p peak (Fig. 3(c)) consisted of two spin- orbit doublets characteristic of Ni21 and Ni31 and two shake-up satellites. The binding energy of Ni2p3/2 was about 855.2 eV, which is consistent with the previously reported value18. The XPS Co2p peak (Fig. 3(d)) consisted of two spin-orbit doublets characteristic of Co21 and Co31 and two shake-up satellites. The binding energy corresponding to Co2p3/2 was about 780.2 eV18. Results h The most of the pores are observed in the range of 2–10 nm, with a centered pore distributions at around 3.57 nm, and 2.29 nm for Ni(OH)2 and Co(OH)2 samples, respectively indicating that pores of hierarchical nanosheets and nanoflakes are mainly mesopores with some micropores. This mesoporous structure of Ni(OH)2 and Co(OH)2 could provide easy access for the ions in the electrolyte as well as short diffusion path for intercalation and de-intercalation. The porosity of samples is an amalgamation of internal space of the agglomerated nanosheets/nanoflakes and the porosity due to internal space of bunch of nanosheets/nanoflakes. The pore size distribution of Ni(OH)2 and Co(OH)2 samples is slightly narrow which further supports the better homogeneity in the surface mor- phology and porosity of samples. These surface properties directly affect the electrochemical properties of electrode materials. with high accessible surface area and macroporous open pore struc- tures were acted as electrode as well as current collector which also provides highly porous channels for the full access of electrolytes to active materials. The cyclic voltammetry (CV) curves (Fig. 5(a)) indi- cate that the SP@rGO devices can be operated over a wide range of scan rates: from 5 up to 500 mV/s. Even at the scan rate of 500 mV/s, the cyclic voltammograms retain the rectangular shape showing the ideal electrochemical double layer behavior of rGO coated sponge electrode. The high scan rate that SP@RGO can achieve implies that ultrahigh power density can be obtained in this device. The galvano- static charge/discharge curve at a specific current of 5 A/g (Fig. 5(b)) shows an ultrafast charge discharge rate, linear dependence on volt- age and time, and very small voltage drop demonstrating that the SP@rGO electrodes can work at high specific current. Thus, the results demonstrate that SP@rGO electrode exhibit high EDLC per- formance and would be a promising energy storage substrate. p g gy g To demonstrate the advantages of hybrid electrodes (SP@rGO@ Ni and SP@rGO@Co) which are obtained by hybridization of pseu- docapacitive and non-faradic materials, we have investigated the electrochemical properties in two electrode configuration devices. The SP@rGO@Ni and SP@rGO@Co served as electrodes as well as current collectors with binder-free, highly porous and conductive framework which provides full access to electrolyte ions. The shapes of the CV curves of SP@rGO@Ni and SP@rGO@Co symmetric devices (Fig. Results h line in nature. Due to the small content of rGO coated on the sponge substrate, the corresponding peak for rGO was not detected in XRD. Further, to investigate the oxidation states of elements, XPS analysis was carried out. The XPS Ni2p peak (Fig. 3(c)) consisted of two spin- orbit doublets characteristic of Ni21 and Ni31 and two shake-up satellites. The binding energy of Ni2p3/2 was about 855.2 eV, which is consistent with the previously reported value18. The XPS Co2p peak (Fig. 3(d)) consisted of two spin-orbit doublets characteristic of Co21 and Co31 and two shake-up satellites. The binding energy corresponding to Co2p3/2 was about 780.2 eV18. Fig. 3(a, b) shows the XRD patterns of SP@rGO@Ni and SP@ rGO@Co, respectively. As seen from the XRD patterns, it is evi- denced that both materials are polycrystalline in nature. The diffrac- tion peaks (001), (100), (101), (102), (110) and (111) in Fig. 3(a) are readily indexed to a pure single phase of b-Ni(OH)2 with the hexa- gonal brucite structure [JCPDS 14-0117] whereas the peaks (100), (101), (110) and (111) correspond to pure hexagonal b-Co(OH)2 according to JCPDS 30-0443 (Fig. 3(b)). Moreover, broadened dif- fraction peaks evidence that both deposited materials are nanocystal- TEM analysis is further carried out to provide more insight into the detailed microstructures of the Ni(OH)2 and Co(OH)2 samples. TEM image shown in Fig. 4(a) presents a panoramic view of thin nanosheets of Ni(OH)2. It is further seen that, the nanosheets are loosely packed and have separate existence. Fig. 4(b) shows the TEM image of a representative flowerlike Co(OH)2 nanostructure which is Figure 3 | (a), (b) XRD patterns and (c), (d) core level XPS of Ni2p and Co2p spectra for SP@rGO@Ni and SP@rGO@Co hybrid materials, respectively. Figure 3 | (a), (b) XRD patterns and (c), (d) core level XPS of Ni2p and Co2p spectra for SP@rGO@Ni and SP@rGO@Co hybrid materials, respectively. d) core level XPS of Ni2p and Co2p spectra for SP@rGO@Ni and SP@rGO@Co hybrid materials, respectively. Figure 3 | (a), (b) XRD patterns and (c), (d) core level XPS of Ni2p and Co2p spectra for SP@rGO@Ni and SP@r (b) XRD patterns and (c), (d) core level XPS of Ni2p and Co2p spectra for SP@rGO@Ni and SP@rGO@Co hybrid ma SCIENTIFIC REPORTS | 4 : 7349 | DOI: 10.1038/srep07349 SCIENTIFIC REPORTS | 4 : 7349 | DOI: 10.1038/srep07349 3 www.nature.com/scientificreports in good agreement with the results from the FESEM image. Results h In addi- tion to this the edge of the hierarchical Ni(OH)2 nanosheets and Co(OH)2 nanoflakes further indicates the ultrathin nature of a single component with almost transparent feature. Fig. 4(c–d) shows the nitrogen adsorption and desorption isotherms and the correspond- ing pore size distributions (inset) of Ni(OH)2 and Co(OH)2 samples, respectively. The specific surface areas were calculated from the Brunauer-Emmett-Teller (BET) method and the pore size distribu- tions (PSD) were obtained by means of the Barrett-Joyner-Halenda (BJH) equation using the adsorption isotherm branch. The BET specific surface areas of Ni(OH)2 and Co(OH)2 samples were found to be 91 and 87 m2/g, respectively. The pore size distribution curves of Ni(OH)2 and Co(OH)2 samples are also shown in inset of Fig. 4(c– d). The most of the pores are observed in the range of 2–10 nm, with a centered pore distributions at around 3.57 nm, and 2.29 nm for Ni(OH)2 and Co(OH)2 samples, respectively indicating that pores of hierarchical nanosheets and nanoflakes are mainly mesopores with some micropores. This mesoporous structure of Ni(OH)2 and Co(OH)2 could provide easy access for the ions in the electrolyte as well as short diffusion path for intercalation and de-intercalation. The porosity of samples is an amalgamation of internal space of the agglomerated nanosheets/nanoflakes and the porosity due to internal space of bunch of nanosheets/nanoflakes. The pore size distribution of Ni(OH)2 and Co(OH)2 samples is slightly narrow which further supports the better homogeneity in the surface mor- phology and porosity of samples. These surface properties directly affect the electrochemical properties of electrode materials. in good agreement with the results from the FESEM image. In addi- tion to this the edge of the hierarchical Ni(OH)2 nanosheets and Co(OH)2 nanoflakes further indicates the ultrathin nature of a single component with almost transparent feature. Fig. 4(c–d) shows the nitrogen adsorption and desorption isotherms and the correspond- ing pore size distributions (inset) of Ni(OH)2 and Co(OH)2 samples, respectively. The specific surface areas were calculated from the Brunauer-Emmett-Teller (BET) method and the pore size distribu- tions (PSD) were obtained by means of the Barrett-Joyner-Halenda (BJH) equation using the adsorption isotherm branch. The BET specific surface areas of Ni(OH)2 and Co(OH)2 samples were found to be 91 and 87 m2/g, respectively. The pore size distribution curves of Ni(OH)2 and Co(OH)2 samples are also shown in inset of Fig. 4(c– d). Results h 6(A, B)) indicate that the capacitance characteristic is different from that of electric double layer capacitance in which the shape is normally close to an ideal rectangular shape. Each CV curve consists of a pair of strong redox peaks, which indicates that the capacitance characteristics are mainly governed by redox reactions. It must also be noted that as the scan rate increased, the potential of the anodic and cathodic peaks shift to more positive and negative To evaluate the electrochemical performance SP@rGO electrodes, a typical two electrode configuration was constructed. Two pieces of SP@rGO sponge as electrodes, one piece of separator and 2 M KOH as electrolyte was used. The sponges based conductive framework Figure 4 | (a), (b) TEM images of Ni(OH)2 and Co(OH)2 samples with corresponding high magnified TEM images (inset), respectively, (c), (d) Nitrogen adsorption/desorption isotherm of Ni(OH)2 and Co(OH)2 samples and inset shows the corresponding BJH pore size distribution plots. Figure 4 | (a), (b) TEM images of Ni(OH)2 and Co(OH)2 samples with corresponding high magnified TEM images (inset), respectively, (c), (d) Nitrogen adsorption/desorption isotherm of Ni(OH)2 and Co(OH)2 samples and inset shows the corresponding BJH pore size distribution plots. SCIENTIFIC REPORTS | 4 : 7349 | DOI: 10.1038/srep07349 SCIENTIFIC REPORTS | 4 : 7349 | DOI: 10.1038/srep07349 4 Figure 5 | (a) Cyclic voltammetry curves of SP@rGO electrodes collected at different scanning rates in 2 M KOH electrolyte, respectively (b) Galvanostatic charge/discharge curves of SP@rGO electrodes at different current densities. Figure 5 | (a) Cyclic voltammetry curves of SP@rGO electrodes collected at different scanning rates in 2 M KOH electrolyte, respectively (b) Galvanostatic charge/discharge curves of SP@rGO electrodes at different current densities. directions, respectively. The possible charge storing mechanisms for Ni(OH)2 and Co(OH)2 are as follows19: directions, respectively. The possible charge storing mechanisms for Ni(OH)2 and Co(OH)2 are as follows19: the inner-pore surfaces of the hybrid electrode materials are effec- tively utilized for the intercalation20. The highest specific capacitance values obtained for symmetric SP@rGO@Ni and SP@rGO@Co devices were 812 F/g (1.7 F/cm2) for mass loading of 4.2 mg/cm2 whereas 752 F/g (1.46 F/cm2) for mass loading of 3.9 mg/cm2 at a potential scan rate of 5 mV/s, respectively. The galvanostatic charge- discharge curves of both hybrid symmetric devices are shown in Fig. 6(C–D) at different current densities. Results h The curves show that these hybrid systems can be operated at a wide range of current densities with small voltage drop, further demonstrating the excellent electro- chemical performance of SP@rGO@Ni and SP@rGO@Co nanos- tructured hybrid electrodes. The charge-discharge curves reflect Ni(OH)2zOH{uNiOOHzH2Oze{ ð1Þ Co(OH)2zOH{uCoOOHzH2Oze{ ð2Þ ð2Þ As expected, there is an increase in current with the scan rate while the CV curves retain the shape when the applied scan rate is very high (200 mV/s). This indicates a proper hybridization of (Ni(OH)2 or Co(OH)2) with rGO which allows efficient ion transport at the inter- face of hybrid electrodes and electrolyte. Moreover, both outer- and Figure 6 | (A), (B) Cyclic voltammetry curves of SP@rGO@Ni and SP@rGO@Co hybrid electrodes collected at different scanning rates in 2 M KOH electrolyte, respectively (C), (D) Galvanostatic charge/discharge curves of SP@rGO@Ni and SP@rGO@Co hybrid electrodes at different current densities, respectively. Figure 6 | (A), (B) Cyclic voltammetry curves of SP@rGO@Ni and SP@rGO@Co hybrid electrodes collected at different scanning rates in 2 M KOH electrolyte, respectively (C), (D) Galvanostatic charge/discharge curves of SP@rGO@Ni and SP@rGO@Co hybrid electrodes at different current densities, respectively. SCIENTIFIC REPORTS | 4 : 7349 | DOI: 10.1038/srep07349 5 Figure 7 | CV curves of the (a) SP@rGO//SP@rGO@Ni and (b) SP@rGO//SP@rGO@Co hybrid devices at different working voltages at a scan rate of 100 mV/s. CV curves of (c) SP@rGO//SP@rGO@Ni and (d) SP@rGO//SP@rGO@Co hybrid devices at different scan rates. Variation of specific and areal capacitances of (e) SP@rGO//SP@rGO@Ni and (f) SP@rGO//SP@rGO@Co devices as function of the scan rate. Figure 7 | CV curves of the (a) SP@rGO//SP@rGO@Ni and (b) SP@rGO//SP@rGO@Co hybrid devices at different working voltages at a scan rate of 100 mV/s. CV curves of (c) SP@rGO//SP@rGO@Ni and (d) SP@rGO//SP@rGO@Co hybrid devices at different scan rates. Variation of specific and areal capacitances of (e) SP@rGO//SP@rGO@Ni and (f) SP@rGO//SP@rGO@Co devices as function of the scan rate. order to reach the highest cell voltage, the charges stored in both electrodes must be balanced by adjusting the mass loading of the electrodes. The positive to negative electrode mass ratio was calcu- lated by the following equation to achieve a charge balance q1 5 q2. the faradic behavior of the Ni(OH)2 and Co(OH)2 samples. It should also be noted that the small voltage-drop at the start of discharge indicates a low internal resistance, excellent conductivity, and the capability for a quick charge. Results h This may be attributed to the inclusion of rGO in Ni(OH)2 and Co(OH)2 nanostructures. The estimated specific capacitance values of SP@rGO@Ni and SP@rGO@Co hybrid electrodes were found to be 806 and 747 F/g at a current density of 2 A/g. The good capacitive behavior of SP@rGO@Ni and SP@rGO@Co electrodes is due to the combined contribution of rGO with Ni(OH)2 and Co(OH)2 nanostructures which provides large internal accessible surface sample matrix, decrease the ion dif- fusion resistance and enhance the electro-active surface utilization during the redox process. Moreover, the 3D macroporous framework of the sponge and numerous nano-channels in Ni(OH)2 nanosheets and Co(OH)2 nanoflakes act as ‘‘ion-buffering reservoirs’’ which reduce the mean free path of the ions and facilitate faster ionic and electronic kinetics, thus maximizing the reversible insertion/de- insertion reactions21. mz m{ ~ (C{|DE{) (Cz|DEz) ð3Þ ð3Þ where q1, q2, m1, m2, C1, C2, E1, and E2 are the charge, mass, specific capacitance and potential windows for the cathode (1) and anode (2). Hence, the mass ratio between the positive and negative electrodes of the SP@rGO//SP@rGO@Ni and SP@rGO//SP@rGO@ Co hybrid devices should be 3.19 and 2.37, respectively. Accordingly, the charge balance between positive and negative electrodes, the optimal positive-to-negative mass ratio was determined to be 4.12 mg/cm251.28 mg/cm2 and 3.23 mg/cm251.33 mg/cm2 for SP@rGO//SP@rGO@Ni and SP@rGO//SP@rGO@Co hybrid devices, respectively. Fig. 7(a–b) show the CV curves at different voltage windows for SP@rGO//SP@rGO@Ni and SP@rGO//SP@ rGO@Co hybrid devices respectively at a scan rate of 100 mV/s. It can be clearly seen that the fabricated hybrid devices show a good capacitive behavior and expand their potential windows up to 1.5 and 1.4 V for SP@rGO//SP@rGO@Ni and SP@rGO//SP@rGO@Co cells, respectively. Further increase in voltage window causes some irreversible reaction since sudden increase in current has been observed (see Fig. 7(a–b)). It was further found that the current and consequently the specific capacitance greatly increase as the operating voltage window is increased, which indicates the improve- ment in stored energy and delivered power. As a consequence, the overall performance of the supercapacitor could also be remarkably improved. As is generally known, operating at higher voltage will be favorable for reducing the number of devices in series required to reach the desired output voltage in practical applications. Results h Very small voltage-drop is observed at initial discharge curve indicating excellent conductivity and fast I–V response. SP@rGO@Ni and SP@rGO//SP@rGO@Co hybrid devices mea- sured at various scan rates of 10–500 mV/s. These CV curves exhibit a non-rectangular current response on voltage reversal with small redox peaks. The shapes of CV curves remain unchanged even at a high scan rate of 500 mV/s, suggesting that the cell possesses excellent rate capability which is desirable for high-power supercapcitors. The specific capacitances of hybrid devices (based on the total mass of the active materials of the two electrodes) at different scan rates calculated from the CV curves is presented in Fig. 7(e–f). The highest gravimetric capa- citance (areal capacitance) for SP@rGO//SP@rGO@Ni and SP@ rGO//SP@rGO@Co hybrid devices were found to be 185 (0.49 F/ cm2) and 158 (0.35 F/cm2) at 10 mV/s, which are greatly larger than the values reported for hydroxide based asymmetric super- capacitors25–28. The specific capacitances decrease gradually with increasing scan rate as diffusion limits the movement of electro- lyte ions at high scan rates because of the time constraint and only the outer active surface can be utilized for charge storage, resulting in a lower electrochemical utilization of electroactive materials29. The higher specific capacitance could be obtained at higher opera- tion voltage windows thanks to the redox reactions of Ni(OH)2 and Co(OH)2. Importantly, it should be pointed out that the specific capacitance is calculated based on the total mass of the active material on both electrodes. The excellent performance of the hybrid devices can thus be attributed to the high capacitance and rate performance as well as the synergistic effects of both the SP@rGO and SP@rGO@Ni/SP@rGO@Co hybrid electrodes. Finally, a long cycling life is an important requirement for super- capacitor applications. Long cycling-life tests were carried out for the SP@rGO//SP@rGO@Ni and SP@rGO//SP@rGO@Co hybrids by repeating the CV test at a scan rate of 100 mV/s for 2000 cycles (see inset of Fig. 8(C–D)). Fig. 8(C–D) shows the capacitance reten- tion ratio of the hybrid devices as a function of the cycle number. It is worth noting that the specific capacitance decreases suddenly after the initial 50 cycles, which is probably related to pulverization and loss of electrical contact between the active material and the current as well as wettability issues. Results h Thus, we chose an operation voltage window of 1.5 V and 1.4 V for SP@rGO// SP@rGO@Ni and SP@rGO//SP@rGO@Co cells in 2 M KOH aque- ous electrolyte to further investigate the electrochemical perform- ance, respectively. Fig. 7(c–d) shows the CV curves of the SP@rGO// In order to further increase the specific energy without sacrificing power delivery and cycle life in the aqueous electrolyte; supercapa- citors have been proposed to reach a higher cell voltage through the asymmetric mode where one electrode is made of a faradaic material and the other of a non-faradaic one. Several reports are available on this approach22,23. Here, we are going to demonstrate somewhat dif- ferent hybrid design which is comprised of one non-faradic electrode (SP@rGO) with other hybrid electrode which combines non-faradic and pseudocapacitive characteristics (SP@rGO@Ni or SP@rGO@ Co) in aqueous system. Due to the high hydrogen evolution over- potential of rGO in aqueous media24 and the excellent capacitive features of SP@rGO@Ni and SP@rGO@Co synthesized in this work, hybrid configuration is tested for achieving the goal of high-energy and high-power densities. In this hybrid device, charges are stored non-faradically in rGO electrodes, and through redox reactions in the hybrid metal hydroxide electrodes. The specific capacitances of the SP@rGO@Ni, SP@rGO@Co and SP@rGO electrode were calcu- lated to be 812 F/g (4.2 mg/cm2), 752 F/g (3.9 mg/cm2) and 127 F/g (1.9 mg/cm2) at the scan rates of 5 mV/s, respectively. Note that in SCIENTIFIC REPORTS | 4 : 7349 | DOI: 10.1038/srep07349 6 www.nature.com/scientificreports Figure 8 | Galvanostatic charge-discharge curves of (A) SP@rGO//SP@rGO@Ni and (B) SP@rGO//SP@rGO@Co hybrid devices at different current densities. Variation of capacity retention of (C) SP@rGO//SP@rGO@Ni and (D) SP@rGO//SP@rGO@Co hybrid devices with number of cycles at 100 mV/s scan rate along with corresponding CV curves (inset) at 1st and 2000th cycles. Figure 8 | Galvanostatic charge-discharge curves of (A) SP@rGO//SP@rGO@Ni and (B) SP@rGO//SP@rGO@Co hybrid devices at different current densities. Variation of capacity retention of (C) SP@rGO//SP@rGO@Ni and (D) SP@rGO//SP@rGO@Co hybrid devices with number of cycles at 100 mV/s scan rate along with corresponding CV curves (inset) at 1st and 2000th cycles. all of the curves are not ideal straight lines, indicating the involve- ment of a redox reaction process. Furthermore, there is an initial drop in potential, which may be caused by the internal resistance as well as different rates of oxidation and reduction reactions. Results h After 2000 cycles, the SP@rGO//SP@ rGO@Ni and SP@rGO//SP@rGO@Co hybrid devices display an excellent long cycle life with 90% and 87% retention of their initial specific capacitance, respectively, demonstrating superior long-term electrochemical stability. Moreover, excellent stability confirms the proper hybridization of two different (pseudocapacitive and non- faradic) materials. Such a stable and connecting structure helps to alleviate the structure damage caused by volume expansion during cycling process, resulting in an enhanced stability. Thus, such a cyc- ling performance is highly competitive with those of some other asymmetric supercapacitors, such as Ni(OH)2//activated carbon (82% retention after 1000 cycles)30, LiNi1/3Co1/3Mn1/3O2//AC (ca. 80% retention after 1000 cycles)31, graphene-MnO2//graphene (79% retention after 1000 cycles)32, and porous NiO//carbon devices (50% after 1000 cycles)33. y Galvanostatic charge/discharge curves of hybrid cells were recorded with various current densities to further evaluate the elec- trochemical performance as shown in Fig. 8(A–B). It can be seen that SCIENTIFIC REPORTS | 4 : 7349 | DOI: 10.1038/srep07349 7 Figure 9 | The power density versus energy density of SP@rGO//SP@ rGO@Ni and SP@rGO//SP@rGO@Co hybrid devices in a Ragone plot. an energy density .10 Wh/kg even at a high power density. The energy density demonstrated in this work is significantly higher than those obtained for other carbon-based symmetric capacitors in aque- ous electrolytes, such as activated carbon (,10 Wh/kg)34, carbon nanotubes (,10 Wh/kg)35, graphene (9.1 Wh/kg)36. The excellent performance is also comparable or superior to other asymmetric cells with mild aqueous electrolytes, such as Co(OH)2//GO (11.94 Wh/ kg)25, Co(OH)2//AC (92.7 Wh/kg)26, Ni(OH)2//AC (32.7 Wh/kg)27, b-Ni(OH)2//AC (36.2 Wh/kg)37, Ni(OH)2-graphene//porous gra- phene (77.8 Wh/kg)38, a-Ni(OH)2//AC (42.3 Wh/kg)28, Ni(OH)2// AC (35.7 Wh/kg)39, Ni(OH)2/ultrathin graphite foam//activated microwave exfoliated graphite oxide (13.4 Wh/kg)40. Discussion b SCIENTIFIC REPORTS | 4 : 7349 | DOI: 10.1038/srep07349 8 The SP@rGO was washed with water to get rid of the surfactant after drying in an electrical oven for 2 h. Then the surfactant-free SP@rGO was put in a vacuum oven for another 3 h to remove the water completely. working potential window for the entire system (DVmax) will be the sum of potentials across rGO electrode (DVNF) and hybrid electrode (DVHy). Thus the total amount of energy stored in this hybrid system is the sum of energy stored in rGO electrode (ENF) and that of the hybrid electrode (EHy). The hybrid electrode system has advantage at high power density due to the structural characteristics of the system compared to those of typical asymmetric capacitors41. In effect, the rGO electrode, which can store electrochemical energy by electro- static force, enhances the electron transfer to the Ni(OH)2 and Co(OH)2 in the hybrid electrode system, causing a better charge transfer reaction at a high rate. To show high energy density of hybrid device for example SP@rGO//SP@rGO@Ni, the device was charged to light an LED as a demonstration for real application (Fig. 10(c–e)). After a quick charge of around 20 seconds the cell can light one LED for ,5 min. This result further demonstrates the successful utility of hybrid devices with aqueous electrolyte to realize high performance supercapacitors. Development of SP@rGO@Ni and SP@rGO@Co hybrid electrodes. Subsequent depositions of nanostructured Ni(OH)2 and Co(OH)2 on to SP@rGO support substrate were carried out by simple chemical bath deposition (CBD) method. Briefly, solutions of 0.1 M NiSO4 and 0.1 M CoCl2 complexed with ammonia solution and 0.1 M hexamethylenetetramine (HMT) were prepared in de-ionized water separately in order to synthesize Ni(OH)2 and Co(OH)2 thin films, respectively. Further the pieces of SP@rGO support substrates were immersed in these baths and the bath was heated. When the bath attained the temperature of 343 K, the precipitation started in the bath. During the precipitation, a heterogeneous reaction occurs and nanostructured Ni(OH)2 and Co(OH)2 thin coatings were deposited on the SP@rGO substrates. The deposition time for both materials placed at 343 K was kept constant at 4 h. At the end, SP@rGO coated with Ni(OH)2 and Co(OH)2 are referred to as SP@ rGO@Ni and SP@rGO@Co, respectively. Materials characterization. Discussion b For a better understanding of the working of hybrid device with hybrid electrode, the schematic of hybrid device is sketched in Fig. 10(a). The characteristic of hybrid electrode is hybridization of pseudocapacitive component (Ni(OH)2 and Co(OH)2) and non- faradic component (rGO) in a single electrode so that energy can be stored through both mechanisms. For example, in case of SP@ rGO@Ni hybrid electrode, the rGO is charged by electrostatic force in the initial state until the electrode potential attains the redox reaction potential of the Ni(OH)2 till q1 as seen in Fig 10(b). Then the Ni(OH)2 is charged by the redox reaction while maintaining the redox reaction potential of the hybrid electrode, until the component reaches the full-charge state (QF). After the full charge of the Ni(OH)2, the rGO is charged again (q2) to reach the maximum potential of hybrid electrode storing the charge on the surface. Hence the total charge stored in the hybrid electrode is due to both components ((q1 1 q2) 1 QF). In addition to this the maximum Figure 9 | The power density versus energy density of SP@rGO//SP@ rGO@Ni and SP@rGO//SP@rGO@Co hybrid devices in a Ragone plot. Energy density and power density are two key factors for evalu- ating the performance of supercapacitors. A high performance supercapacitor is expected to provide high energy density or high specific capacitance at high discharge rates. Fig. 9 presents the Ragone plot of the SP@rGO//SP@rGO@Ni and SP@rGO//SP@ rGO@Co hybrid devices. The as-assembled SP@rGO//SP@rGO@ Ni hybrid device with a cell voltage of 1.5 V can deliver a high specific energy of 42.02 Wh/kg with maximum specific power of 11 kW/kg whereas SP@rGO//SP@rGO@Co cell exhibits 33.01 Wh/kg with specific power of 8 kW/kg. Furthermore, these devices can retain Figure 10 | (a), (b) Schematic representation of hybrid combination (device) of hybrid electrode (SP@rGO@Ni or SP@rGO@Co) and SP@rGO electrode with propylene carbonate paper as separator along with charge-potential profile. (c)–(e) Demonstration of the use of the SP@rGO//SP@rGO@Ni hybrid device is given with a light emitting diode (LED). Figure 10 | (a), (b) Schematic representation of hybrid combination (device) of hybrid electrode (SP@rGO@Ni or SP@rGO@Co) and SP@rGO electrode with propylene carbonate paper as separator along with charge-potential profile. (c)–(e) Demonstration of the use of the SP@rGO//SP@rGO@Ni hybrid device is given with a light emitting diode (LED). Discussion b The X-ray diffraction (XRD) of samples was carried out using a Bruker AXS D8 Advance diffractometer with copper radiation (Ka of l 5 1.54 A˚). The X-ray photoelectron spectra (XPS) data were obtained by X-ray photoelectron spectroscopy (XPS, Perkin-Elmer model PHI 1600). The microstructures of the samples were investigated by scanning electron microscope (SEM, Nova NanoSEM 200). Transmission electron microscopy observations were conducted using JEOL JEM-2100 operated at 200 kV. N2 adsorption/desorption was determined by Brunauer-Emmett-Teller (BET) measurements using Micromeritics instrument. p p The superior electrochemical performance of the fabricated hybrid devices (SP@rGO//SP@rGO@Ni and SP@rGO//SP@rGO@ Ni) can be reasonably attributed to the synergistic contribution of both materials (hybrid electrode) as well as that between the positive and negative electrodes in single device. The energy density of the hybrid devices is significantly improved because of the extended wide operation voltage window. On the other hand, the rGO in both electrodes demonstrate their distinctive features such as excellent electrochemical stability and superior conductivity due to the fact that rGO not only acts as the support for the Ni(OH)2 nanosheets and Co(OH)2 nanoflakes, but also maintains the mechanical integ- rity and high electrical conductivity of the overall electrode. Additionally, 3D macroporous framework as the negative electrode facilitates the transport of electrolyte ions and provides a larger sur- face area for charge-transfer reactions, ensuring high power density and excellent rate performance. Thus, pairing up SP@rGO@Ni or SP@rGO@Co and SP@rGO hybrid materials for hybrid devices represents a new approach to high-performance energy storage devices. Our future research will be focused on the use of organic and ionic liquid based electrolytes for such hybrid-electrode based hybrid devices. Supercapacitors cell fabrication and electrochemical characterization. Two identical pieces of hybrid electrode (SP@rGO@Ni and SP@rGO@Co) each with an area of 1 cm2 were sandwiched by porous polypropylene film separator soaked with 2 M KOH electrolyte and assembled into a coin cell. The mass of the active materials was calculated by the mass difference method. Hybrid devices were assembled with hybrid electrode (SP@rGO@Ni and SP@rGO@Co) as the positive electrode (cathode) and SP@rGO as the negative electrode (anode). The electrodes were separated by polypropylene film separator soaked with 2 M KOH electrolyte and assembled into a coin cell. All electrochemical characteristics were evaluated by cyclic voltammetry (CV) and galvanostatic charge/discharge (CD) measurements on IviumStat and Atlas-0961 multichannel battery interface. 1. Dubal, D. P. et al. Discussion b p p p 11. Yu, G. et al. Solution-processed Graphene/MnO2 nanostructured textiles for high- performance electrochemical capacitors. Nano Lett. 11, 2905–2911 (2011). 12. Shen, L., Che, Q., Li, H. & Zhang, X. Mesoporous NiCo2O4 nanowire arrays grown on carbon textiles as binder-free flexible electrodes for energy storage. Adv. Funct. Mater. 24, 2630–2637 (2014). 13. Lu, X. et al. Oxygen-deficient hematite nanorods as high-performance and novel negative electrodes for flexible asymmetric supercapacitors. Adv. Mater. 26, 3148–3155 (2014). 14. Weng, Z. et al. Graphene-cellulose paper flexible supercapacitors. Adv. Energy Mater. 1, 917–922 (2011). 15. Dubal, D. P. & Holze, R. Self-assembly of stacked layers of Mn3O4 nanosheets using a scalable chemical strategy for enhanced, flexible, electrochemical energy storage. J. Power Sources 238, 274–282 (2013). Discussion b Supercapacitors based on flexible substrates: An overview. Energy Technol., 2, 325–341 (2014). 2. Simon, P. & Gogotsi, Y. Materials for electrochemical capacitors. Nat. Mater. 7, 845–854 (2008). 3. Lu, X., Yu, M., Wang, G., Tong, Y. & Li, Y. Flexible solid-state supercapacitors: design, fabrication and applications. Energy Environ. Sci., 7, 2160–2181 (2014). y With our earnest efforts, we have designed stunning hybrid mate- rials based on sponge-supported rGO and transition metal hydro- xides for hybrid energy storage devices. By taking into account, the processing and fabrication cost of hybrid materials and hybrid devices, inexpensive and scalable ‘dip and dry’ and CBD methods with aqueous electrolyte system have been utilized. The synergic combination of non-faradic (rGO) and redox (Ni(OH)2 and Co(OH)2) materials provides dual charge-storing mechanisms whereas 3D porous framework of sponge support allows good accessibility of electrolyte to hybrid electrodes resulting in excellent electrochemical performance. Moreover, hybrid devices using SP@ rGO and SP@rGO@Ni or SP@rGO@Co as the negative and positive electrodes, respectively have been demonstrated as a bolstering approach. The proper hybridization of rGO and Ni(OH)2/ Co(OH)2 improves interfacial contacts which delivers high energy as well as sustains the volume expansion during the long term char- ging/discharging reactions in an extended operating voltage. These encouraging findings open up the possibility of hybrid electrodes for numerous applications in hybrid energy storage devices to meet the diverse demands where high power and energy storage systems are required. 4. Arico, A. S. et al. Nanostructured materials for advanced energy conversion and storage devices. Nat. Mater. 4, 366–377 (2005). 5. Zhang, L. L. & Zhao, X. S. Carbon-based materials as supercapacitor electrodes. Chem. Soc. Rev. 38, 2520–2531 (2009). 6. Suarez-Guevara, J., Ruiz, V. & Gomez-Romero, P. Hybrid energy storage: high voltage aqueous supercapacitors based on activated carbon-phosphotungstate hybrid materials. J. Mater. Chem. A, 2, 1014–1021 (2014). 7. Fedorkova, A., Nacher-Alejos, A., Gomez-Romero, P., Orinakova, R. & Kaniansky, D. Structural and electrochemical studies of PPy/PEG-LiFePO4 cathode material for Li-ion batteries. Electrochim. Acta 55, 943–947 (2010 8. Dubal, D. P., Gund, G. S., Lokhande, C. D. & Holze, R. Controlled growth of CoSx nanostrip arrays (CoSx-NSA) on nickel foam for asymmetric supercapacitors. Energy Technol. 2, 401–408 (2014). 9. Chen, W. et al. High performance nanostructured supercapacitors on a sponge. Nano Lett. 11, 5165–5171 (2011). 10. Lu, X. et al. WO3-x@Au@MnO2 core-shell nanowires on carbon fabric for high- performance flexible supercapacitors Adv. Mater. 24, 938–944 (2012). Methods Fabrication of sponge@rGO (SP@rGO) composite. At first, rGO was prepared by the modified Hummer’s method (supporting information S1) reported elsewhere14. Briefly, rGO ink with concentration of 2 mg/ml in 10 mg/ml of sodium dodecylbenzenesulfonate (SDBS) water solution was prepared by applying a 30 min bath sonication. A piece of commercially available macroporous cellulose sponge previously cleaned with water and ethanol several times was dipped into this rGO ink. 16. Chen, F. et al. Large-scale and shape-controlled syntheses of three-dimensional CdS nanocrystals with flowerlike structure. J. Phys. Chem. C 112, 1001–1007 (2008). ( ) 17. Gund, G. S., Dubal, D. P., Jambure, S. B., Shinde, S. S. & Lokhande, C. D. Temperature influence on morphological progress of Ni(OH)2 thin films and its SCIENTIFIC REPORTS | 4 : 7349 | DOI: 10.1038/srep07349 9 subsequent effect on electrochemical supercapacitive properties. J. Mater. Chem. A 1, 4793–4803 (2013). 36. Wang, H. et al. Advanced asymmetrical supercapacitors based on graphene hybrid materials. Nano Res. 4, 729–736 (2011). subsequent effect on electrochemical supercapacitive properties. J. Mater. Chem. A 1, 4793–4803 (2013). 18. Chen, W., Yang, Y. & Shao, H. Cation-exchange induced high power electrochemical properties of core-shell Ni(OH)2@CoOOH. J. Power Sources 196, 488–494 (2011). y 37. Huang, J. et al. Asymmetric supercapacitors based on b-Ni(OH)2 nanosheets and activated carbon with high energy density. J. Power Sources 246, 371–376 (2014). 38. Yan, J. et al. Advanced asymmetric supercapacitors based on Ni(OH)2/graphene and porous graphene electrodes with high energy density. Adv. Funct. Mater. 22, 2632–2641 (2012). 19. Salunkhe, R. R., Jang, K., Lee, S. & Ahn, H. Aligned nickel-cobalt hydroxide nanorod arrays for electrochemical pseudocapacitor applications. RSC Advances 2, 3190–3193 (2012). 39. Li, H. B. et al. Amorphous nickel hydroxide nanospheres with ultrahigh capacitance and energy density as electrochemical pseudocapacitor materials. Nat. Commun. 4, 1894 (2013). 20. Zhang, G. Q. & Lou, X. W. Controlled growth of NiCo2O4 nanorods and ultrathin nanosheets on carbon nanofibers for high-performance supercapacitors. Sci. Rep. 3, 1470 (2013). 40. Ji, J. et al. Nanoporous Ni(OH)2 thin film on 3D ultrathin-graphite foam for asymmetric supercapacitor. ACS Nano 7, 6237–6243 (2013). 21. Dubal, D. P., Kim, J. G., Kim, Y., Holze, R. & Kim, W. B. Demonstrating the highest supercapacitive performance of branched MnO2 nanorods grown directly on flexible substrates using controlled chemistry at ambient temperature. Energy Technol. 1, 125–130 (2013). 41. Choi, H. S. & Park, C. R. Author contributions D.P.D. and P.G.R. designed the experiments, analyzed the data and wrote the manuscript. D.P.D. carried out synthesis and characterization of hybrid thin films. D.P.D. and R.H. designed and carried out electrochemical measurements. To the preparation and reviewing manuscript, all authors contributed equally. 28. Lang, J. W., Kong, L. B., Liu, M., Luo, Y. C. & Kang, L. Asymmetric supercapacitors based on stabilized a-Ni(OH)2 and activated carbon. J. Solid State Electrochem. 14, 1533–1539 (2010). 29. Shinde, S. K., Dubal, D. P., Ghodake, G. S., Kim, D. Y. & Fulari, V. J. Nanoflower- like CuO/Cu(OH)2 hybrid thin films: Synthesis and electrochemical supercapacitive properties. J. Electroanal Chem. 732, 80–85 (2014). Acknowledgments 24. Hsu, C. T. & Hu, C. C. Synthesis and characterization of mesoporous spinel NiCo2O4 using surfactant-assembled dispersion for asymmetric supercapacitors. J. Power Sources 242, 662–671 (2013). Authors appreciate the award to DPD of a Humboldt Fellowship of the Alexander von Humboldt Foundation (AvH), Germany and the award of a Marie-Curie Fellowship through Beatriu de Pinos Program (BP-DGR-2013) for Catalan system of science and technology, Spain. Partial funding from Spanish Grant MAT2012-39199-C02-01 is gratefully acknowledged. ICN2 acknowledges support from the Severo Ochoa Program (MINECO, Grant SEV-2013-0295). Authors appreciate the award to DPD of a Humboldt Fellowship of the Alexander von Humboldt Foundation (AvH), Germany and the award of a Marie-Curie Fellowship through Beatriu de Pinos Program (BP-DGR-2013) for Catalan system of science and technology, Spain. Partial funding from Spanish Grant MAT2012-39199-C02-01 is gratefully acknowledged. ICN2 acknowledges support from the Severo Ochoa Program (MINECO, Grant SEV-2013-0295). 25. Salunkhe, R. R. et al. Direct growth of cobalt hydroxide rods on nickel foam and its application for energy storage. Chem. Eur. J. 20, 3084–3088 (2014). 26. Kong, L. B., Liu, M., Lang, J. W., Luo, Y. C. & Kang, L. Asymmetric supercapacitor based on loose-packed cobalt hydroxide nanoflake materials and activated carbon. J. Electrochem. Soc. 156, A1000–A1004 (2009). 27. Tang, Y. et al. Hydrothermal synthesis of a flower-like nano-nickel hydroxide for high performance supercapacitors. Electrochim. Acta 123, 158–166 (2014). Methods Theoretical guidelines to designing high performance energy storage device based on hybridization of lithium-ion battery and supercapacitors. J. Power Sources 259, 1–14 (2014). 22. Wang, F. et al. Electrode materials for aqueous asymmetric supercapacitors. RSC Adv. 3, 13059–13084 (2013). 23. Zhai, T. et al. A New Benchmark Capacitance for Supercapacitor Anodes by Mixed-Valence Sulfur-Doped V6O13-x. Adv. Mater. 26, 5869–5875 (2014). Additional information 30. Lang, J. W., Kong, L. B., Liu, M., Luo, Y. C. & Kang, L. Asymmetric supercapacitors based on stabilized a-Ni(OH)2 and activated carbon. J. Solid State Electrochem. 14, 1533–1539 (2010). Supplementary information accompanies this paper at http://www.nature.com/ scientificreports Supplementary information accompanies this paper at http://www.nature.com/ scientificreports Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. 31. Zhao, Y. et al. Pseudocapacitance properties of AC/LiNi1/3Co1/3Mn1/3O2 asymmetric supercapacitor in aqueous electrolyte. Synth. Met. 159, 331–337 (2009). How to cite this article: Dubal, D.P., Holze, R. & Gomez-Romero, P. Development of hybrid materials based on sponge supported reduced graphene oxide and transition metal hydroxides for hybrid energy storage devices. Sci. Rep. 4, 7349; DOI:10.1038/srep07349 (2014). 32. Wu, Z. S. et al. High-energy MnO2 nanowire/graphene and graphene asymmetric electrochemical capacitors. ACS Nano 4, 5835–5842 (2010). 33. Wang, D. W., Li, F. & Cheng, H. M. Hierarchical porous nickel oxide and carbon as electrode materials for asymmetric supercapacitor. J. Power Sources 185, 1563–1568 (2008). 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 permissionfrom the licenseholderin order toreproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 34. Wang, D. W., Li, F., Liu, M., Lu, G. Q. & Cheng, H. M. 3D aperiodic hierarchical porous graphitic carbon material for high-rate electrochemical capacitive energy storage. Ang. Chem. International Ed. 47, 373–376 (2008). g g 35. Yu, C., Masarapu, C., Rong, J., Wei, B. & Jiang, H. Stretchable supercapacitors based on buckled single-walled carbon-nanotube macrofilms. Adv. Mater. 21, 4793–4797 (2009). SCIENTIFIC REPORTS | 4 : 7349 | DOI: 10.1038/srep07349 10
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Discovering General Multidimensional Associations
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a11111 RESEARCH ARTICLE Discovering General Multidimensional Associations Ben Murrell1*, Daniel Murrell2, Hugh Murrell3 1 Department of Medicine, University of California San Diego, San Diego, United States of America, 2 Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, Cambridge, United Kingdom, 3 Computer Science, University of KwaZulu-Natal, Pietermaritzburg, South Africa * bmurrell@ucsd.edu OPEN ACCESS OPEN ACCESS Citation: Murrell B, Murrell D, Murrell H (2016) Discovering General Multidimensional Associations. PLoS ONE 11(3): e0151551. doi:10.1371/journal. pone.0151551 Editor: Raya Khanin, Memorial Sloan Kettering Cancer Center, UNITED STATES Received: June 27, 2015 Accepted: March 1, 2016 Published: March 18, 2016 Copyright: © 2016 Murrell 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. Citation: Murrell B, Murrell D, Murrell H (2016) Discovering General Multidimensional Associations. PLoS ONE 11(3): e0151551. doi:10.1371/journal. pone.0151551 Editor: Raya Khanin, Memorial Sloan Kettering Cancer Center, UNITED STATES Received: June 27, 2015 Accepted: March 1, 2016 Published: March 18, 2016 Abstract a1111 When two variables are related by a known function, the coefficient of determination (denoted R2) measures the proportion of the total variance in the observations explained by that function. For linear relationships, this is equal to the square of the correlation coefficient, ρ. When the parametric form of the relationship is unknown, however, it is unclear how to estimate the proportion of explained variance equitably—assigning similar values to equally noisy relationships. Here we demonstrate how to directly estimate a generalised R2 when the form of the relationship is unknown, and we consider the performance of the Maximal Information Coefficient (MIC)—a recently proposed information theoretic measure of dependence. We show that our approach behaves equitably, has more power than MIC to detect association between variables, and converges faster with increasing sample size. Most importantly, our approach generalises to higher dimensions, estimating the strength of multivariate relationships (Y against A, B, . . .) as well as measuring association while con- trolling for covariates (Y against X controlling for C). An R package named matie (“Measur- ing Association and Testing Independence Efficiently”) is available (http://cran.r-project.org/ web/packages/matie/). Introduction Measures of association between variables are useful across the sciences. Often the form of the association is unknown and traditional methods for association mining that assume particular functional forms (usually linearity) can miss or underestimate associations that are non-linear. When large numbers of variables are examined and where associations are expected to be com- plex, as is common in the biological sciences in particular, tools that automatically quantify associations without restrictive assumptions are particularly useful, since the number of pair- wise associations grows quadratically with the number of variables, and higher order relation- ships grow even faster. Thus, finding good methods for association mining should be prioritised. Data Availability Statement: Code is open source and available from http://cran.r-project.org/web/ packages/matie/index.html. Funding: BM is supported by K99AI120851 from the National Institute of Allergy and Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Reshef et al. describe [1] desired properties of a measure of bivariate association: generality and equitability. A measure that is general will discover, with sufficient sample size, any Competing Interests: The authors have declared that no competing interests exist. 1 / 10 PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 Discovering General Multidimensional Associations departure from independence, while a measure that is equitable will assign similar scores to equally noisy relationships of different kinds. A further attractive property is that a measure should scale like the coefficient of determination (R2): the proportion of variance explained. Reshef et al. demonstrate [1] that other measures of association (including Spearman’s rank correlation, mutual information, maximal correlation and principal curve-based correlation) are not equitable; different functional forms with similar amounts of noise can produce vastly different estimates of association strength. Here, we explore a conceptually simple approach to quantifying and testing for relationships between variables, showing that generality and equability can be achieved by estimating a gen- eralised R2 through density approximation. We first describe the approach, then proceed to benchmark it on multiple simulated scenarios, examining how it behaves and what power it has as a statistical test. We demonstrate some desirable properties of our method, including the ability to characterise higher order associations between more than two variables, and we high- light some pathologies of MIC. First consider a function with additive noise, y ¼ f ðxÞ þ N . PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 Introduction The coefficient of determina- tion is the proportion of variance in y “explained” by the deterministic component f(x) relative to the total variance in y, which is inflated by unexplained stochastic noise, N . This notion of variance is defined in terms of average squared deviations—the distance between a data point and a model. The explained variance, R2, is 1  s2 Error=s2 Total, where the total variance (s2 Total) is the average squared deviation from a flat “null” model and the error variance (s2 Error) is the aver- age squared deviation from f(x), the “alternative” model. Least squares regression assumes that observations are normally distributed about the explanatory function. The deviation of a point from the regression line can thus be expressed as a probability density, and R2 has an equivalent form [2–4]: R2 ¼ 1  Y i Pðxi; yijnullÞ Pðxi; yijaltÞ  2=n ð1Þ ð1Þ This formulation of R2 asserts that the proportion of unexplained variance is the geometric mean of the squared ratio of the probability of observing a data point under the null model over the probability of that data point under the alternative model. The explained variance is 1 minus the unexplained proportion. See Fig 1 for a visual depiction and see section 1 of S1 File for a derivation. This formulation of R2 asserts that the proportion of unexplained variance is the geometric mean of the squared ratio of the probability of observing a data point under the null model over the probability of that data point under the alternative model. The explained variance is 1 minus the unexplained proportion. See Fig 1 for a visual depiction and see section 1 of S1 File for a derivation. Since this R2 now depends only on the probability density ratio between two models, it is applicable even when the assumptions behind least squares regression are violated. This is a powerful rethinking of R2. The idea of “explained variance” is generalised away from the restrictive assumptions of normally distributed noise, and, most importantly, the very notion of a regression curve is no longer required. This generalised R2 can be calculated as long as the probability distributions for the null and alternative models can be evaluated. Since this R2 now depends only on the probability density ratio between two models, it is applicable even when the assumptions behind least squares regression are violated. Introduction Marginal distributions of X and Y are represented above and to the right. The classical R2 is calculated using deviations of the samples from the blue and white lines. Panel B depicts the probability distribution over xi, yi for the alternative (red) and null (blue) models. Panel C shows the height of the observations on the alternative distribution, relative to the null distribution. The generalised R2 is calculated from the ratio of these heights, and does not require an explicit regression line (white), which is included only as a guide for the eye. See section 2 of S1 File for numerical examples of varying noise levels doi:10.1371/journal.pone.0151551.g001 model for classical R2—a constant function with Gaussian errors—is a particularly bad fit (see section 3 of S1 File). A also has an information theoretic interpretation: for known distributions it is a sample estimate that converges to Linfoot’s ‘Informational Measure of Correlation’ [5] when the number of observations tends to infinity (see section 4 of S1 File). So far, the computation of A requires a known distribution. Estimating ^A  A for a number of observations from an unknown distribution thus reduces to the problem of estimating the density at each point for an independent null and (potentially) dependent alternative model. We adopt a kernel density approach [6, 7], where the density of the distribution at each point is approximated by the sum over a number of Gaussian ‘kernel’ distributions centered at nearby points (see Fig 2). For the null model, we constrain the joint density to be the product of Fig 2. Estimating an unknown distribution. The distribution for the alternative model (red—where X can depend on Y) is constructed by adding two dimensional Gaussian “kernel” distributions centered at each observation. As more of these kernels are added, the distribution comes to resemble the true distribution from which the observations are sampled. We can use a similar approach to estimating a null model that expressly disallows any dependence between X and Y (blue) by constructing one dimensional marginal distributions (the blue lines to either side) by summing one dimensional Gaussian kernels, and then creating the joint distribution as the product of these estimated marginals. Fig 2. Estimating an unknown distribution. The distribution for the alternative model (red—where X can depend on Y) is constructed by adding two dimensional Gaussian “kernel” distributions centered at each observation. Introduction This is a powerful rethinking of R2. The idea of “explained variance” is generalised away from the restrictive assumptions of normally distributed noise, and, most importantly, the very notion of a regression curve is no longer required. This generalised R2 can be calculated as long as the probability distributions for the null and alternative models can be evaluated. We base our measure of dependence between variables upon this generalised R2. Even when a known distribution generates our data, we still need to specify the null distribution before R2 can be computed, but this generalised definition of R2 is agnostic about a choice of null model. An attractive property for a measure of dependence is that it is 0 if and only if X and Y are inde- pendent. A sensible choice of null model is thus where P(X, Y) = P(X)P(Y), enforcing indepen- dence. Since explicitly choosing a null distribution places a restriction on the generalised R2, we distinguish our measure of association, calling it A. Classical R2 from least squares regression assumes a different choice of null model (a constant function with normally distributed errors), so A can be thought of as a sister to classical R2. They are equivalent for bivariate Gaussian dis- tributions where the marginals are also normally distributed, but will differ when the null 2 / 10 Discovering General Multidimensional Associations Fig 1. Illustrating the generalised R2. Panel A: Data is normally distributed about the alternative model—the white regression line Y = sin(X). The null model is the blue Y = 0. Marginal distributions of X and Y are represented above and to the right. The classical R2 is calculated using deviations of the samples from the blue and white lines. Panel B depicts the probability distribution over xi, yi for the alternative (red) and null (blue) models. Panel C shows the height of the observations on the alternative distribution, relative to the null distribution. The generalised R2 is calculated from the ratio of these heights, and does not require an explicit regression line (white), which is included only as a guide for the eye. See section 2 of S1 File for numerical examples of varying noise levels. Fig 1. Illustrating the generalised R2. Panel A: Data is normally distributed about the alternative model—the white regression line Y = sin(X). The null model is the blue Y = 0. doi:10.1371/journal.pone.0151551.g002 PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 Introduction As more of these kernels are added, the distribution comes to resemble the true distribution from which the observations are sampled. We can use a similar approach to estimating a null model that expressly disallows any dependence between X and Y (blue) by constructing one dimensional marginal distributions (the blue lines to either side) by summing one dimensional Gaussian kernels, and then creating the joint distribution as the product of these estimated marginals. doi:10.1371/journal.pone.0151551.g002 doi:10.1371/journal.pone.0151551.g002 3 / 10 PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 Discovering General Multidimensional Associations estimates of the marginal densities, enforcing independence. We wish to constrain ^A to vary between 0 and 1, so we cannot allow the null to outperform the alternative model, lest ^A become negative. We thus define the density of the alternative model at each sample point to be a weighted mixture of dependent (full joint) and independent (product of marginal) models, with a single mixture parameter controlling the proportion for all points. Therefore, the alter- native model can reduce to the null model as a special case, ensuring non-negativity. We esti- mate the model parameters—and thus the densities—by maximizing the cross-validation likelihood (see Methods). We also introduce a statistical test for non-independence associated with ^A, computing p- values through a randomization procedure (See S1 File section 6 for details). Briefly, we use the cross-validation likelihoods for both the null and alternative models to produce a cross-valida- tion likelihood ratio statistic (cvLRS). The null test statistic distribution can be obtained by ran- domly permuting the variables to break the association and induce independence, but we provide a fast approximation to this distribution for bivariate relationships, obtained by fitting a mixture of χ2 distributions to the empirical permutation distribution. As pointed out in Speed [8], an important question is how much of the variance in Y can be explained by X, after controlling for C. Here, we introduce a non-linear analogue of the semi- partial correlation coefficient, which is one approach to ‘controlling’ for covariates in the linear model setting. In the linear case, the semipartial (squared) correlation between Y and X con- trolling for C, denoted here as R2 Y;X;C, is the proportion of variance in Y that can be uniquely explained by X, after the contribution of C has been accounted for. Introduction This has a simple interpre- tation in terms of the coefficients of determination: R2 Y;X;C ¼ R2 Y;XC  R2 Y;C, where R2 Y;XC is the proportion of variance in Y accounted for by X and C together, and R2 Y;C is the proportion of variance in Y accounted for by C alone. Here, we consider the analogous quantity defined in terms of our nonlinear measure of association: ^AY;X;C ¼ ^AY;XC  ^AY;C. PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 Discovering General Multidimensional Associations Fig 3. Equitability and convergence of ^A. Top: For functions of 2 variables, ^A is approximately equitable, as demonstrated with 16 example functions, with N = 1000. Each function is marked with a different color. ^A (left) is closer to the classical R2 than MIC (right), especially for associations near independence. Bottom: Estimates of association from ^A (left) and MIC (right) as sample size (N) increases for three different relationships: a noiseless circle (red), a bivariate normal distribution with expected R2 = 0.5 (green), and independent noise (blue). MIC converges very slowly. doi:10 1371/journal pone 0151551 g003 Fig 3. Equitability and convergence of ^A. Top: For functions of 2 variables, ^A is approximately equitable, as demonstrated with 16 example functions, with N = 1000. Each function is marked with a different color. ^A (left) is closer to the classical R2 than MIC (right), especially for associations near independence. Bottom: Estimates of association from ^A (left) and MIC (right) as sample size (N) increases for three different relationships: a noiseless circle (red), a bivariate normal distribution with expected R2 = 0.5 (green), and independent noise (blue). MIC converges very slowly. doi:10.1371/journal.pone.0151551.g003 association [9] for all non-linear relationships tested. The ^A test was comparable in perfor- mance to the recently proposed HHG test [10], having greater power on 4 out of 7 tested rela- tionships, being particularly more powerful for circular relationships, and low-noise high- complexity non-linear relationships potentially embedded in background noise. We note that MIC has a user-tunable parameter, and this has recently been shown to influence its power to detect relationships [11]. Our results were obtained with the default MIC parameter settings, and may vary with different parameter choices. As shown in Fig 4, ^A generalises well to multiple dimensions, producing equitable estimates very similar to classical R2 for functions of two dimensions. If desired, it can assess the strength of association between vector valued variables, indicating what proportion of the variance in (X, Y) is explained by (A, B, C), for example. It also generalises to more than two variables (with each variable being possibly vector valued), which could be used to discover lower dimensional manifolds embedded in a higher dimensional space (see S1 File section 7). PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 Results Fig 3 demonstrates that ^A is approximately equitable across a number of relationships (see S1 File section 5 for details of the relationships tested—the sample size was n = 1000 in all cases), and is in greater agreement with classical R2 than is MIC, especially for relationships where R2 is close to 0. The noise model was Gaussian in all simulations, and other noise models could exhibit different behavior. When the marginal distribution of a variable departs substantially from a nor- mal distribution, ^A (like MIC) may produce more conservative estimates of association than clas- sical R2 (see S1 File section 3). This is because the null model for ^A makes less restrictive assumptions (only independence is assumed, without a parametric form), describing the data better than the null model for classical R2, which is a constant function with Gaussian errors. ^A converges faster with increasing sample size than MIC (Fig 3—bottom panels). For exam- ple, despite having a theoretical large-sample limit of 1 for a noiseless circle [1], MIC  0.74 when N = 10000. In contrast, ^A≳0:99 when N  200. When testing for significance of non-linear associations, we should expect no free lunch— the power of each method could vary depending on the form of the relationship. Methods should be chosen based on their performance on the kinds of relationships expected from the empirical data. Doing this rationally would require a thorough characterisation of the space of possible relationships which is beyond the scope of this paper. We do, however, compare four tests on 7 different relationships, for illustrative purposes (See S1 File section 6 for details), and find the ^A significance test to be attractive. It has greater power to detect associations than MIC for all but one of the relationships we tested, and outperforms Székely’s dCov test for 4 / 10 PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 We do observe that performance degrades in higher dimensions (data not shown), with ^A underesti- mating the association we would expect from a fixed R2 as the dimensionality increases, which is likely a result of the inefficiency of kernel density estimation in higher dimensions. We show (see S1 File section 8) that our ability to control for covariates is well behaved, agreeing with the linear semipartial correlation when all relationships are linear. Semipartial association becomes more interesting when the data are non-linear. When relationships are 5 / 10 PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 Discovering General Multidimensional Associations Fig 4. Equitability of ^A in higher dimensions. ^A against R2 (left) for multivariate datasets (N = 4000 for all) generated by adding normally distributed noise to 16 different functions of two variables (right—see S1 File section 5 for more detail). d i 10 1371/j l 0151551 004 Fig 4. Equitability of ^A in higher dimensions. ^A against R2 (left) for multivariate datasets (N = 4000 for all) generated by adding normally distributed noise to 16 different functions of two variables (right—see S1 File section 5 for more detail). doi:10 1371/journal pone 0151551 g004 Fig 4. Equitability of ^A in higher dimensions. ^A against R2 (left) for multivariate datasets (N = 4000 for all) generated by adding normally distributed noise to 16 different functions of two variables (right—see S1 File section 5 for more detail). doi:10.1371/journal.pone.0151551.g004 doi:10.1371/journal.pone.0151551.g004 non-linear, the standard linear semipartial correlation can severely underestimate semipartial association (only seeing the linear dependence of Y upon X, regardless of the control variable C) but, more interestingly, it can also overestimate the semipartial association between Y and X by ignoring a non-linear dependence of Y on the control variable C, returning values close to 1 when in fact Y is conditionally independent of X given C. Our non-linear semipartial associa- tion has no such difficulty, returning values close to 0 for such cases. non-linear, the standard linear semipartial correlation can severely underestimate semipartial association (only seeing the linear dependence of Y upon X, regardless of the control variable C) but, more interestingly, it can also overestimate the semipartial association between Y and X by ignoring a non-linear dependence of Y on the control variable C, returning values close to 1 when in fact Y is conditionally independent of X given C. Our non-linear semipartial associa- tion has no such difficulty, returning values close to 0 for such cases. Consider, for example, the following data generating process (Fig 5, panel A): C  Uð1; 1Þ X  C2 þ N ð0; Þ Y  C2 þ N ð0; Þ Y depends quadratically on the control variable C, and is conditionally independent of X given C, so discovering X tells us nothing about Y if we already know the value of C. X and Y are strongly linearly related, but all the association can be explained through the common influ- ence of C. When computing the linear semipartial correlation R2 Y;X;C, Y appears to depend strongly on X even after controlling for C (R2 Y;X;C ¼ 0:90), but this is because the quadratic dependence of Y upon C is missed by a linear model. The non-linear semipartial association discovers the quadratic dependence of Y on C, and correctly declares that X provides no addi- tional influence: ^AY;X;C ¼ 0. If we “correct” the linear semipartial correlation by controlling for C2 rather than C, transforming the association back into linearity, R2 Y;X;C2 ¼ 3:5  105, validat- ing the nonlinear semipartial association of 0. By contrast, consider the following process (Fig 5, panel B): C  Uð1; 1Þ X  C2 þ N ð0; 2Þ Y  X þ N ð0; Þ 6 / 10 Discovering General Multidimensional Associations Fig 5. Semipartial correlation examples. In panel A, Y is conditionally independent of X given C. Missing the quadratic dependence of Y on C, linear R2 Y;X;C ¼ 0:90. Non-linear semipartial association correctly identifies C as explaining all of the variance in Y, yielding ^AY;X;C ¼ 0. In panel B, Y also depends on the noise term in X (see text), and non-linear semipartial association captures this: ^AY;X;C ¼ 0:28. Fig 5. Semipartial correlation examples. In panel A, Y is conditionally independent of X given C. Missing the quadratic dependence of Y on C, linear R2 Y;X;C ¼ 0:90. Non-linear semipartial association correctly identifies C as explaining all of the variance in Y, yielding ^AY;X;C ¼ 0. In panel B, Y also depends on the noise term in X (see text), and non-linear semipartial association captures this: ^AY;X;C ¼ 0:28. doi:10.1371/journal.pone.0151551.g005 doi:10.1371/journal.pone.0151551.g005 Again, there is a quadratic dependence between Y and C, but this time Y also depends partly on the noise component in X, which means that, while C can account for much of the variance in Y, C cannot account for all of it. The linear semipartial correlation again misses the quadratic dependence of Y on C, incorrectly deducing that C cannot account for any of the variance in Y, and asserting the X explains almost all of the variance in X (R2 Y;X;C ¼ 0:90). The non-linear semipartial association behaves sensibly, asserting that 28% of the variance in Y is explained by X after controlling for C (^AY;X;C ¼ 0:28). This result is appropriate given the value of the linear semipartial correlation correcting for the quadratic dependence on C (using C2 as the control variable): R2 Y;X;C2 ¼ 0:28. Section 7 of S1 File demonstrates the use of ^A on a dataset of relationships obtained from Markov-Chain Monte Carlo samples from a phylogenetic analysis of Influenza evolution [12]. Section 7 of S1 File demonstrates the use of ^A on a dataset of relationships obtained from Markov-Chain Monte Carlo samples from a phylogenetic analysis of Influenza evolution [12]. PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 Discussion While this paper represents the initial practical contribution, further work remains to charac- terise the theoretical properties of A and ^A. A is clearly invariant to monotonic transformations of variables, but its estimate ^A is not, although it may be as N tends to infinity (in our particular implementation, we artificially enforce it through a rank transformation). Simulations suggest that ^A tends to 0 wherever variables are independent, and 1 whenever a relation is noiseless and nowhere flat, but perhaps there are other circumstances under which 1 will be the large sample limit (MIC, for example, can achieve 1 at large samples for noisy relationships—see S1 File section 10). Is the ^A test for independence consistent against all alternatives, achieving a power of 100% as N tends to infinity whenever independence is violated in any way? ^A appears to be robust to outliers (see S1 File section 11), but is it possible to design outlier distributions that mislead it? ^A could also be improved by more sophisticated techniques to estimate the density ratio of the joint and independent distributions [13, 14], which may improve the con- vergence of ^A for smaller sample sizes, but at a computational cost. A can be viewed as a PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 7 / 10 Discovering General Multidimensional Associations transformed mutual information, and the performance and equitability of mutual information estimators needs to be examined and compared to our approach for estimating ^A, but this is beyond the scope of the present paper. Also, it may be possible to use our approach for estimat- ing ^A as a means of estimating mutual information itself. This could turn out to perform simi- larly to kernel density-based estimates of mutual information (especially [15] which also uses cross-validation to estimate the kernel bandwidth), but none of those, to our knowledge, use the mixture approach we employ here when estimating the alternative model, so some differ- ences are expected. Kinney and Atwal [16] have recently asserted that “No nontrivial dependence measure can satisfy R2-equitability”, providing a theorem to support this. However, as we show [17], their result hinges on a peculiar definition of “noise”, which allows a trend to be embedded in the noise term, effectively introducing an un-identifiability in their definition which they then exploit to prove the notion incoherent. Discussion When you take the trend out of the noise term, you are left with a perfectly sensible notion of “R2-equitability”. Under the resulting notion of “R2-equitability”, however, R2 is sensitive to non-linear transformations of variables, and isn’t symmetric, as one would expect of any measure defined in terms of squared departure from a trend line. Here we have shown how to inherit the good properties of measures of dependence that are based on the ratio of full joint over independent distributions, which are not sensitive to transformations of variables and are symmetric, while still behaving like an R2, and giving results similar (but not identical) to canonical R2 for reasonable bivariate relationships. Methods Consider two (possibly) vector valued variables, X and Y, with n observations {x1, . . ., xn} and {y1, . . ., yn}. Each xi itself may be a vector xa i ; . . . ; xz i , as may each yi. Further, imagine three ker- nel distributions, KX(x), KY(y) and KXY(<x,y >), where the kernels are symmetric, non-nega- tive, and integrate to 1, and where angle brackets indicate vector concatenation. Our null model assumes that X and Y are independent, and so we define the leave-one-out cross valida- tion likelihood as the product of marginal kernel density estimates: LCVðnullÞ ¼ Y n i¼1 Pðxijx8j6¼iÞPðyijy8j6¼iÞ ð2Þ ð2Þ  Y n i¼1 X 8j6¼i KXðxj  xiÞ n  1  X 8j6¼i KYðyj  yiÞ n  1 " # ð3Þ ð3Þ The alternative model allows Y to depend on X for a proportion of points, (1 −w), with a leave-one-out cross validation likelihood defined as: LCVðaltÞ ¼ Y n i¼1 ½ð1  wÞ  Pðxi; yijx8j6¼i; y8j6¼iÞ þ w  Pðxijx8j6¼iÞPðyijy8j6¼iÞ ð4Þ LCVðaltÞ ¼ Y n i¼1 ½ð1  wÞ  Pðxi; yijx8j6¼i; y8j6¼iÞ þ w  Pðxijx8j6¼iÞPðyijy8j6¼iÞ ð4Þ  Y n i¼1 ð1  wÞ X 8j6¼i KXYð< xj  xi; yj  yi >Þ n  1 þ w X 8j6¼i KXðxj  xiÞ n  1 X 8j6¼i KYðyj  yiÞ n  1 " # ð5Þ ð4Þ  Y n i¼1 ð1  wÞ X 8j6¼i KXYð< xj  xi; yj  yi >Þ n  1 þ w X 8j6¼i KXðxj  xiÞ n  1 X 8j6¼i KYðyj  yiÞ n  1 " # ð5Þ ð5Þ ð5Þ In our particular implementation, the values of each variable are replaced with their ranks (this is for computational convenience and should have little effect since A itself is invariant to order preserving transformations of variables) with ties broken randomly, and the kernels are PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 8 / 10 Discovering General Multidimensional Associations isotropic Gaussians, with KX and KY sharing an ‘independent’ kernel variance parameter s2 I , and KXY having a distinct ‘dependent’ variance parameter, s2 D. With the rank-transformed observations, the data are now integers, and we discretize our Gaussian kernels by evaluating them on a bounded grid, and re-normalizing them. S1 File. Supporting text, figures and tables. pp g g 1. Generalized R2 generalizes classical R2. 2. Sinusoidal example. 3. Classical R2, generalized R2, A, ^A and Linfoots Informational Measure of Correlation. 4. A is a sample approximation of Linfoots Informational Measure of Correlation. 5. List of functions used for the equitablity plots. 6. Significance tests. 7. ^A can detect manifolds. 8. Semipartial association—controlling for variables. 9. Example: BEAST analysis. 10. MIC can return 1 for noisy relationships. 11. ^A is robust to outliers. 12. A small samples bias correction. 13. matie, An R package for computing ^A 14. Execution time: matie versus MIC. (PDF) 1. Generalized R2 generalizes classical R2. 1. Generalized R2 generalizes classical R2. 2. Sinusoidal example. 2. Sinusoidal example. 3. Classical R2, generalized R2, A, ^A and Linfoots Informational Measure of Correlation. 4. A is a sample approximation of Linfoots Informational Measure of Correlation. 5. List of functions used for the equitablity plots. 8. Semipartial association—controlling for variables. 9. Example: BEAST analysis. 9. Example: BEAST analysis. 10. MIC can return 1 for noisy relationships. 11. ^A is robust to outliers. 11. ^A is robust to outliers. 12. A small samples bias correction. 12. A small samples bias correction. 13. matie, An R package for computing ^A 13. matie, An R package for computing ^A 14. Execution time: matie versus MIC. (PDF) 14. Execution time: matie versus MIC. (PDF) Methods The null model thus has a single parameter, s2 I , and the alternative model has 3 parameters: s2 I , s2 D, and w. These parameters are optimised numerically to maximise the cross-validation likelihood, yielding ^A after employing Eq (1). We found this estimate to be slightly biased down (when calibrated relative to classical R2 for bivar- iate Gaussians, where A = classical R2) for small samples, so we included an empirically-esti- mated small samples correction (see S1 File section 12). An R package named matie (“Measuring Association and Testing Independence Efficiently”—see S1 File section 13) for estimating ^A is available on CRAN (http://cran.r-project.org/web/packages/matie/). For effi- ciency, core routines, such as the leave-one-out cross-validation likelihood calculation, are written in C, with everything else in R. The optimization itself is done numerically, using the dfoptim package. Like MIC, estimating ^A is quadratic in the sample size, but with a much lower growth rate than MIC (see S1 File section 14). Acknowledgments We would like to thank Joel Wertheim for sharing BEAST log files with us, and Miguel Lacerda and Konrad Scheffler for helpful comments on the manuscript. BM is supported by K99AI120851 from the National Institute of Allergy and Infectious Diseases. 9 / 10 PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 Discovering General Multidimensional Associations Author Contributions Conceived and designed the experiments: BM DM HM. Performed the experiments: BM DM onceived and designed the experiments: BM DM HM. Performed the experiments: BM DM Conceived and designed the experiments: BM DM HM. Performed the experiments: BM DM HM. Analyzed the data: BM DM HM. Contributed reagents/materials/analysis tools: BM DM HM. Wrote the paper: BM DM HM. HM. Analyzed the data: BM DM HM. Contributed reagents/materials/analysis tools: BM DM HM. Analyzed the data: BM DM HM. Contributed reagents/materials/analysis tools: BM DM HM Wrote the paper BM DM HM M. Analyzed the data: BM DM HM. Contributed reagents/materials/analysis tools: BM DM M. Wrote the paper: BM DM HM. HM. Wrote the paper: BM DM HM. PLOS ONE | DOI:10.1371/journal.pone.0151551 March 18, 2016 References 1. Reshef DN, Reshef YA, Finucane HK, Grossman SR, McVean G, Turnbaugh PJ, et al. Detecting Novel Associations in Large Data Sets. Science. 2011; 334(6062):1518–1524. doi: 10.1126/science.1205438 PMID: 22174245 2. Cox DR, Snell EJ. Analysis of Binary Data, Second Edition (Chapman & Hall/CRC Monographs on Sta- tistics & Applied Probability). 2nd ed. Chapman and Hall/CRC; 1989. 3. Magee L. R 2 Measures Based on Wald and Likelihood Ratio Joint Significance Tests. The American Statistician. 1990; 44(3):250–253. doi: 10.2307/2685352 4. Nagelkerke NJD. A note on a general definition of the coefficient of determination. Biometrika. 1991; 78 (3):691–692. doi: 10.1093/biomet/78.3.691 5. Linfoot EH. An informational measure of correlation. Information and Control. 1957; 1(1):85–89. doi: 10. 1016/S0019-9958(57)90116-X 6. Rosenblatt M. Remarks on Some Nonparametric Estimates of a Density Function. The Annals of Math- ematical Statistics. 1956; 27(3):832–837. doi: 10.1214/aoms/1177728190 7. Parzen E. On Estimation of a Probability Density Function and Mode. The Annals of Mathematical Sta- tistics. 1962; 33(3):1065–1076. doi: 10.1214/aoms/1177704472 8. Speed T. A Correlation for the 21st Century. Science. 2011; 334(6062):1502–1503. doi: 10.1126/ science.1215894 PMID: 22174235 9. Székely GJ, Rizzo ML. Brownian distance covariance. The Annals of Applied Statistics. 2009; 3 (4):1236–1265. doi: 10.1214/09-AOAS312 10. Heller R, Heller Y, Gorfine M. A consistent multivariate test of association based on ranks of distances. Biometrika. 2012 11. Reshef DN, Reshef YA, Sabeti PC, Mitzenmacher MM. An Empirical Study of Leading Measures of Dependence; 2015. Available from: http://arxiv.org/abs/1505.02214 12. Wertheim JO. The Re-Emergence of H1N1 Influenza Virus in 1977: A Cautionary Tale for Estimating Divergence Times Using Biologically Unrealistic Sampling Dates. PLoS ONE. 2010; 5(6):e11184+. doi: 10.1371/journal.pone.0011184 PMID: 20567599 13. Suzuki T, Sugiyama M, Tanaka T. Mutual information approximation via maximum likelihood estimation of density ratio. In: 2009 IEEE International Symposium on Information Theory. IEEE; 2009. p. 463– 467. 14. Vincent P, Bengio Y. Manifold Parzen Windows. In: Advances in Neural Information Processing Sys- tems 15. MIT Press; 2002. p. 825–832. 15. Härdle W, Werwatz A, Müller M, Sperlich S. Nonparametric Density Estimation. In: Nonparametric and Semiparametric Models. Springer Series in Statistics. Springer Berlin Heidelberg; 2004. p. 39–83. Available from: http://dx.doi.org/10.1007/978-3-642-17146-8_3 16. Kinney JB, Atwal GS. Equitability, mutual information, and the maximal information coefficient. Pro- ceedings of the National Academy of Sciences. 2014; 111(9):3354–3359. doi: 10.1073/pnas. 1309933111 17. Murrell B, Murrell D, Murrell H. R2-equitability is satisfiable. Proceedings of the National Academy of Sciences. 2014; 111(21):E2160. doi: 10.1073/pnas.1403623111 10 / 10
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Zur Verbrennung des Kohlenoxyds
Berichte der Deutschen Chemischen Gesellschaft
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I) H. 6, 244. 9) Dabei sollte nach einer damals von Hoppe-Seyler vertretenen Theorie der Wuserstoff den molekularen Sauerstoff aktivieren, 2 H + 03 -+ HsO + 0. 3) Am. 4, 50. Rerlchte d. D. Cbem. Gesellschaft Jahrg. XXXXY. 4 -5 I) H. 6, 244. 9) Dabei sollte nach einer damals von Hoppe-Seyler vertretenen Theorie der Wuserstoff den molekularen Sauerstoff aktivieren, 2 H + 03 -+ HsO + 0. 3) Am. 4, 50. Rerlchte d. D. Cbem. Gesellschaft Jahrg. XXXXY. 679 Um die gleiche Zeit ist von Remsen3) durch wiederholte Versuche festgestellt worden, daB O z o n selbst bei 300° diese Oxy- 4 -5 4 -5 680 dation nicht zu leisten vermag, also selbst nicht unter Bedingungen, unter denen infolge seines Lerfalls atomarer SauerstoFE auftritt. Da demnach offenbar die durch Palladium gesteigerte Oxydationskraft des Sauerqtoffs uicht nus seiner Spaltung in Atome abgeleitet werden kann, so sucbt hloritz Traube’), in eingehender Beschaftigung mit den] vorliegeoden Thema, die Erscbeinung so z u erklaren, da13 durch eine katalytische M’irkung des Palladiums Wasse rs t o f f p e r o x y d entstebe, das dem gleichen Katalysator seine merkwiirdige Oxydationskraft verdanke. Dieser recht wenig klare Erkliirungsversuch stiitzte sich auf die Tatsachte, da13 bei der Reaktion wirklich Wasserstoffperoxyd zu finden war. Da13 der im Palladium gebundene Wasserstoff bei der Reaktion keine Rolle spielt, zeigt T r a u b e dadurch, dn13 der Versuch mit wasserstofffreiem Palladium norh besser gelingt. g g Die gleiche Auffassung ubertrug T r a u be zwei Jahre spater 2, auf die von D i x o n 3, gemnchte Beobnchtung, daB zur Verbrennung des Kohlenoxyds auch bei hohen Temperatimen die Anwesenheit von Spuren von Wasser unerl5Blich ist. Dauach sol1 dieses Wasser durch Kohlenoxyd und Sauerstoff zu Kohlendioxyd und Wasserstoffperoryd umgesetzt werden : CO +H,O +-O, __ + COa+HO.OH. Die letzte Verbiudung sollte ein zweites hiolekul CO zu COY oxydieren und dabei gleirhzeitig das fiir die Wiederholung der Um- setzuug notige Wasser ziiriickliefern. - Im Zusammenbang mit an- deren Untersuchungen*) babe ich mich auch mit der durch Pall n d’ LUDl katalysierten Verbrennung des Kohlenoxyds beschaftigt und bin dabei, entsprecbend meiner Erwartung, zu einer wesentlich anderen Auf- fassung gekommen. A19 wichtigstes Ergebnis fnnd icb, d a13 K o hlen - o x y d a u c h u n t e r volligeni AusschluB von Luftsauerstoff d n r c h P a l l a d i u m s c h w a r z bei G e g e n w a r t v o n Wasser i n d e r KBlte zu K o h l e n d i o x y d v e r b r a n n t wird. Der frei werdende Wasserstoff wird dabei voni Palladium aufgenommen. 679 679 stallisiercn die Substanzcn schwierig und undeutlich, aus Toluol in kleinen Nadeln. stallisiercn die Substanzcn schwierig und undeutlich, aus Toluol in kleinen Nadeln. Aus der Identitiit der beiden Azokcrper ergibt sich die Pormel der Sulfo- shre und die Verdringung der Carboxylguppc durch den Azorest, wie bei der Oxynaphthoesiiure sclbst. N a p h t h 0- p i k r insau r e a u s 1 - 0 x y -4-n a p h th o e siiure. 1.9 g Oxynaphthoesaure wurden in die Sulfosaure iibergefiihrt und unter Eiskiihlung 1 Mol. Salpetersaure in Form von Nitriersiiure zu- gegeben. Unter Kohlensiureentwicklung farbt sich die Masse dunkel. Naoh mehrstundigem Stehen wurde mit der gleichen Menge Wasser versetzt und das in der Kalte ausgeschiedene Pradukt abgesogen. Es ist in Wasser vollstandig liislich, da es groBtenteils ans Sulfosaure besteht, die aber etwa 0.2 g Naphthopikrinsaure (2.4.5-Trinit r o - l - naphthol) in Losung halten. Zur Isolierung salzt man mit Koch- salz aus; beim nochmaligem Behandeln mit Wasser blieb jetzt das Trinitronaphthol ungelost und wurde durch das N a t r i u m s a l z , sowie KrystsUisation aus Essigester und Zusatz von Ligroin gereinigt. Es zeigte den Schmelzpunkt, die .Loslichkeitsverhaltnisse und Schwer- loslichkeit des K a l i u m s a l z e s wie Naphthopikrinsaure. 0.1057 g Sbst.: 14.15 ccm N (20.50, 742 mm). CloH50,Ns. Ber. N 15.05. Gef. N 14.87. 0.1057 g Sbst.: 14.15 ccm N (20.50, 742 mm). CloH50,Ns. Ber. N 15.05. Gef. N 14.87. Durch Zusatz von mehr Nitriersaure wurde die Ausbeute nicht wesentlich erhiibt. Durch Zusatz von mehr Nitriersaure wurde die Ausbeute nicht wesentlich erhiibt. 90. Heinrich Wieland: Zur Verbrennung des Koblenoxyds. [Mitteilung aus dem Chem. Lnborat. der Akad. der Wissensch. zu M~inchen.] (Eingegangen am 24. Fcbruar 1912.) I m Laboratorium von H o p p e - S e y l e r hat \'or 31 Jahren E. Baunirtnn I) die Entdeckung gernacht, da13 K o h l e n o x y d durch den Luftsauerstoff bei Gegenwart von Palladiumwasserstoff und Wasser bei gewiihnlicher Temperatur zu K o h l e n d i o x y d verbrannt wirdz). 9 In meiner letzten Abhsndlung babe ich gizeigt, d 3 bei erhoher Tem- peratur die Reaktion durch Palladium umgekehrt werden kann, indem Pal- ladiumwaaseistoff und CO1 Kohlenoxyd und Wasser geben. *) B. 16, 123 [1883]. 3) Chem. N. 46, 151; vergl. auch B. 38, 2426 [1905]. ‘) Vergl. B. 46, 484 [1912]. 9 In meiner letzten Abhsndlung babe ich gizeigt, d 3 bei erhoher Tem- peratur die Reaktion durch Palladium umgekehrt werden kann, indem Pal- ladiumwaaseistoff und CO1 Kohlenoxyd und Wasser geben. a) B. 18, 1890 [1885]. *) B. 16, 123 [1883]. 3) Chem. N. 46, 151; vergl. auch B. 38, 2426 [1905]. a) B. 18, 1890 [1885]. 679 (Die Zufuhr von Luft bei deu Versuchen von B a u m a n n - T r a u b e hat nur den Zweck, den Wasserstoff zu verbrennen und dadurch die katalytische Kraft des Metalls aufrecht zu erhalten.) Die Reaktion geht also nach dern eiufachen Schema: CO +H10 __ + CO? + Hg’) CO +H10 __ + CO? + Hg’) *) B. 16, 123 [1883]. a) B. 18, 1890 [1885]. *) B. 16, 123 [1883]. a) B. 18, 1890 [1885]. 3) Chem. N. 46, 151; vergl. auch B. 38, 2426 [1905]. 9 In meiner letzten Abhsndlung babe ich gizeigt, d 3 bei erhoher Tem- peratur die Reaktion durch Palladium umgekehrt werden kann, indem Pal- ladiumwaaseistoff und CO1 Kohlenoxyd und Wasser geben. 9 In meiner letzten Abhsndlung babe ich gizeigt, d 3 bei erhoher Tem- peratur die Reaktion durch Palladium umgekehrt werden kann, indem Pal- ladiumwaaseistoff und CO1 Kohlenoxyd und Wasser geben. 68 1 ~ 68 1 \ o r sich und bildet, wie beknnnt, die eine Richtung der sonst nur bei h6beren Temperaturen verlaufenden W a s s e r g a s - R e a k t i o n '). An hi& sngt diese Gleichung uber den chernischen Gang der Reaktion nichts. Ich konnte nun zeigen, da13 ihr erstes Produkt Atneisen- s a u r e ist, die sich, wenn auch in geringer Menge, isolieren liek Palladiumschwarz benirkt daher in der Kalte die Additiou von Wasser an Kohlenoxyd, eine Reaktion, die bekanntlich bisher nur unter Bei- hilfe von starkem Alkali iind in der Warme ausgefuhrt werden konnte. Durch die Entdeckung des geeigneteri Katalysators ist also dem Me- tbylenderivat C: 0 seine so auffallende Rerktionstrlgheit gegenuber cler einfachsten Bdditionsreaktion genomrnen. Ameisensaure wird nun, \vie schon seit langem bekannt ist2), durch fein verteilte Metalle der Platingruppe rasch gespalten in Koblendioxyd und Wasserstoff: 0 : C<::H + COa + HI (im Metall gebunden), und n u r der Urnstand, da13, wie ich fand, diese Reaktion For einem, die rechte Seite allerdings stark begiinstigenden Gleichgewicht Halt Inncht, ermijglichte ihren Nachweis bei der vorliegenden Reaktion. I) Die Rcaktion zwischen Kohlenoxyd und Wasserdampf beginnt bei etme 4000; unter Druck (im EioschlnBrohr) gcht sie bei Gegenwart von Pla- tinschwamm bei 150° vor sich (Maquenne, B1. r2] 39, 308 [1883]). Auch ooter der Wirkung der dunklen elektrischen Entlndung geben Koblenoxyd und Wasserdampf Kolrlensiure und Wasserstoff (loc. cit.). ') Die Bildnng von Knallgas wird auch TOU Dixon zur Erklirung seiner , [ ] Beobachtung angenommen. *) Die Arneisensiiure sieht auch Armstrong a l b Zwischenprodukt an. Zit. nach Dixon, B. 38, 3432 [1905]. Vergl. dazu spater. I) Die Rcaktion zwischen Kohlenoxyd und Wasserdampf beginnt bei etme 4000; unter Druck (im EioschlnBrohr) gcht sie bei Gegenwart von Pla- tinschwamm bei 150° vor sich (Maquenne, B1. r2] 39, 308 [1883]). Auch ooter der Wirkung der dunklen elektrischen Entlndung geben Koblenoxyd und Wasserdampf Kolrlensiure und Wasserstoff (loc. cit.). Vergl. dazu spater. *) Die Arneisensiiure sieht auch Armstrong a l b Zwischenprodukt an. ') Die Bildnng von Knallgas wird auch TOU Dixon zur Erklirung seiner Zit. nach Dixon, B. 38, 3432 [1905]. Beobachtung angenommen. 679 Es liegt nun keio Grund vor, fur die Verbrennung des Kobleo- oxiyds bei hoher Ternperatur (und ohne Katalysator) einen anderen lleaktionsverlauf anzunehmeo, und man wird daber die Unentbebr- lichkeit von Wasserspuren dahin zu deuten haben, da13 sie, wie bei dem in der Kiilte katalysierten ProzeB, das Kohlenoxyd iiber die A r n e i ~ e n s l u r e ~ ) in Kohlendioxyd irnd Wasserstoff verwandeln. Der verbrennende Wasserstoff ') gibt das Wasser irnmer wieder zuriick, das in diesem Sinn als echter Katltlysator wirkt. Es istdenu auch moglich, die B i l d u n g d e r A m e i s e n s a u r e b c i d e r h e i o e n V e r b r e n n u n g n a c h z u w e i s e n , wenn man die Kohlen- oxyd-Flamme, in der Brennsthke unterstutzt durch eine an der Robr- miindung untergestellte kleine Sparflamme, gegeo Eis richtet. l m abschmelzenden Wasser findet man die AmeisensHure, indem nian mit einigen Tropfen Rarytwasser neutralisiert, auf dem Wasser- I) Die Rcaktion zwischen Kohlenoxyd und Wasserdampf beginnt bei etme 4000; unter Druck (im EioschlnBrohr) gcht sie bei Gegenwart von Pla- tinschwamm bei 150° vor sich (Maquenne, B1. r2] 39, 308 [1883]). Auch ooter der Wirkung der dunklen elektrischen Entlndung geben Koblenoxyd und Wasserdampf Kolrlensiure und Wasserstoff (loc. cit.). I) Die Rcaktion zwischen Kohlenoxyd und Wasserdampf beginnt bei etme 4000; unter Druck (im EioschlnBrohr) gcht sie bei Gegenwart von Pla- tinschwamm bei 150° vor sich (Maquenne, B1. r2] 39, 308 [1883]). Auch ooter der Wirkung der dunklen elektrischen Entlndung geben Koblenoxyd und Wasserdampf Kolrlensiure und Wasserstoff (loc. cit.). 45 682 bad einengt und dann nach dem Abfiltrieren des ausgeschiedenen Carbonats die Restlosung zur Trockne verdampft. Der Ruckstand enthalt eine zwar kleine, aber durch das Silbersalz absolut scharl nachweisbare Menge von Bariumformiat. Bei der hier beschriebenen Oxydations-Katalyse des Kohlen- oxyds - und auch bei der normalen Verbrennung - handelt es sich also, streng genommen, nicht um eiuen Oxydationsvorgang, sondern urn eine D e h y d r i e r u n g des Hydrats, der Ameisensriure. Die Verbrennung des Wasserstoffs ist ein sekundlirer Vorgang, der nur den Zweck hat, den Katalysator in Kraft tind den rucklaufendeii ProzeB hintan zu halten. Dies kBnnte man ebensogut durch Weg- pumpen, Diffusioniereu u. dgl. erreichen. 679 Fur die heiWe V e r b r e n - n ung liefert der Knallgas-ProzeW natiirlich die fiir die erste Phase er- forderliche Reaktionswiirme. Was endlich das Wasserstoffperoxyd anlangt, dem T r a u be einr so groBe Bedeutung beigemessen hat, so bildet es iiiir ein unwesent- liches Begleitprodukt; es entsteht nebenbei als erste Strife der Wasser- stoffverbrennung, Ha + Os -+ HO.OH. Dies gilt auch fur sein Vorkommen in der Kohlenoxyd-Flamme. Der Dehpdrieruogswasserstoff aus der vorhio besprochenen Keaktion co + Ha0 -* (COaHa) -+ COa + H, wird durch plotzliches Ab- kuhlen a d dem Weg der Wnsserbilaung als HpO2 festgehalten. D i e B i l d u n g v o u K o h l e n d i o s y d und Wasser bei G e g e n w a r t Y O u Pa 11 a di u m s c h w a r z. I) Das Priiparat enthalt pro Gramm 1.8 ccm Wa.sserstoff. 9, Z. a. Ch. 16, 50. 9 Aus der Summe der:Reaktionswarme CO +Ha0 -* (CO2Ha) --f CO:, +R, 3) Vgl. dam B. 45, 490 [1912]. + 10 OOO cal und der Rildnugswiirme des' PalladiumwasserstolIs. D i e B i l d u n g v o u K o h l e n d i o s y d und Wasser bei G e g e n w a r t Y O u Pa 11 a di u m s c h w a r z. Um den Versuch absolut einwandfrei z u gestalten, d. h. jede Miiglichkeit einer Sauerstoffwirkung auszuschlieBen, wurden 0.76 g mit Wasserstoff angeatztes Schwarz, dessen Darstelluog in1 letzteu Heft dieser Berichte (S. 489) beschrieben ist, bei AusschluB von Luft in eirie mit Stickstoff gefiillte C4lasflasche (50 ccm Inhalt) ge- bracht und mit 4 ccm Wasser iiberschichtet. Dann wurde mit reinem, aus Ameisensriure uud konzentrierter Schwefelsaure entwickeltem und durch 50-proz. Kalilnuge geschicktem Kokleuoxyd der Stickstoff ver- drangt und die Flasche, tinter schwschem CO-Druck stehend, auf der Maschine geschiittelt. Der CO-Vorrat befand sich in einem Azotometor, das vom oberen Rohr aus durch einen Schlauch nit der Schuttelflasche verbunden und schon von Anfang an in dan Apparat eingeschaltet war. Das Ableitungsrohr der Flasche wurde wiihrend der Heaktion mit einem Quetschhahn geschlossen. Nach siebeustundigem Schutteln hatte das Volumen im Azoto- meter urn 22.S ccrn abgenommen, teils infolge der Gasbindung im Palladium, teils infolge der gr6Beren Wasserliislichkeit der wahrend 683 der Reaktion gebildeteo KohlensHure. Es wurde jetzt der Inhalt des ganzen Systems durch Stickstoft in zwei Vorlagen mit Barytwasser getrieben und die gebildete Kohlensaure indirekt als B a r i u m s u l f a t bestimmt. Erhalten wurden 0.203 g SulIat, entsprechend 0.0383 g oder etwa 22ccm (feuchten) Kohlendioxyds. Das Wasser, das bei der Reaktion zugegen war, reagierte deutlich sauer. Es wurde mit einigen Tropfe'n Barytwasser neutralisiert rind dann auf dem Wasser- bade zur Trockne verdampft. Der Ruckstand bestand linter den1 Mikroskop aus den gleichen Krystallen, wie ein eigena dargestelltes Priiparat von B a r i u m f o r m i a t ; in wenig Wasser aufgenommen, gab er, wie dieses? ein schwer losliches farbloaes S i l b e r s a l z , das beini Erwiirmen auf dem Wasserbade schwarz wurde. der Reaktion gebildeteo KohlensHure. Es wurde jetzt der Inhalt des ganzen Systems durch Stickstoft in zwei Vorlagen mit Barytwasser getrieben und die gebildete Kohlensaure indirekt als B a r i u m s u l f a t bestimmt. Erhalten wurden 0.203 g SulIat, entsprechend 0.0383 g oder etwa 22ccm (feuchten) Kohlendioxyds. Das Wasser, das bei der Reaktion zugegen war, reagierte deutlich sauer. Es wurde mit einigen Tropfe'n Barytwasser neutralisiert rind dann auf dem Wasser- bade zur Trockne verdampft. D i e B i l d u n g v o u K o h l e n d i o s y d und Wasser bei G e g e n w a r t Y O u Pa 11 a di u m s c h w a r z. Der Ruckstand bestand linter den1 Mikroskop aus den gleichen Krystallen, wie ein eigena dargestelltes Priiparat von B a r i u m f o r m i a t ; in wenig Wasser aufgenommen, gab er, wie dieses? ein schwer losliches farbloaes S i l b e r s a l z , das beini Erwiirmen auf dem Wasserbade schwarz wurde. Bei eineni zweiten Versuch wurde ebenfalls die K o h l e n s a u r e bestimrnt, die Anieisensiiure nachgewiesen, au(3erdern such nocb der rom Palladium aulgenomniene Wnsserstoff gemessen. Ange- wandt 0.35 g Schwarz'). 0.32 g davon enthielteu nach der Reaktion 8 ccm Gas; davon waren nach der (hsanalyse 1.5 ccni CO, der Rest r e i n e r Wasserstoff. Da die Absorptionsfahigkeit des Palladium: fur CO nach H e r b e c k uod L u n g e ? ) sehr gering ist, so durfte das gefundene CO auch fur Wasserstoff einzusetzen sein, entstanden nacli COa + HS -+ CO + Ha09). Rei der Reaktion waren also 9 ccni Wasserstoff entstanden, denen 9.6 ccm CO, (Ref. 0.093 g BaSO,) gegenuberstehen. g g Ein Vergleich mit der COa-Meoge des ersten Versuchs zeigt, daU der Umsatz ziemlich genau proportional ist dem Gewicht des ange- wandten Palladiums. Ein dritter Versuch wurde mit einem absichtlich einige Zeit der Luft ausgesetzten Priiparat gernacht. Auch hier wurdeii a m 0.24 g 5 ccni Wasserstoff (mit 40°/0 CO) erhalten. Will man die geschilderte Rerktion nur qualitativ ausfiihren, z. B. a h Vorlesungsversuch, so bringt man das Palladiomschware (ca. 0.3-0.5 6) unter AusschluB von Luft in ein von Stickstoff durchstromtes, umgebogenes Kugelrohr, wie es zum Abschlu5 von Kippschen Apparaten gebraucht wird. Der obere Trichter ist abgesprengt; hier ist dic Virbindung mit dem Kohlen- oxyd-Generator. Das untere Ende sitzt auf einer mit Barytwasser gefallten Sangflasche. Das Kohlenoxyd wird durch 50-proz. Kalilauge und dam durch Wasser geleitet. Beim Eiotritt des Gases erwiirmt sich das Palladium '), und alsbald beginnt die Triibuog des Barytwaasers; nach wcnigen Minuten hat sich ein betrachtlicher Niederschlag von Bariumcarbonat gebildet. Ersetzt man jetzt das carbonathaltige Barytwasser durch frisches klares, ao bemerkt man, dab fortan die Triibung nur langsam mieder eintritt. Das Schwsrx ist init WasserstoFf beladen und hat dadurch stark an hktivitit vcrloren. I ) B. 7, 1038 [1874]. B. 44, 2309 [1911]. D i e B i l d u n g v o u K o h l e n d i o s y d und Wasser bei G e g e n w a r t Y O u Pa 11 a di u m s c h w a r z. Nimmt man jetzt durch Einleiten von LuFt (durch ein schuo vor dem Ver- suche eingeschaltetes Dreiwegrohr) den Wasserstoff weg, so stellt sich die katalptische Wirkung sofort wieder her, und man kann nun lingere Zeit dichte Wolken von Bariumcarbonat in tler Vorlagc erzeugcn. Infolgo der Wlrme- tijnung aus der Knallgas-Verbrennung nimmt der Katalysator eine Temperatnr ron 40-500 an. Der Versuch verliuft analog, nur etwas langsamer, wenn man das Palladiumschwarz in Wasser suspendiert. Dabei bietet das Kugelrohr den Vorteil, daS beim Heben und Senken immer neue Teile des Schwarzes mit dem Gas in Beriihrung kommen; man hat so cinen gewissen Ersatz Fur die Schiittslmaschine. Streut mau in ein Reagensglas, das niit feucbtem Kohlenoxyd u n d Luft gefiilit ist, Palladiumschwarz, in feioern Regen ein so iiuRert sicli rler kntdysierte Vorgang in hiibschem, lebhaftem Puokenspruben. A m ei s e n s ii u r e a u s I< oh 1 en d i o s y d u d W a s s e r s t o f f. A m ei s e n s ii u r e a u s I< oh 1 en d i o s y d u d W a s s e r s t o f f. Es ist schon seit langem bekannt, daB die zweite Stufe der im yorhergebenden behandelten Reaktion, die Spalt.ung der Ameisen- scture in Kohlendioxyd und Wasserstoff, durch fein verteilte Platin- metalle auRerordentlicb bescbleunigt wird. St. Claire-Deville und Debray’) haben die Reaktion mit Rhodium- ubd Iridiummohr zu- erst beobachtet. Irrtunilicherweise geben sie an, daB Phtinmohr UII- wirksam sei. Vielleicbt war ibr Priiparat gegliiht worden, denn da- (lurch werden, wie ich fand, sehr wirksame Palladiumpraparate vollig passiviert. Fiir das Palladiumschwarz ist die gleicbe debydrierende Wirkung kijrzlich von Z el i s k y ’) angegeben worden. Von allen l)ehydrierungen, die icb bis jetzt untersucbt habe, unterscheidet sicb die vorliegende dadurch, da13 die katalytische Kraft des Palladiums iiber seine Wasserstoff-Kapazitat binausgebt, daB sich also freier Wnsserstoff entwickelt. So konnten mit 0.32 g Palladiumscbwsrz a m 2 ccm einer 20-proz. A m e i s e n s a u r e - L o s u n g in wenigen Minuten 30 ccni reineu Wasserst.offs (analysiert) aufgefangen werden. Dann nber geht die katalytische Wirkung sebr rssch zuruck, und nacb 3 Tagen entbalt die uber dem Metal1 stebende Losung nocb bedeu- tende Mengen der Saure. Es wurde oben verrnutet, daS der NachweiJ der Smeisensaure Iwi der Katalyse von Kohlenoxyd und Wasser dem Umstand zu ver- clxnken sei, daB die Debydrierung an ein Gleicbgewicht gebunden sei, iind zwar nahm ich dies deshalb ao, weil sich K o b l e n d i o x y d durcb IC’asserstoff (und Palladium), w n n auch sebr langsam, zu Ameisen- 685 s i i u r e r e d u z i e r e n liibt. Bei diesen Versuchen, bei denen mich Hr. Dr. E r n s t Waser niit Eifer und Geschick unterstiitzt hat, wurde eiue kolloidale Palladiumlosung nrit den beiden Gasen, die in Azcito- metern mit der Flasche in Verbindung standen, jeweils 20 Stunden lang geschiittelt und die in sehr geringer Menge entstandene A m e i s e n - s i u r e wie oben isoliert und nachgewiesen. Bei einem letzten Ver- such wurden etwa 0.5 g Palladiumschwarz in 20 ccm Wasser mit Wasserstoff gesiittigt, daon Fliissigkeit und Gasraum mit Kohlensiure erfullt und in Verbindung mit der Wasserstoffbombe bei l’h Atrn. oberdruck 8 Stunden l a g geschiittelt. Hierbei wurde etwas mehr Ameisenslure gewonnen, aber immerhin nur eioe Menge, die einige Milligramm nicht uberatieg. 1) Cr. r. 162, 1212 [1911]. A m ei s e n s ii u r e a u s I< oh 1 en d i o s y d u d W a s s e r s t o f f. Die vollstandige U m k e h r u n g d e r W a s s e r g a s - R e a k t i o n , also die Spaltung der Anieivensilure in Kohlenoxyd und Wasser, scheint das Palladiumschwarz bei gewiiholicher Temperatur nicht zu kataly- sieren. Jedoch ist zu erwiihnen, daB S a b a t i e r und Mailhe’) im l i t a n o x y d eine Substanz gefunden haben, die bei 250-300° Ameisen- saure in CO + &C) spaltet, und da8 nach denselben Autoren T h o r - CO H o x y d die ainphotere Katalyse COzHg <: ~ ~ 2 unter ahnlichen o x y d die ainphotere Katalyse COzHg <: ~ ~ 2 unter ahnlichen Sedingungen zu leisten vermag. o x y d die ainphotere Katalyse COzHg <: ~ ~ 2 unter ahnlichen Sedingungen zu leisten vermag. Sedingungen zu leisten vermag. Sedingungen zu leisten vermag. 91. Heinrich Wieland: mer die katalytieche Urnwandlung von Sohwefeidioxyd in Schwefelaliure. (Vorliufige Mitteilung.) (Eingegnngen am 24. Februar 1912.) 91. Heinrich Wieland: mer die katalytieche Urnwandlung von Sohwefeidioxyd in Schwefelaliure. (Vorliufige Mitteilung.) (Eingegnngen am 24. Februar 1912.) (Eingegnngen am 24. Februar 1912.) (Eingegnngen am 24. Februar 1912.) Die vorstehend beschriebenen Walirnehrnungen veranlahten mich, die Katalyse yon Schwefeidiox’yd u n d W a s s e r , d. h. von Schweflig- siiurehydrat, vom gleichen Gesichtspunkt aus zu untersuchen. Leitet man uber Palladiumschwarz u n t e r v o l l k o m m e n e m A u s s c h l u B von Luftsauerstoff feuchtes Schwefeldioxyd, so fiodet tinter be- triichtlicher Warmeentwicklnng sofortige Reaktion statt. Nachdeni hierauf dles Schwefeldioxyd durch Kohlensiiure ausgetrieben ist, findet man nach dem Aufnehmen in Wasser im Palladium eine be- trachtliche Menge YOU Schwefelsiiure. Da die Reaktion ohne Sauerstoff vor sich gegangen ist, k a n n d i e Schwefelsiiure n u r 1) Cr. r. 162, 1212 [1911].
https://openalex.org/W2473899497
https://jmla.pitt.edu/ojs/jmla/article/download/16/11
English
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Patron perception and utilization of an embedded librarian program
Journal of the Medical Library Association
2,016
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SURVEYS AND STUDIES SURVEYS AND STUDIES SURVEYS AND STUDIES Patron perception and utilization of an embedded librarian program Lindsay Blake, MLIS, AHIP; Darra Ballance, MLIS, AHIP; Kathy Davies, MLS; Julie K. Gaines, MLIS; Kim Mears, MLIS, AHIP; Peter Shipman, MLIS; Maryska Connolly-Brown, MLIS; Vicki Burchfield, MLIS See end of article for authors’ affiliations. DOI: http://dx.doi.org/10.3163/1536-5050.104.3.008 Lindsay Blake, MLIS, AHIP; Darra Ballance, MLIS, AHIP; Kathy Davies, MLS; Julie K. Gaines, MLIS; Kim Mears, MLIS, AHIP; Peter Shipman, MLIS; Maryska Connolly-Brown, MLIS; Vicki Burchfield, MLIS See end of article for authors’ affiliations. Objective: The study measured the perceived value of an academic library’s embedded librarian service model. Setting: The study took place at the health sciences campuses of a research institution. Methods: A web-based survey was distributed that asked respondents a series of questions about their utilization of and satisfaction with embedded librarians and services. Results: Over 58% of respondents reported being aware of their embedded librarians, and 95% of these were satisfied with provided services. Conclusions: The overall satisfaction with services was encouraging, but awareness of the embedded program was low, suggesting an overall need for marketing of services. Keywords: Library Surveys[MeSH], Perception[MeSH], Embedded Librarians, Evaluation, Library Services, Awareness, Satisfaction as data management planning, research impact measurement, and scholarly communication. Embedded librarianship is a service model that moves librarians physically away from the library building and places them where patrons work. ‘‘Embedded’’ can mean physical space within a school, academic department, or research group, or librarians may be virtually embedded in online courses. Working in close proximity to patrons in specific disciplines allows the librarian to more accurately assess needs and develop customized programs and services beyond traditional library reference services. New roles for embedded librarians may include becoming partners for scholarly research, pursuing grant funding, or being appointed to college governing committees. Embedded librarians can expand their roles by learning and incorporating nontraditional skills such The implementation of the embedded service model at Augusta University has already been reported [1]. While there are descriptions of many facets of embedded librarian service, there is no comprehensive tool evaluating the activities of embedded librarians that can answer the question: how do patrons perceive the value of embedded librarian services? We sought to measure the awareness and perception of the new service model among clinicians, faculty, and students with a survey instrument. A supplemental appendix is available with the online version of this journal. RESULTS The survey was distributed to more than 4,000 students, faculty, and residents and was completed by 381 respondents, resulting in a response rate of 10%. Over 58% of total respondents reported being aware of their colleges’ or departments’ embedded librarians. Respondents who did not recognize an embedded librarian by name or photograph or had not worked with an embedded librarian accounted for 45% of the total responses, while the remaining 55% of respondents chose 1 or more librarians who had worked with them. Most respondents who had indicated awareness of their colleges’ or departments’ embedded librarians had contacted an embedded librarian 1–5 times within the last 12 months (70%). Contact occurred primarily through email (77%) and in person (64%). Respondents who were aware of their colleges’ or departments’ embedded librarians were asked to identify their primary appointment or college (Figure 1). Changes to the survey were made to reflect the responses received from librarian reviews and campus interview sessions. The final web-based survey was distributed by email in April 2015 to all Augusta University students, full-time faculty, clinicians, and residents in areas where embedded librarians were assigned: four colleges, two hospital departments, and one institute. Responses were solicited for four weeks; weekly reminder emails were sent; and the embedded librarians personally encouraged participation from their embedded areas. g departments’ embedded librarians had contacted an embedded librarian 1–5 times within the last 12 months (70%). Contact occurred primarily through email (77%) and in person (64%). Respondents who were aware of their colleges’ or departments’ embedded librarians were asked to identify their primary appointment or college (Figure 1). The survey included both Likert-scale and open- ended questions and began by defining ‘‘embedded librarian.’’ Respondents were then asked about their awareness of the embedded librarian program. Thumbnail photographs of each embedded librarian assisted respondents with identifying the librarians with whom they have worked. Respondents who were unfamiliar with the program and unable to identify a librarian were directed to open-ended questions to explore why. The remaining respondents self-identified as a student, resident, clinician, or faculty member and then were routed to questions specific to that role. The question set varied according to role: student questions related to classwork and use of library resources, faculty questions related to teaching and research, and clinician or resident questions related to patient care and clinical training. RESULTS Because most faculty also fulfill clinical roles, respondents who identified as faculty or clinician had the opportunity to answer both sets of questions. The survey included both Likert-scale and open- ended questions and began by defining ‘‘embedded librarian.’’ Respondents were then asked about their awareness of the embedded librarian program. Student responses Nearly 82% of the 106 students who indicated working with a librarian were also aware that a librarian was embedded in their colleges. Eleven students (10.6%) had worked with 2 or more embedded librarians. Embedded librarian services used most frequently by students included assistance with classwork or homework assignments (58.7%), group or classroom instruction (52.9%), and literature searching (53.9%). A breakdown of results by college revealed unique perspectives on the value of library services. Allied health sciences students strongly agreed that librarian assistance helped them complete assignments and improved their grades and projects. Dental students felt that assistance from librarians improved their literature searching skills and projects and increased their confidence in completing research. Nursing students felt more confident in their literature searches, completion of assignments, and research. They also felt that librarian assistance led to an improvement in their grades. Medical students agreed, but did not strongly agree, that working with a librarian helped them in class and Survey This article has been approved for the Medical Library Association’s Independent Reading Program ,http://www.mlanet. org/page/independent-reading-program.. A web-based survey was distributed to health sciences students, residents, faculty, and clinicians using Qualtrics software (Appendix, online only). The study team began survey development by f hi f hi A supplemental appendix is available with the online version of this journal. J Med Libr Assoc 104(3) July 2016 226 An embedded librarian program (frequency and percent) were calculated for survey responses regarding awareness, use, and satisfaction with embedded librarians for students, faculty, and clinician or resident respondents. collaborating with experts from the university’s Educational Innovation Institute (EII). Input from EII consultants helped the study team apply established principles to increase the internal validity of survey results. Specific validity components that were examined included content and internal structure [2]. Institutional review board approval was granted for the study. The survey was distributed by email to a sample of health sciences librarians, both embedded and traditional, for input and critique. Additionally, focus interviews were conducted on campus with students, faculty, and residents to obtain feedback on the questions and survey format. collaborating with experts from the university’s Educational Innovation Institute (EII). Input from EII consultants helped the study team apply established principles to increase the internal validity of survey results. Specific validity components that were examined included content and internal structure [2]. Institutional review board approval was granted for the study. The survey was distributed by email to a sample of health sciences librarians, both embedded and traditional, for input and critique. Additionally, focus interviews were conducted on campus with students, faculty, and residents to obtain feedback on the questions and survey format. Analysis All statistical analysis was performed by a statistician from the Department of Biostatistics and Epidemiology using SAS 9.4. Descriptive statistics J Med Libr Assoc 104(3) July 2016 227 Blake et al. Figure 1 Survey respondents by college and institution Figure 1 Survey respondents by college and institution used included online teaching of a course (41.9%), publication (34.9%), and scholarly support (27.9%). with completion of assignments, improvement of grades and projects, and confidence in their literature searches and research. The majority of faculty agreed or strongly agreed (97.6%) that embedded librarians saved them time and were an integral part of their groups (95.3%). Most faculty agreed or strongly agreed (96.9%) that literature searches conducted by the embedded librarian helped them make better decisions about their work. All faculty agreed that they would contact their embedded librarian again in the future. Overall, most students (94%) agreed or strongly agreed that they were satisfied with the services that the embedded librarians provided, and 84% agreed or strongly agreed that they would seek out the help of an embedded librarian for future projects or assignments. Clinician and resident responses Of note, one respondent indicated that ‘‘all the embedded librarians have made a significant impact in my research, teaching, and service to the [university] community.’’ When asked to identify additional services that embedded librarians should provide, respondents indicated that embedded librarian services at satellite campuses were needed, as well as assistance with literature searching for systematic reviews. The survey suggests that perception—or how our patrons understand our role and value—may be the area needing the most improvement. To gauge perception of the program, the term ‘‘embedded librarian’’ was first clearly defined. It is important to note that all recipients of the survey belonged to a college or department with an embedded librarian. Of 381 responses, nearly 55% were able to correctly identify an embedded librarian by photograph. This suggested that the embedded librarians were familiar faces in those colleges, but that librarians should work harder to promote themselves and the range of services that they provide through the embedded program. Accurate assessment regarding perceptions of an embedded program is hard to obtain and measure, yet is vitally important to giving a more complete picture of the overall success of a program. Deficiency in any area can impact the effectiveness of an embedded program. For example, failure to inform users of available services or lack of follow- through when providing those services can both negatively impact perception of the program and the library in general. To this end, many of the questions in our survey were designed to measure how librarians were viewed and the impact that they were perceived to have on research quality, learning objectives, and work outcomes. The overall satisfaction with services was encouraging, with 95% of respondents who had worked with a librarian agreeing or strongly agreeing that embedded services added value to projects, embedded librarians were an integral part of the group, and the services of embedded librarians would be used in the future. The majority of contact with embedded librarians was conducted through email and in person, rather than through the learning management system, phone calls, or text messaging. Surprisingly, despite the ease and availability of electronic means of contact, respondents indicated that they initiated contact with a librarian in person nearly 64% of the time. This would indicate that having a physical presence prompts more frequent use of the librarian and library services. Clinician and resident responses Around 93% of the 43 faculty respondents were aware that embedded librarians worked with them in their colleges. Email was the most popular form of contact (93.0%), followed by in person (58.1%) and phone (41.9%). This was also the only group in the survey to use text messaging. Sixty-three clinicians, which included residents and faculty with clinical appointments, completed the survey. Respondents indicated using librarian assistance for providing education on rounds (67%), managing patient care (55%), and aiding their choice of tests (36%) or treatments (36%). Information provided by the librarian most commonly resulted in changes in education during rounds (44%), management of patient care (36 5%), and choice of Literature search services were by far the most popular among faculty, with 83.7% saying they had worked with an embedded librarian for these services. Additional services that faculty frequently management of patient care (36.5%), and choice of test (24%). J Med Libr Assoc 104(3) July 2016 228 An embedded librarian program DISCUSSION services. Others stated that they had not had a project that would require the use of embedded librarian services. When asked what would help respondents consult their embedded librarians more often, the participants indicated that increased advertising of embedded services would be advantageous, as well as an increased presence in clinical departments or colleges. Our team’s goal for the survey was to measure patron perception and use of embedded librarians at our institution. We accomplished this by assessing patron knowledge of the overall embedded program and individual embedded librarians, patron use of specific embedded services, and the perceived value of embedded services and librarians. Ultimately, this survey asked respondents to expand upon their experiences by providing information such as how users customarily contacted embedded librarians, what services they used, and what impact—positive or negative—they felt that embedded librarians had on their work or research. Additional open-ended questions were evaluated for overall themes. When asked in what ways an embedded librarian added value to classes and/or sessions, respondents indicated that the librarian assisted with class projects and taught valuable literature searching skills in both the academic and clinical setting. J Med Libr Assoc 104(3) July 2016 AUTHORS’ AFFILIATIONS Lindsay Blake, MLIS, AHIP, lblake@augusta.edu, Clinical Librarian; Darra Ballance, MLIS, AHIP, dballance@ augusta.edu, Area Health Ed- ucation Centers (AHEC) Li- brarian; Kathy Davies, MLS, kadavies@augusta.edu, Asso- ciate Director of Research; Robert B. Greenblatt M.D. Li- brary, Augusta University, 1120 15th Street, Augusta, GA 30912; Julie K. Gaines, MLIS, jkgaines@uga.edu, Head, Augusta University/University of Georgia Partnership Campus Library, Augusta University, Health Science Campus, Russell Hall, Room 222, Athens, GA 30602; Kim Mears, MLIS, AHIP, kmears@augusta.edu, Scholarly Communications Li- brarian; Peter Shipman, MLIS, pshipman@ augusta.edu, Dental Medicine and Cancer Librarian; Robert B. Greenblatt M.D. Library, Augusta Univer- sity, 1120 15th Street, Augusta, GA 30912; Maryska Connolly-Brown, MLIS, mconnolly-brown@ hsc.edu, Technical Services Librarian, Walter M. Bortz III Library, Hampden-Sydney College, P.O. Box 7, Hampden-Sydney, VA 23943; Vicki Burch- field, MLIS, vburchfield@augusta.edu, Nursing In- formation Librarian, Robert B. Greenblatt M.D. Library, Augusta University, 1120 15th Street, Au- gusta, GA 30912 Limitations One major limitation in our study was a response rate of just 10%. The majority of respondents may represent the population who have had a positive experience with the program in the past or have a preexisting relationship due to ongoing research or collaboration. This population would be more likely to have daily contact with embedded librarians and, as such, have received more reminders about the survey and had more motivation to participate. In contrast, other potential respondents would have received only the initial and follow-up emails. The online format was selected for ease of distribution and anonymity but may have presented another limitation as technological aptitude, browser compatibility, and other unforeseen technical challenges could have prevented some potential respondents from participating. Clinician and resident responses Indeed, Clyde and Lee noted that a physical presence in buildings was associated with increased student and faculty unscheduled consultations, increased requests for class instruction and research support, and increased invitations to departmental events [3]. This supports the value of having a librarian on site to answer questions as they arise. Circumstances vary from one college to the next, making embedded librarians and the services that they provide quite different even within an institution. Because college and departmental needs vary, the survey could not list all basic and specialized services provided by each embedded librarian. Qualitative research has also not been done in each embedded area. While this type of research would give a more unbiased picture of the value of Individuals who did not identify an embedded librarian in the survey were asked if there was a reason that they had not had the opportunity to work with an embedded librarian. Many stated that they were unaware of the embedded librarians and expressed uncertainty over the extent of provided J Med Libr Assoc 104(3) July 2016 229 Blake et al. Administration. 2011;51(4):389–402. DOI: http://dx.doi. org/10.1080/01930826.2011.556963. embedded librarians, it would necessarily have to be tailored to all librarians and their embedded areas. Since the goal is a unified embedded evaluation, a more inclusive approach was required. Received July 2015; accepted February 2016 REFERENCES 1. Blake L, Mears K, Davies K, Ballance D, Shipman P, Connolly-Brown M, Gaines JK. Adapting an embedded model of librarianship, college by college. Med Ref Serv Q. 2014;33(3):264–77. DOI: http://dx.doi.org/10.1080/ 02763869.2014.925668. 2. Downing SM. Validity: on meaningful interpretation of assessment data. Med Educ. 2003 Sep;37(9):830–7. 2. Downing SM. Validity: on meaningful interpretation of assessment data. Med Educ. 2003 Sep;37(9):830–7. 3. Clyde J, Lee J. Embedded reference to embedded librarianship: 6 years at the University of Calgary. J Libr 3. Clyde J, Lee J. Embedded reference to embedded librarianship: 6 years at the University of Calgary. J Libr 230 J Med Libr Assoc 104(3) July 2016
https://openalex.org/W2145494382
https://europepmc.org/articles/pmc1459173?pdf=render
English
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Mining, compressing and classifying with extensible motifs
Algorithms for molecular biology
2,006
cc-by
5,885
Published: 23 March 2006 Published: 23 March 2006 Algorithms for Molecular Biology2006, 1:4 doi:10.1186/1748-7188-1-4 This article is available from: http://www.almob.org/content/1/1/4 © 2006Apostolico et al; licensee BioMed Central Ltd. © 006 posto co et a ; ce see o e Ce t a t . This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central Algorithms for Molecular Biology Open Access Abstract Background: Motif patterns of maximal saturation emerged originally in contexts of pattern discovery in biomolecular sequences and have recently proven a valuable notion also in the design of data compression schemes. Informally, a motif is a string of intermittently solid and wild characters that recurs more or less frequently in an input sequence or family of sequences. Motif discovery techniques and tools tend to be computationally imposing, however, special classes of "rigid" motifs have been identified of which the discovery is affordable in low polynomial time. Results: In the present work, "extensible" motifs are considered such that each sequence of gaps comes endowed with some elasticity, whereby the same pattern may be stretched to fit segments of the source that match all the solid characters but are otherwise of different lengths. A few applications of this notion are then described. In applications of data compression by textual substitution, extensible motifs are seen to bring savings on the size of the codebook, and hence to improve compression. In germane contexts, in which compressibility is used in its dual role as a basis for structural inference and classification, extensible motifs are seen to support unsupervised classification and phylogeny reconstruction. Conclusion: Off-line compression based on extensible motifs can be used advantageously to compress and classify biological sequences. Page 1 of 7 (page number not for citation purposes) Research Mining, compressing and classifying with extensible motifs Alberto Apostolico*1,2, Matteo Comin1 and Laxmi Parida3 Address: 1Dipartimento di Ingegneria dell'lnformazione, Università di Padova, Padova, Italy, 2College of Computing, Georgia Institute of Technology, 801 Atlantic Drive, Atlanta, GA 30332, USA and 3IBM T. J. Watson Research Center, Yorktown Heights, NY 10598, USA Email: Alberto Apostolico* - axa@dei.unipd.it; Matteo Comin - ciompin@dei.unipd.it; Laxmi Parida - parida@us.ibm.com * Corresponding author Received: 06 March 2006 Accepted: 23 March 2006 Received: 06 March 2006 Accepted: 23 March 2006 Background Main() Iterate(m, B, Result) { { Result ← {}; G:l m' ← m; B ← {mi|mi is a cell}; G:2 For each b = Extract(B) with For each m = Extract(B) G:3 ((b ~-compatible m' Iterate(m, B, Result); OR (m' ~-compatible b)) Result ← Result; G:4 If (m' ~-compatible b) } G:5 mt ← m' ~ b; G:6 If Nodelnconsistent(mt) exit; G:7 If (| m'| = | b|) B ← B - {b}; G:8 If (| | ≤ K) G:9 m' ← mt; G:10 Iterate(m', B, Result); G:11 If (b ~-compatible m') G:12 mt ← b ~ m'; G:13 If Nodelnconsistent(mt) exit; G:14 If (| m'| = | b|) B ← B - {b}; G:15 If (| | ≥ K) G:16 m' ← mt; G:17 Iterate(m', B, Result); G:18 For each r ∈ Result with r = m' G:19 If (m' is not maximal w.r.t. r) return; G:20 Result ← Result ∫ {m'}; }   mt   mt   m. A motif mi is maximal in extension if no annotated dot character of mi can be replaced by a fixed length substring (without annotated dot characters) that appears in all the locations in m. A maximal motif is maximal in compo- sition, in extension and in length. For an exhaustive description of these properties we refer the reader to [1].   there exists a realization m' of m that occurs at l. Note than an extensible string m could possibly occur more than once at a location on a sequence s. Throughout in the dis- cussion we are interested mostly in the (unique) first left- most occurrence at each location. there exists a realization m' of m that occurs at l. Note than an extensible string m could possibly occur more than once at a location on a sequence s. Throughout in the dis- cussion we are interested mostly in the (unique) first left- most occurrence at each location. For a sequence s and positive integer k, k ≤ |s|, a string (extensible or rigid) m is a motif of s with |m| > 1 and loca- tion list m = (l1, l2, ..., lp), if both m[1] and m[|m|] are solid and m, | m| ≥ k, is the list of all and only the occurrences of m in s. Given a motif m let m[j1], m[j2], ... Background number of consecutive dots allowed in a string. In such cases, for clarity of notation, we use the extensible wild card denoted by the dash symbol "-" instead of the annotated dot character, .[1,d] in the string. Note that '-' ∉ Σ. Thus a string of the form a.[1,d]b will be simply written as a-b. A motif m is extensible if it contains at least one annotated dot, otherwise m is rigid. Given an extensible string m, a rigid string m' is a realization of m if each annotated dot .α is replaced by l ∈ α dots. The collection of all such rigid realizations of m is denoted by R(m). A rigid string m occurs at position l on s if m[j]  s[l + j - 1] holds for 1 ≤ j ≤ |m|. An extensible string m occurs at position l in s if g Let s be a sequence of sets of characters from an alphabet Σ ∫ {·}, where '.' ∉ Σ denotes a don't care (dot, for short) and the rest are solid characters, we use σ to denote a sin- gleton character. For characters e1 and e2, we write e1  e2 if and only if e1 is a dot or e1 = e2. Allowing for spacers in a string is what makes it extensible. Such spacers are indi- cated by annotating the dot characters. Specifically, an annotated "." character is written as .α where α is a set of positive integers {α1, α2, ..., αk} or an interval α = [αl, αu], representing all integers between αl and αu including αl and αu. Whenever defined, d will denote the maximum Page 1 of 7 (page number not for citation purposes) http://www.almob.org/content/1/1/4 Algorithms for Molecular Biology 2006, 1:4 Table 1: The pseudocode of the motif extraction algorithm. Table 1: The pseudocode of the motif extraction algorithm. Background m[jl] be the l solid elements in the motif m. Then the sub- motifs of m are given as follows: for every ji, jt, the sub- motif m[ji ... jt] is obtained by dropping all the elements before (to the left of) ji and all elements after (to the right of) jt in m. We also say that m is a condensation for any of its sub-motifs. We are interested in motifs for which any condensation would disrupt the list of occurrences. For- mally, let m1, m2, ..., mj be the motifs in a string s. A motif mi is maximal in length if there exists no ml, l ≠ i with and mi is a sub-motif of ml. A motif mi is max- imal in composition if no dot character of mi can be replaced by a solid character that appears in all the locations in    | | | |   m m i l = Page 2 of 7 (page number not for citation purposes) Results and discussion In the following experiment we construct a phylogeny of the Eutherian orders using complete unaligned mitochon- drial genomes of the following 15 mammals from Gen- Bank: human (Homo sapiens [GenBank:V00662]), chimpanzee (Pan troglodytes [GenBank:D38116]), pigmy chimpanzee (Pan paniscus [GenBank:D38113]), gorilla (Gorilla gorilla [GenBank:D38114]), orangutan (Pongo pygmaeus [GenBank:D38115]), gibbon (Hylo- bates lar [GenBank:X99256]), sumatran orangutan (Pongo pygmaeus abelii [GenBank:X97707]), horse (Equus caballus [GenBank:X79547]), white rhino (Cera- totherium simum [GenBank:Y07726]), harbor seal (Phoca vitulina [GenBank:X63726]), gray seal (Hali- choerus grypus [GenBank:X72004]), cat (Felis catus [Gen- Bank:U20753]), finback whale (Balenoptera physalus [GenBank:X61145]), blue whale (Balenoptera musculus [GenBank:X72204]), rat (Rattus norvegicus [Gen- Bank:X14848]) and house mouse (Mus musculus [Gen- Bank:V00711]). In this paper, we present lossy off-line data compression techniques by textual substitution in which the patterns used in compression are chosen among the extensible motifs that are found to recur in the textstring with a min- imum pre-specified frequency. Motif discovery and motif- driven parses of various kinds have been previously intro- duced and used in [5]. Whereas the motifs considered in those studies are "rigid", here we assume that each sequence of gaps present in a motif comes endowed with some individually prescribed degree of elasticity, whereby a same pattern may be stretched to fit segments of the source that match all the solid characters but are otherwise of different lengths. This is expected to save on the size of the codebook, and hence to improve compression. The figure of compression achieved by our algorithm shows good sensitivity in telling apart veritable families of proteins from spurious blends. This sets forth an approach to classification that does away with alignment. The data used for the test consists of protein sequences, which are known to be hardly compressible at all [6]. The experiment reported below uses three different families which were picked at random from the PROSITE reposi- tory: AP endonucleases (acnucl), G-protein coupled receptors (gprot) and Succinyl-CoA ligases (succ). Table 2 summarizes the results of lossy and lossless compression for various values of the parameters. The artificial groups are marked "-mix", the last column shows the lossless compression ratio of fake over faithful families, when using motifs with the same parameter values. In all cases, the artificial families show compression ratios that are poorer by 10/20%, and the superiority of the lossy vari- ants manifests itself throughout. The experiments thus verify the discrimination potential of data compression by extensible motifs. Results and discussion Several measures of distance have been proposed and used to classify documents of diverse nature and to infer relationships among them. In practice, each measure translates in a computational task which might be more or less of a burden. In domains such as genome analysis and natural language processing, the increasing availability of longer and longer sequences and more and more massive data sets is playing havoc with similarity measures based on edit computations and the likes [2]. As an alternative, succinct scores related to compressibility -interpreted as a measure of structural complexity or information contents- have been deployed, of which the lineage may be traced back to Kolmogorov's complexity. The Kolmogorov com- plexity of a string x, denoted K(x), is the length of the short- est program that would cause a standard universal computer to output x. Along the same lines, the conditional Kolmogorov complexity K(x|y) for strings x and y is defined as the length of the shortest program that, given y as input, Page 2 of 7 (page number not for citation purposes) Page 2 of 7 (page number not for citation purposes) Algorithms for Molecular Biology 2006, 1:4 http://www.almob.org/content/1/1/4 will output x as the result. Intuitively, the conditional complexity expresses the information difference between the strings x and y. We refer the reader to, e.g., [3] for a detailed treatment of the theory. Whereas the original Kol- mogorov complexity is hardly computable, important emulators have been developed since [4], which conju- gate compressibility and ease of computation. Following in these steps, we now test the discriminating power of the data compression method that is based on our Off-line steepest descent paradigm with extensible motifs. D x y Off line xy Off line x Off line y Off l ( , ) ( ) { ( ), ( )} { = − - - - - min max ine x Off line y ( ), ( )} , - where (xy) denotes the concatenation of x and y. Hence, D(x, y) measures the improvement over Off-line(y) that is brought about by using x as a "dictionary" when com- pressing y. Results and discussion File File len param density Lossy Lossless Compr ratio % K D acnucl 4197 10 3 1717 2425 4197 5 3 1757 2370 acnucl-mix 4149 10 3 1864 2629 +8.4 4149 5 3 1972 2621 +10.6 gprot 25482 30 3 7003 9879 25482 20 3 7399 10276 gprot-mix 25335 30 3 8179 11945 +20.9 25335 20 3 8386 11978 +16.5 succ 16297 20 3 4994 7449 16297 10 3 4977 7466 succ-mix 16410 20 3 5929 8803 +18.2 16410 10 3 6415 8962 +19.2 Table 2: Comparing sensitivity of lossy versus lossless compression by Off-line with Extensible Motifs, as applied to real and fake protein families. 2: Comparing sensitivity of lossy versus lossless compression by Off-line with Extensible Motifs, as applied in families. is very close to ests that even a type of regular- account palin- support good was applied to logy with what ee obtained in et of languages Declaration of tree is coherent secutive solid characters are allow maximal extensible (with D spacers least K times in s. Incidentally, adapted to extract rigid motifs as a suffices to interpret D as the max characters between two consecutiv The algorithm works by convert sequence of possibly overlapping c est substring in any pattern on s, th characters: one at the start and the tion of this substring. A maximal sequence of cells. Initialization phase K D 0 3 1717 2425 3 1757 2370 0 3 1864 2629 3 1972 2621 0 3 7003 9879 0 3 7399 10276 0 3 8179 11945 0 3 8386 11978 0 3 4994 7449 0 3 4977 7466 0 3 5929 8803 0 3 6415 8962 secutive solid characters are allowed. The output is all maximal extensible (with D spacers) patterns that occur at least K times in s. Incidentally, the algorithm can be adapted to extract rigid motifs as a special case. For this, it suffices to interpret D as the maximum number of dot characters between two consecutive solid characters. The phylogeny obtained in our experiment is very close to the commonly accepted ones, which suggests that even a method of compression based on a single type of regular- ity, as opposed to those that take into account palin- dromes and other structures may support good comparative genomics. Conclusion i The cell is the smallest extensible component of a maximal pattern and the string can be viewed as a sequence of over- lapping cells. If no don't care characters are allowed in the motifs then the cells are non-overlapping. The initializa- tion phase has the following steps. Comparisons of the compression ratios respectively achieved by rigid and extensible motifs displays that the latter bring about additional savings in compression. This suggests that extensible motifs may be preferred to rigid ones also in those cases where they are used as bases for similarity measure and classification among sequences. The unsupervised classification method built on top of such measures have been shown here to consistently pro- duce phylogenic trees for species genomes as well lan- guage classifications built on text documents. Step 1: Construct patterns that have exactly two solid char- acters in them and separated by no more than D spaces or "." characters. This is done by scanning the string s from left to right. Further, for each location we store start and end position of the pattern. For example, if s = abzdabyxd and K = 2, D = 2, then all the patterns generated at this step are: ab, a.z, a..d, bz, b.d, b..a, zd, z.a, z..b, da, d.b, d..y, a.y, a..x, by, b.x, b..d, yx, y.d, xd, each with its occurrence list. Thus ab = {(1, 2), (5, 6)}, a.z = {(1, 3)} and so on.   Results and discussion The algorithm works by converting the input into a sequence of possibly overlapping cells: A cell is the small- est substring in any pattern on s, that has exactly two solid characters: one at the start and the other at the end posi- tion of this substring. A maximal extensible pattern is a sequence of cells. For a comparison, the same treatment was applied to human language text classification, in analogy with what is found in [7,8]. Figure 2 displays the tree obtained in experiments performed with a small subset of languages on the widely translated "Universal Declaration of Human Rights". Once more, the resulting tree is coherent with commonly accepted ones. Results and discussion Conclusion Comparisons of the compression ratios respectively achieved by rigid and extensible motifs displays that the latter bring about additional savings in compression. This suggests that extensible motifs may be preferred to rigid ones also in those cases where they are used as bases for secutive solid characters are allowed. The output is all maximal extensible (with D spacers) patterns that occur at least K times in s. Incidentally, the algorithm can be adapted to extract rigid motifs as a special case. For this, it suffices to interpret D as the maximum number of dot characters between two consecutive solid characters. The algorithm works by converting the input into a sequence of possibly overlapping cells: A cell is the small- est substring in any pattern on s, that has exactly two solid characters: one at the start and the other at the end posi- tion of this substring. A maximal extensible pattern is a sequence of cells. Initialization phase The cell is the smallest extensible component of a maximal pattern and the string can be viewed as a sequence of over- lapping cells. If no don't care characters are allowed in the motifs then the cells are non-overlapping. The initializa- tion phase has the following steps. Table 2: Comparing sensitivity of lossy versus lossless compression by Off-line with Extensible Motifs, as applied to real and fake protein families. File File len param density Lossy Lossless Compr ratio % K D acnucl 4197 10 3 1717 2425 4197 5 3 1757 2370 acnucl-mix 4149 10 3 1864 2629 +8.4 4149 5 3 1972 2621 +10.6 gprot 25482 30 3 7003 9879 25482 20 3 7399 10276 gprot-mix 25335 30 3 8179 11945 +20.9 25335 20 3 8386 11978 +16.5 succ 16297 20 3 4994 7449 16297 10 3 4977 7466 succ-mix 16410 20 3 5929 8803 +18.2 16410 10 3 6415 8962 +19.2 Table 2: Comparing sensitivity of lossy versus lossless compression by Off-line with Extensible Motifs, as applied to real and fake protein families. Results and discussion It seems thus meaningful to build a classifier on top of this measure. Compressibility by extensible motifs may be used to set up a similarity meas- ure on sequences to be used in the inference of phylogeny. The measure could be extended into a metric distance, along the lines of [7]. Specifically, we denote by Off-line(z) the output size obtained when compressing a string z using the lossless variant of our paradigm, and compute the quantity: The evolutionary tree in Figure 1 is generated by a variant of the classical neighbor-join where instead of minimiz- ing the distances between nodes we maximized the sepa- ration. Specifically, for each pair (x, y) of sequences, the quantity D(x, y) is computed. Next, the neighbor-join algorithm is used to build the tree from the matrix of dis- tances. This algorithms selects a pair of (x, y) among those achieving the minimum value for D, and creates an inter- nal node as their father. It then coalesces x and y into a combined sequence the D value of which is computed as the maximum (instead of the average) of those of x and y. The process is continued until the D-matrix has shrunk to a scalar. The first notable finding is that closely related species are indeed grouped together, e.g., grayseal with harboseal, orangutan with sumatranorang, etc. Whereas there is no gold standard for the entire tree, biologists do suggest the following grouping for this case: • Eutheria-Rodens: housemouse, rat. • Primates: chimpa, gibbon, gorilla, human, orangutan, pigmychimpa, sumatranorang. • Ferungulates: bluewhale, finbackwhale, grayseal, har- boseal, horse, whiterhino. Page 3 of 7 (page number not for citation purposes) Page 3 of 7 (page number not for citation purposes) Algorithms for Molecular Biology 2006, 1:4 http://www.almob.org/content/1/1/4 The phylogeny obtained in our experiment is very close to the commonly accepted ones, which suggests that even a method of compression based on a single type of regular- ity, as opposed to those that take into account palin- dromes and other structures may support good comparative genomics. For a comparison, the same treatment was applied to human language text classification, in analogy with what is found in [7,8]. Figure 2 displays the tree obtained in experiments performed with a small subset of languages on the widely translated "Universal Declaration of Human Rights". Once more, the resulting tree is coherent with commonly accepted ones. The evolut Figure 1 Th l The evolutionary tree built from complete mammalian mtDNA sequences of 15 species Figure 1 The evolutionary tree built from complete mammalian mtDNA sequences of 15 species. [width = 450 pt]tree1.eps The evolutionary tree built from complete mammalian mtDNA sequences of 15 species Figure 1 The evolutionary tree built from complete mammalian mtDNA sequences of 15 species. [width = 450 pt]tree1.eps the concatenation of m1 and m2 with an overlap at the common end and start character and m = {(x, y)|(x, l) ∈ }. For example if m1 = ab and m2 = b.d then m1 is ~-compatible with m2 and m1 ~ m2 = ab.d. How- ever, m2 is not ~-compatible with m1.    m m l,y 1 2 ,( ) ∈ Continuing the previous example, b-d is generated at this step with b-d = {(2, 4), (6, 9)}. All cells m with | m| <K are discarded. In the example, the only surviving cells are ab, b-d with ab = {(1, 2), (5, 6)} and b-d = {(2, 4), (6, 9)}     9)} Mining extensible motifs The procedure of motif extraction that is described in Table 1 essentially constructs the inexact suffix tree of [1] implicitly, in a different order. The input is a string s of size n and two positive integers, K and D. Step 2: The extensible cells are constructed by combining all the cells with at least one dot character and the same start and end solid characters. The location list is updated to reflect the start and end position of each occurrence. The extensibility parameter D is interpreted in the sense that up to D (or 1 to D) dot characters between two con- Page 4 of 7 (page number not for citation purposes) Algorithms for Molecular Biology 2006, 1:4 http://www.almob.org/content/1/1/4 The evolutionary tree built from complete mammalian mtDNA sequences of 15 species Figure 1 The evolutionary tree built from complete mammalian mtDNA sequences of 15 species. [width = 450 pt]tree1.eps The evolutionary tree built from complete mammalian mtDNA sequences of 15 species Figure 1 The evolutionary tree built from complete mammalian mtDNA sequences of 15 species. [width = 450 pt]tree1.eps Page 5 of 7 (page number not for citation purposes) Iteration phase NodeInconsistent(m) is a routine that checks if the new motif m is non-maximal w.r.t. earlier non-ancestral nodes by checking the location lists. The procedure is best described by the pseudocode shown in Table 1. Steps G:18–19 detect the suffix motifs of already detected max- imal motifs. Result is the collection of all the maximal extensible patterns. Let B be the collection of cells. If m = Extract(B), then m ∈ B and there does not exist m' ∈ B such that m' ∗ m holds: m1 ∗ m2 if one of the following holds: (1) m1 has only solid characters and m2 has at least one non-solid character (2) m2 has the "-" character and m1 does not, and, (3) m1 and m2 have d1, d2 > 0 dot characters respectively and d1 <d2. Further, m1 is ~-compatible with m2 if the last solid char- acter of m1 is the same as the first solid character of m2. Further if m1 is ~-compatible with m2, then m = m1 ~ m2 is A tight time complexity for the procedure is not easy to come by, however, if we consider M to be the number of extensible maximal motifs and S to be the size of the out- Page 5 of 7 (page number not for citation purposes) Page 5 of 7 (page number not for citation purposes) http://www.almob.org/content/1/1/4 Algorithms for Molecular Biology 2006, 1:4 A partial tree of languages using a distance based on com- pression by extensible motifs Figure 2 A partial tree of languages using a distance based on com- pression by extensible motifs. [width = 280 pt]tree2.eps two stages are, as a rule, carried out somewhat independ- ently. The notion of a motif that we adopt tightly combines the structure of the motif pattern, as described by its syntactic specification, with the statistical measure of its occurrence count. This supports a notion of saturation that finds nat- ural use in the dual contexts of structural inference and compression. As said, this saturation condition mandates that motifs that could be made more specific without altering their set of occurrences do not bear interest and may be discarded. In this Section, we present lossy off-line data compression techniques by textual substitution in which the patterns used in compression are chosen among the extensible motifs that are found to recur in the textstring with a min- imum pre-specified frequency. Iteration phase As mentioned, motif dis- covery and motif-driven parses of various kinds have been previously introduced and used in [5], however, the motifs considered in those studies are "rigid". A partial tree of languages using a distance based on com- pression by extensible motifs Figure 2 A partial tree of languages using a distance based on com- pression by extensible motifs. [width = 280 pt]tree2.eps A partial tree of languages using a distance based on com- pression by extensible motifs Figure 2 A partial tree of languages using a distance based on com- pression by extensible motifs. [width = 280 pt]tree2.eps The transition from rigid to extensible motifs requires a complete restructuring of the combinatorial and compu- tational tools for their extraction and implementation. Specifically, one needs: put – i.e. the sum of the sizes of the motifs and the sizes of the corresponding location lists – then the time taken by the algorithm is O(SM log M). In experiments of the kind described later in the paper, at 3 GHz clock, time ranged typically from few minutes to half an hour. • An algorithm for the extraction of flexible motifs. • An algorithm for the extraction of flexible motifs. • A criterion for choosing and encoding the motifs to be used in compression. Compression by extensible motifs • A new suite of software programs implementing the whole. • A new suite of software programs implementing the whole. Traditionally, the design of codebooks used in compres- sion proceeds from specifications that are either statistical or syntactic. The quintessential statistical approach is rep- resented by Huffman codes, in which symbols are ranked according to their frequencies and then assigned in order of decreasing probability to longer and longer codewords. In a syntactic approach, the codebook is built out of pat- terns that display certain features, e.g., of robustness in the face of noise, loss of synchronization, etc. The focal point in these developments is the structure of the codewords. For instance, a codeword is a pattern w of length m such that any other codeword must be at a distance of d from w, the distance being measured in terms of errors of a cer- tain type. We can have only substitutions in the Hamming variant, substitutions, insertions and deletions in the Lev- ensthein variant, and so on. Of course, the two aspects blend in the final code. With Huffmann codes, for instance, once the characters are statistically ranked a code with certain syntactic characteristics, notably, obeying the prefix property, is built. Likewise, once the codebook of an error correcting code is designed, the statistics of the source is taken into account for encoding. However, these The orchestration of these ingredients are briefly described next. We regard the motif discovery process as distributed on two stages, where the first stage unearths motifs endowed with a certain set of properties and the second implements them in the compression. The first part was dealt with in the preceding section. Like with rigid motifs in [5], the flexible ones presented here may be restored at the receiver using information about gap fill- ing, to be transmitted separately. In images, for instance, a tremendous amount of compression is attained, albeit with a large loss such as 40% or so, yet simple predictors in the form of linear interpolation restores more than 95% of the original. The methods presented here belong to a class of off-line textual substitution that try to reap through greedy approximation the benefits of otherwise intractable opti- mal macro schemes [9]. Page 6 of 7 (page number not for citation purposes) All authors contributed equally to this work. All authors contributed equally to this work. Compression by extensible motifs The specific heuristic followed here is based on a greedy iterative selection (see e.g., [10]) which consists of identifying and using, at each iteration, Page 6 of 7 (page number not for citation purposes) Page 6 of 7 (page number not for citation purposes) Algorithms for Molecular Biology 2006, 1:4 http://www.almob.org/content/1/1/4 a substring w of the text x such that encoding all instances of w in x yields the highest possible contraction of x. This process may be also interpreted as learning a "straight line" grammar of minimum description length for the sourcestring, for which we refer to [5,11,12] and refer- ences therein. Off-line methods are not always practical and can be computationally imposing even in approxi- mate variants. They do find use in contexts and applica- tions, such as mass production of CD-ROMs, backup archiving, etc. (see, e.g., [13]). Paradigms of steepest descent approximations have delivered good perform- ances in practice and also appear to be the best candidates in terms of the approximation achieved to optimum descriptor sizes [14]. copy, and we need to add one bit to distinguish original characters from pointers. The original encumbrance posed by m on the text is in this case (log |Σ| + 1)sm, from which we subtract |m| log |Σ| + fmDm log D + log |m| + fm(log r + 1), where r is the size of the dictionary, in itself a parameter to be either fixed a priori or estimated. Acknowledgements Work Supported in part by the Italian Ministry of University and Research under the National Projects FIRB RBNE01KNFP, PRIN "Combinatorial and Algorithmic Methods for Pattern Discovery in Biosequences", and PRIN "Data mining methods for e-business applications", and by Italy-Israel Inter- nationalization FIRB Project n. RBIN04BYZ7. Our steepest descent paradigm performs a number of phases consisting each in the selection of the pattern to be used for compression followed by the actual substitution and encoding. The process stops when no further com- pression is achieved. The sequence representation at the outset is finally pipelined into some of the popular encod- ers and the best one among the overall scores thus achieved is retained. Clearly, at any stage it is impossible to choose the motif on the basis of the actual compression eventually conveyed by that motif. The decision must be based on an estimate, that takes in to account the mechan- ics of encoding. In practice, we estimate at log(i) the number of bits needed to encode the integer i (we refer to, e.g., [4] for reasons that legitimate this choice). In one scheme [10], one eliminates all occurrences of m, and record in succession m, its length, and the total number of its occurrences followed by the actual list of such occur- rences. Letting |m| to denote the length of m, Dm denotes the number of extensible characters in m, fm the number of occurrences of m in the textstring, sm the number of char- acters occupied by the motif m in all its occurrences on s, |Σ| the cardinality of the alphabet and n the size of the input string, the compression brought about by m is esti- mated by subtracting from the sm log |Σ| bits originally encumbered by this motif on s, the expression |m| log |Σ| + log |m| + fmDm log D + fm log n + log fm charged by encod- ing, thereby obtaining: References 1. Chattaraj A, Parida L: An inexact suffix tree based algorithm for extensible pattern discovery. Theoretical Computer Science 2005, 335:3-14. 2. Vinga S, Almeida J: Alignment-free sequence comparison – a review. Bioinformatics 2003, 19(4):513-523. 3. Li M, Vitanyi P: An Introduction to Kolmogorov Complexity and Its Applica- tions Springer Verlag; 1997. 4. Lempel A, Ziv J: On the Complexity of Finite Sequences. IEEE Transactions on Information Theory 1976, 22:75-81. 5. Apostolico A, Comin M, Parida L: Motifs in Ziv-Lempel-Welch Clef. Proceedings of IEEE DCC Data Compression Conference 2004:72-81. 6. Nevill-Manning C, Witten I: Protein is Incompressible. Proceed- ings of IEEE DCC Data Compression Conference 1999:257-266. g p 7. Li M, Chen X, Li X, Ma B, Vitanyi P: The Similarity Metric. IEEE Transactions on Information Theory 2004, 50(12):3250-3254. 8. Li M, Badger J, Chen X, Kwong S, Kearney P, Zhang H: An Informa- tion-based Sequence Distance and its Application to whole Mitochondrial Genome Phylogeny. Bioinformatics 2001, 17(2):149-154. 9. Storer JA: Data Compression: Methods and Theory Computer Science Press; 1988. 10. Apostolico A, Lonardi S: Off-line Compression by Greedy Tex- tual Substitution. Proceedings of the IEEE 2000, 88(11):1733-1744. 11. Kieffer J, Yang E: Grammar-based Codes: a New Class of Uni- versal Lossless Source Codes. IEEE Transactions on Information Theory 2000, 46:737-754. y 12. Neville-Manning C, Witten IH, Maulsby D: Compression by Induc- tion of Hierarchical Grammars. Proceedings of IEEE DCC Data Compression Conference 1994:244-253. p f 13. DeAgostino S, Storer JA: On-line Versus Off-line Computation in Dynamic Text Compression. Inform Process Lett 1996, 59(3):169-174. 14. Lehman E, Shelat A: Approximation Algorithms for Grammar Based Compression. Proceedings of the eleventh ACM-SIAM Sympo- sium on Discrete Algorithms (SODA) 2002:205-212. G(m) = (sm - |m|) log |Σ| - log |m| - fm(Dm log D + log n) - log fm G(m) = (sm - |m|) log |Σ| - log |m| - fm(Dm log D + log n) - log fm This is accompanied by a loss L(m) represented by the total number of don't cares introduced by the motif, expressed as a percentage of the original length. If dm is the total number of such gaps introduced across all its occur- rences, this would be: L(m) = dm/sm. Other encodings are possible (see, e.g., [10]). References In one scheme, for example, every occurrence of the chosen pat- tern m is substituted by a pointer to a common dictionary Page 7 of 7 (page number not for citation purposes) Page 7 of 7 (page number not for citation purposes) (page number not for citation purposes)
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Supplementary material 2 from: Tomaskinova J, Geneletti D, Dicks LV, Grace M, Collier M, Longato D, Stoev P, Sapundzhieva A, Balzan MV (2021) Capacity-building as an instrument to foster the implementation of nature-based solutions. Research Ideas and Outcomes 7: e77666. https://doi.org/10.3897/rio.7.e77666
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ssessing the effectiveness of urban osystems to prioritise nature-based utions in a high-density urban area Mario V Balzan Malta College of Arts, Science and Technology, Malta oting research excellence in nature-based solutions for innovation, sustainable economic growth and human well-being in Malta. This project receives funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 809988. Promoting research excellence in nature-based solutions for innovation, sustainable economic growth and human well-being in Malta. Assessing the effectiveness of urban ecosystems to prioritise nature-based solutions in a high-density urban area Mario V Balzan Malta College of Arts, Science and Technology, Malta This project receives funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 809988. een Infrastructure hat can be considered as being effective n infrastructure? e: Balzan (2020) Source: Balzan (2020) • What can be considered as being effective green infrastructure? • What can be considered as being effective green infrastructure? zan (2020) Source: Balzan (2020) Green infrastructure is a strategically planned etwork of natural and semi-natural areas with ther environmental features designed and managed to deliver a wide range of ecosystem ervices such as water purification, air quality, pace for recreation and climate mitigation and daptation. This network can improve environmental onditions and therefore citizens' health and uality of life whilst supporting a green economy nd enhances biodiversity. Source: Balzan (2020) een infrastructure is a strategically planned ork of natural and semi-natural areas with r environmental features designed and aged to deliver a wide range of ecosystem ces such as water purification, air quality, e for recreation and climate mitigation and tation. s network can improve environmental itions and therefore citizens' health and ty of life whilst supporting a green economy enhances biodiversity. Source: Balzan (2020) • Green infrastructure is a strategically planned network of natural and semi-natural areas with other environmental features designed and managed to deliver a wide range of ecosystem services such as water purification, air quality, space for recreation and climate mitigation and adaptation. • This network can improve environmental conditions and therefore citizens' health and quality of life whilst supporting a green economy and enhances biodiversity. Source: Balzan (2020) Source: presentation J. Rockström and P. Sukhdev at EAT conference Source: presentation J. Rockström and P. Sukhdev at EAT confere Source: presentation J. Rockström and P. Sukhdev at EAT conference Urban ecosystem services Source: cocity se Source: cocity.se ort st n Prioritising nature-based solutions • How to prioritise and plan for NbS? • How to prioritise and plan for NbS? • Ecosystem service assessments can support planning processes by identifying the most effective decisions on the protection, enhancement and establishment of green infrastructure to lead to measurable improvement in human well-being. • However, the implementation of ecosystem services concepts in urban planning has remained low, and actions aimed at supporting ecosystem services are often not supported by an appropriate knowledge- base (e.g. Cortinovis and Geneletti, 2018; La Rosa et al., 2016). Balzan et al. (2018; Land Use Policy) The ReNature NbS Compendium : http://renature-project.eu/compendium Balzan et al (2018; Land U Balzan et al. (2018; Land Use Policy) Crop (Provisioning ES) Aesthetic value (Cultural ES) Source: Balzan et al., 2018, Land Use Policy Source: Balzan et al., 2018, Land Use Policy Source: Balzan et al., 2018, Land Use Policy Habitats of Conservation value (Supporting ES) Air pollution [NO2] removal (Provisioning ES) Habitats of Conservation value (Supporting ES) Habitats of Conservation value (Supporting ES) Crop (Provisioning ES) , , Habitats of Conservation value (Supporting ES) Crop (Provisioning ES) Habitats of Conservation value (Supporting ES) Air pollution [NO2] removal (Provisioning ES) Aesthetic value (Cultural ES) Air pollution [NO2] removal (Provisioning ES) Aesthetic value (Cultural ES) Source: Balzan et al., 2018, Land Use Policy Source: Balzan et al., 2018, Land Use Policy Habitats of Conservation value (Supporting ES) Crop (Provisioning ES) Air pollution [NO2] removal (Provisioning ES) Aesthetic value (Cultural ES) Source: Balzan et al., 2018, Land Use Distribution of ecosystem service supplies? Habitats of Conservation value (Supporting ES) Crop (Provisioning ES) Air pollution [NO2] removal (Provisioning ES) Aesthetic value (Cultural ES) Source: Balzan et al., 2018, Land Use Policy Distribution of ecosystem service supplies? Habitats of Conservation value (Supporting ES) Habitats of Conservation value (Supporting ES) Habitats of Conservation value (Supporting ES) Air pollution [NO2] removal (Provisioning ES) Aesthetic value (Cultural ES) Linking ES capacity to GI availability a)ES capacity is directly associated with GI land cover b)GI availability declines with an increase in population density Balzan et al. (2020; Research Ideas and Outcomes) Linking ES capacity to GI availability Linking ES capacity to GI availability apacity is directly ciated with GI cover vailability declines an increase in ulation density with an increase in population density ( ) Balzan et al. (2020; Research Ideas and Outcomes) nking ES Capacity and Flow to Population Parameters Scatterplots presenting the association between (a) ES capacity and population density, (b) ES capacity and population size and (c) ES flow and population size for local councils in Malta. Lines represent the linear regression function and 95% confidence intervals plotted on the scatterplot. Population Density Population Size Population Size ES Capacity ES Capacity ES Flow king ES Capacity and Flow to Population Parameters Population Density Population Size Population Size p y ES Capacity ES Flow Flow to Population Parameters Population Size Populatio ES Flow Parameters Population Size nking ES Capacity and Population Density ES Capacity ES Capacity Flow to Populatio Population Size ES Flow Population Size Population Density Population Size Population Size Scatterplots presenting the association between (a) ES capacity and population density, (b) ES capacity and population size and (c) ES flow and population size for local councils in Malta. Lines represent the linear regression function and 95% confidence intervals plotted on the scatterplot. Source: Balzan 2017 Source: Balzan et al., 2018, Land Use Policy • Urban areas as coldspots of ES capacities? • Urban areas as coldspots of ES capacities? • Principal component analysis used to assess the ability of different land use land cover (LULC) categories to deliver multiple ecosystem services (red) • Principal component analysis used to assess the ability of different land use land cover (LULC) categories to deliver multiple ecosystem services (red) • Principal component analysis used to assess the ability of different land use land cover (LULC) categories to deliver multiple ecosystem services (red) • Length of arrow is proportional to correlation between environmental variable and ordination. Source: Balzan et al., 2018, Land Use Policy • But, the rate of ecosystem service use (ES flows) is much higher in urban cores and the peri-urban area! • Importance of assessing different dimensions of ES supplies and use • But, the rate of ecosystem service use (ES flows) is much higher in urban cores and the peri-urban area! • Importance of assessing different dimensions of ES supplies and use Source: Balzan & Debono, 2018, One Ecosystem Source: Balzan & Debono, 2018, One Ecosystem Recreational value Recreational va Mean number of favourite points (± standard error of the mean) for the reclassified Urban Atlas land use categories Mean number of favourite points (± standard error of the mean) for the reclassified Urban Atlas land use categories • But, the rate of ecosystem service use (ES flows) is much higher in urban cores and the peri-urban area! • Importance of assessing different dimensions of ES supplies and use Source: Balzan et al., submitted Source: Balzan et al., submitted Source: Balzan et al., submitted Source: Balzan et al., submitted Source: Balzan et al., submitted Urban Ecosystem Services Source: Balzan et al., submitted y rface area of the a urban meration case- s 22.21 Km2 (or 03% of the land e of the Maltese s). The total ation of the es overlapping he study area is 0, with an e population y of 6,658 ± 4,629 s per km2. of 15 ecosystem es mapped. • The surface area of the Valletta urban agglomeration case- study is 22.21 Km2 (or just 7.03% of the land surface of the Maltese Islands). The total population of the localities overlapping with the study area is 129,760, with an average population density of 6,658 ± 4,629 persons per km2. • A total of 15 ecosystem services mapped. Source: Balzan et al., submitted Source: Balzan et al., submitted Source: Balzan et al., submitted Urban Ecosystem Services Source: Balzan et al., submitted Source: Balzan et al., submitted Source: Balzan et al., submitted Source: Balzan e Source: Balzan Relative contribution of UGI types to ES supplies within the study area (Source: Balzan et al., submitted.) Category Noise Abatement Carbon Storage NO2 Removal capacity Cooling Effect ed agriculture 2.17% 5.48% 5.03% 9.50% Arable 4.56% 7.13% 6.28% 11.91% rmanent Crops 4.70% 3.20% 2.87% 3.63% metery 0.10% 0.10% 0.12% 0.12% bed Ground 3.90% 4.11% 3.48% 5.48% Garden 29.26% 22.91% 23.08% 15.47% & Shrubland 26.44% 18.84% 14.77% 16.66% anent Crops 0.75% 0.53% 0.54% 0.55% Quarry 0.00% 0.05% 0.02% 0.03% dy Beach 0.00% 0.00% 0.01% 0.09% gle Beach 0.00% 0.01% 0.01% 0.06% y vegetated 0.00% 0.21% 0.22% 0.78% & Garrigue 6.82% 6.04% 23.52% 13.70% tercourse 0.00% 2.10% 2.08% 4.24% Wetland 0.00% 0.10% 0.14% 0.08% oodland 21 29% 29 19% 17 82% 17 68% Noise Abatement Cooling Effect UGI Category Abandoned agriculture 9% Arable 16% Arable-Permanent Crops 6% Cemetery 0% Disturbed Ground 5% Garden 15% Shrubland 8% Permanent Crops 0% Quarry 0% Sandy Beach 0% Shingle Beach 0% Sparsely vegetated 5% Steppe&Garrigue 23% Watercourse 0% Wetland 0% Woodland 11% Abandoned agriculture 9% Arable 16% Arable-Permanent Crops 6% Cemetery 0% Disturbed Ground 5% Garden 15% Shrubland 8% Permanent Crops 0% Quarry 0% Sandy Beach 0% Shingle Beach 0% Sparsely vegetated 5% Steppe&Garrigue 23% Watercourse 0% Wetland 0% Woodland 11% Abandoned agriculture 9% Arable 16% Arable-Permanent Crops 6% Cemetery 0% Disturbed Ground 5% Garden 15% Shrubland 8% Permanent Crops 0% Quarry 0% Sandy Beach 0% Shingle Beach 0% Sparsely vegetated 5% Steppe&Garrigue 23% Watercourse 0% Wetland 0% Woodland 11% Source: Balzan et al., submitted Woodland 11% Abandoned agriculture 9% Arable-Permanent Crops 6% Source: Balzan et al., subm Source: Balzan et al., submitted Source: B Source: Balzan et al., submitted Source: Balzan et al s Source: Balzan et al., submitted Source: Balzan et al., submitted Sour Source: Balzan et al., submitted Source: Balzan et al., submitted Regulating Ecosystem Services Regulating Ecosystem Services Index of Effectiveness Index of Effectiveness Index of Effectiveness ndex of Effectiveness Regulating Ecosystem Services dex of effectiveness of urban ecosystems is a measure of relative contribution of urban ecosystems to ecosystem e provision based primarily on ecosystem condition (preliminary data shown here). ss land required to produce an urban ecosystem service, the more effective the urban ecosystem. Source: Balzan et al., submitted High index of effectiveness values recorded for: This Photo by Unknown Aut Private gardens Urban woodlands and areas Green streets This Photo by Unknown Author is licensed under CC BY-SA This Photo by Unknown Author is licensed under CC BY-SA-NC This Photo by Unknown Author is licensed under CC BY-NC-ND Permanent crops Private gardens Green streets This Photo by Unknown Author is licensed under CC BY-SA This Photo by Unknown Author is licensed under CC BY-SA-NC This Photo by Unknown Author is licensed under CC BY-SA This Photo by Unknown Author is licensed under CC BY-SA This Photo by Unknown Author is licensed under CC BY-SA Urban woodlands and afforested areas This Photo by Unknown Author is licensed under CC BY-NC-ND Permanent crops This Photo by Unknown Author is licensed under CC BY-SA Green streets This Photo by Unknown Author is licensed under CC BY-SA-NC Permanent crops Urban woodlands and afforested areas This Photo by Unknown Author is licensed under CC BY-SA-NC Source: Balzan et al. (2020) Prioritising NbS interventions • Prioritise policies that protect existing urban gardens, and nature-based interventions that increase tree and soil cover to increase regulating ecosystem services in high- density urban cores; • Prioritise policies that protect existing urban gardens, and nature-based interventions that increase tree and soil cover to increase regulating ecosystem services in high- density urban cores; • Prioritise the protection of private gardens, which contributed significantly to ecosystem service capacities; • Prioritise the protection of private gardens, which contributed significantly to ecosystem service capacities; • However, these measures cannot be considered as a replacement of the existing green infrastructure network as, because of their diversity, urban ecosystems lead to different non-material uses of ecosystems. Thank you! Mario.balzan@mcast.edu.mt
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THE PLAGUE IMMUNITY OF THE CITY OF MADRAS.
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489 man and it is scarcely paradoxical to say that without it Simon would not have been the great hygienic and social reformer that he was. With it, incidentally, he made sanitary reform a branch of English literature. There are certain passages in Simon’s " Sanitary Institutions," a book that deserves to be read side by side with his friend Buckle’s " History of Civilisation," on which any student of English might love to dwell. Chief among these is the whole grand passage dealing with St. Francis of Assisi and the Franciscan Order, which begins on page 49 of the " Introduction." and standard memoirs to considerable advantage, for any- thing in the nature of malice was far from the minds of those ideal hosts of the old school. Sir John Simon sprang from a good French emigr&eacute; stock that had taken to commerce in London and he was sent into the medical profession as so many of his neighbours were sent by merchant fathers too timid to venture to give their sons a university education and yet unwilling to bring all their boys up to the counting- house. As long ago as 1833 he began the study of medicine under the famous Joseph Henry Green of St. Thomas’s Hospital. The elder Greens and Simons both belonged to the City, but Joseph Henry Green, born on London Wall in 1791, was an idealist, as it were, escaping from mercantile associations, and young Simon was from the first strongly influenced by his tall, languid, dreamy master. Green was the intimate friend of Samuel Taylor Coleridge, who ended his opiated career in the house of Mr. Gillman, the Highgate surgeon, and was known also to Wordsworth. Simon in his "Private Recollections " tells with enthusiasm of a morning spent, by Green’s introduction, with William Wordsworth, who read to him a sonnet. Simon and his fellow pupil and afterwards life-long friend, my uncle, the late John Newton Tomkins, F.R.C.S., read Shelley together and were amateurs of literature, English and foreign. Newton Tomkins cherished a lock of Coleridge’s hair which he brought from the necropsy, which was performed by Green. It must be remembered that the medical students of that far-off day certainly furnished the then youthful Charles Dickens with the models for Bob Sawyer and Richard Allen. To the Editors of THE LANCET. SIRS,-It is with much interest and not a little regret that I have read in THE LANCET of July 23rd, p. 211, Dr. W. H. Willcox’s article on the above subject. My object in replying is simply to uphold the delicacy and accuracy of the test in question, for this is my sincere conviction-a conclusion arrived at after nearly seven years of patient labour. It would, indeed, be deplorable if some of your readers, influenced by Dr. Willcox’s article, should be induced to abandon what has been proved one of the most reliable tests that we possess. No doubt some experience is necessary not only in the mode of its application but also in interpreting the results. interpreting I will briefly endeavour to show that the attack on the merits of this method of identification of glucose in the urine has been made upon an erroneous basis-to wit: 1. The test as applied to the urine was originally proposed by Professor von Jaksch and was never intended to be used subsequent to hydrolysis of the urine by hydrochloric acid. 2. Dr. Willcox shows by his own experiments that normal urine when treated by the test in question does not yield characteristic crystals. 3. It is quite possible crystals were obtained from urine after hydrolysis with hydrochloric acid similar to those obtained from diabetic urine ; but this is altering the condition of things. A test is only of value when applied under certain conditions. 4. Supposing the crystals obtained after hydrolysing the urine are similar in appear- ance to those obtained from diabetic urine, the melting point will not be the same unless the hydrolysis produces glucose from some as yet unknown substance or substances. any, living lips. Simon’s general culture advanced apace. He travelled ex- tensively in the home countries of Europe and at an early date became familiar with many notabilities in France and Germany. He spent a day with Liebig as he had spent one with Wordsworth. In Berlin in 1841 he enjoyed the con- versation of Ludwig Tieck and his brother the sculptor, of Rauch the sculptor, Waagen the art curator, and so forth. At Dresden he visited Retsch, the illustrator of "Faust," and at Munich discussed philosophy with the great Schelling. He ex- presses his pleasure at having seen and heard Felix Mendels- sohn in London. To the Editors of THE LANCET. He paid no visit anywhere, in fact, without gaining an insight into all that was best in the artistic and literary life of the place visited. Yet he never forgot his own proper business of science, for at an early age he made the acquaintance in his travels of Vrolik, Schroder von der Kolk, Flourens, Tiedemann, and Henle, and continued throughout life this custom of adding to his circle of friends all that was most eminent in the surgical and medical science of Europe and America. glucose yet It follows, therefore, that the mere production of apparently identical, or even identical, crystals obtained under altered conditions does not in any way detract from the merits of the test. The experiments of Dr. Willcox are of much interest inasmuch as they show the possible exist- ence of other substances in the urine not yet recognised. yet T am Sirs voiirq faithfnllv. yet eco T am Sirs voiirq faithfnllv. A. F. BILDERBECK GOMESS. Drayton-gardens, S.W., July 31st, 1904. A. F. BILDERBECK GOMESS. Drayton-gardens, S.W., July 31st, 1904. In the homes of their staid Puritanical elders the medical students of the " thirties " were apt to be regarded as savages-medicine men dealing in human bones and addicted to body-snatching and the abuse of tobacco. Quite lately Sir John Simon humorously con- fessed that in the house of my maternal grandfather he had been relegated to side doors and back stairs and rooms specially set apart. Such treatment implied no specific insult and no offence was taken by the young men them- selves, but it is certain that Simon, though he might have had the tobacco habit, in no other way resembled the medical students of Dickens or Albert Smith. These details of student life are trivial enough, but now that Mr. Edmund Image, Mr. John Birkett, and Sir John Simon, the three senior Fellows of the Royal College of Surgeons, have all passed away within a year we shall hear them from few, if any, living lips. begins page 9 t I am, Sirs, yours faithfully, I am, Sirs, yours faithfully, August 2ncl, 1904. VICTOR G. PLARR, Librarian of the Royal College of Surgeons of England. m, Sirs, yours faithfully, VICTOR G. PLARR, Librarian of the Royal College of Surgeons of England. am, Sirs, yours faithfully, August 2ncl, 1904. VICTOR G. PLARR, Librarian of the Royal College of Surgeons of England. August 2ncl, 1904. To the -Editors of THE LANCET. Errors of judgment or breakdown of methods have led to indigenous plague in certain districts. But it. has spread very slowly, and instead of there being widely scattered areas infected throughout the presidency ultimately fatecl to coalesce consequent upon the yielding of the intermediate areas, the curious result has been seen of plague being cou- fined to the districts bordering upon the great centre-- namely, the Alysore Territory, whence the infection is constantly imported and where the repetition of chances is consequently greatest. Year after year a gradual addition to the numbers of these districts has occurred and an advance towards the central and southern clistricts is at the present time threatened. It can scarcely be hoped that Madras city will escape being indigenously infected, but in the mean- time the struggle by Government and local authorities in this presidency has been well sustained and the result has been, I think, to earn a grade of protection of life and trade that would not have been feasible had plague been let rip." I am. Sirs. vours faithfullv. / morbific agent may be required to exhibit spread or duration of vitality may not be favourable, a small fact well worth remembering in fighting plague. The sanitarian may, with confidence, apply the principle that every chance of success is of solid advantage to him, and may remember that favouring accidents or chances affecting the disposal of infectious matter need not always be with the enemy y y When plague made itself felt in Hong-Kong in 1894 I issued a circular to port medical officers warning them of its possible entrance to this presidency. The moment it was ascertained that plague had entered Bombay, instead of adopting a system of quarantine and observation in camps I at once advised local bodies to employ passport sur- veillance of the type contemplated by the Local Govern- ment Board in England with reference to cholera. This was subsequently legalised by the Madras Government using the Epidemic Diseases Act; so that, by taking advantage of the revenue system of village organisation, every village and town in the area of 141,726 square miles of this presidency became a centre of sanitary action and surveillance. All persons proceeding from towns or villages notified as infected are required to take passports binding them to undergo surveillance during ten days in whatever localities they halt at. To the -Editors of THE LANCET. The first actual advent of plague in the city of Madras was in October, 1897, and although, as a matter of fact, in this first experience there was neglect of one day in surveillance which nearly led to a disaster, the tracing of cases was due to the existence of the passport system in its yet incomplete and non-legalised form. Since then, in the city of Madras 67 cases of imported plague have occurred, of which 59 have been directly, and the rest indirectly, detected under this system. Last year 79,488 passports were regis- tered and only 2 per cent. of the holders were not traced. The trouble involved up to date to the municipal staff must have been great, but so far as one could perceive the result has been of enormous benefit to the 500,000 inhabitants of the city of Madras. Necessarily, the system is far from infallible. Irrespective of the possible arrival of infected material there must at times be evasions in the case of persons from infected areas who may or may not be incubating plague, the chances always, however, being heavily in favour of their not being infected, as shown by the large average number of cases that pass through the period of surveillance before a single case of plague is detected’- namely, 5000. Indeed, that the passport system has materially diminished without removing absolutely the chances of infection of the city is shown by the fact that in April this year Madras went through a small outbreak of indigenous plague, in all probability as a result of an evasion of the passport rules. The actual origin of infection was never ascertained, although the most reason- able hypothesis was the native rumour that the first death was in the case of a person who had been in the infected area of Bangalore and had evaded the passport rules on arrival. The suspicious death was fortunately reported by an anonymous petition to the medical officer of health who was able to warn in time the district medical officer in charge of a suburb beyond municipal limits to which certain of the contacts adjourned. The result was that the contacts were traced and on all the usual measures being applied to the infected localities the disease was stamped out, but not until 11 cases had occurred in the suburbs and five in the city of Madras. To the -Editors of THE LANCET. SIRS,-In your leading article in the issue of June 18th, on the subject of " Immunity from Plague enjoyed by Shanghai," you remark that a similar immunity of Madras " is not understood." As this is not the first time that this subject has been referred to by professional papers it may be of interest to some of your readers to know what at least are certain of the factors concerned in bringing this protection about. I quite agree with you in thinking that there is nothing inherent in Madras to protect against this disease. Sanitary conditions and the death-rate which results therefrom are phenomenal. Before, how- ever, 1 proceed to state why I consider the disease has been kept at bay I should like it to be understood that I am perfectly aware that, in dealing with a free population, no method of prevention is infallible. And even when recognising the merits of any special method it is necessary also to remember that in respect of plague as of any other infectious disease, it does not follow that, because one or more cases are introduced in a population, an epidemic ’III’lIst ensue. The conditions under which the sc e ce Europe America. We must not forget to mention two studies which, if pursued, might have led astray from science one whose leanings were essentially literary. When Simon was giving anatomical demonstrations in the then very insanitary dissecting rooms of King’s College, London, he would turn during moments of leisure to the study of Oriental languages, particularly of Persian. In the "forties the belief began strongly to prevail that German literature contained founts of esoteric wisdom. Simon with medical friends and others formed themselves into a society for the study of German metaphysics. What he drew thence it is hard to imagine-certainly not the pellucid literary style in which his famous "Reports," his treatises, his polemical letters to the Times, and his numerous articles are so happily couched. He aimed, as we know from himself, at clearness, at perfect lucidity, the perfect expression of the thought in his mind for beauties of style as such he cared naught. Yet Simon’s balanced old-world style is beautiful and remains a monu- ment of English at its best. The style was, indeed, the 490 in these cases no spread has taken place. 1 In an indigenously infected district the examination of each 1600 passport holders arriving in uninfected areas secured one ease of plague.&mdash;W. G. K. LAYMEN AND ELECTRICAL TREATMENT. To the Editors of THE LANCET. LAYMEN AND ELECTRICAL TREATMENT. To the Editors of THE LANCET. SIRS,&mdash;In answer to Dr. E. Stanley Smith’s letter in THE LANCET of August 6th, p. 423, T should like to bring before him the following’ considerations. First, the expense. 1 am sure that any medical man work- ing at electro-therapeutics would willingly treat cases at whatever fee their medical adviser thought or knew they could afford. In suitable cases properly vouched for by their practitioner no fee for treatment in private would, I am sure, he charged. From what I know of the charges of my registered confr&egrave;res I am of opinion that they would agree with me that, as a rule, skiagrams from f:3 to Z10 are the peculiar perquisite of the unregistered quack. I shall be pleased to send to anv registered medical practitioner the list of the British Electro-Therapeutic Society, upwards of 200 medical men in London and the provinces, who would I am certain charge the fee that the medical attendant thought proper. Regarding the ‘ absurdity of contending that there can be anything harmful to a patient in getting a layman to take a skiagraph." I would contend, Sirs, that it most assuredly depends on what the medical man wants. Does he want a picture ’ or Does he want to have skilled medical advice on the interpretation of the skiagraph.&mdash;things which, as you are well aware, are totally different in nature. aware, totally Again, we should be pleased to hear of the "layman." electro-therapeutic institute, &c., who, possessing a certain amount of elementary knowledge of anatomy, is still capable of knowing when to stop in treating any case with x rays, high-frequency, or any other form of electrical treatment. If this does not require a full medical training how is it that our hospital statistics are better than those of laymen or lay institutes ? Why is it that the history of our hospital and, alas, often our private patients is very generally that they have tried this or that quack or quack institute and paid good fees before they come to us or our out-patient department for cure ? This can be easily proved by a visit to any hospital with the requisite electrical department. department. Secondly, regarding Dr. To the -Editors of THE LANCET. Luckily rats did not get in- fected. Not one of the cases was of the bubonic variety ; they were toxic, pneumonic, and intestinal-showing, I think, that the Madras environment is capable of nurturing a very virulent strain of plague. The diagnosis of the last pneumonic case-a contact of a previous pneumonic case-was bacteriologically confirmed by Lieutenant-Colonel Bannerman, I.M.S., the then superintendent of the King Institute, Madras. As, then, you have remarked, there is nothing in the sanitary condition of Madras per se that has hitherto effected its protection. The chief factor in bringing about that result has been the activity of the municipal staff in keeping persons from infected areas under surveil- lance, aided by the passport system. As a result of this method of fighting plague from the com- mencement of danger of importation numerous imported cases in hitherto uninfected areas hav ; been detected in all parts of the presidency. Following prompt action by local officers I faithfullv. W. G. KING, Lieutenant-Colonel I.M.S. Madras, July 13th, 1904. / W. G. KING, Lieutenant-Colonel I.M.S. Madras, July 13th, 1904. W. G. KING, Lieutenant-Colonel I.M.S. Madras, July 13th, 1904. LAYMEN AND ELECTRICAL TREATMENT. To the Editors of THE LANCET. Smith’s statement that medical men shrink from sending their patients to another medical man owing to their diagnosis and treatment being overhauled by another," I would only ask the following question : Will the diagnosis and past treatment, good or bad, be upheld by a quack or quack institute rather than by one’s own registered colleague’? registered co eague Mr. H. Valentine Kuaggs’s letter in the same issue contains a somewhat alluring but dangerous suggestion. It is that medical practitioners who practise electro-therapeutics should advertise to obtain the support of some medical men and also to act as a foil to the numerous electrical quacks. This would entail de-registration. Does Mr. Knaggs seriously consider what this means to a duly registered medical practitioner? Would he give the same advice to the dermatologist, surgeon aurist, surgeon oculist, and laryngologist, or any other of our special practitioners ’ 1 venture to think, Sirs, that should we attempt to follow Mr. Knaggs’s advice he would probably be the first to cast at us a stone. lance, by passport system. As a result of this method of fighting plague from the com- mencement of danger of importation numerous imported cases in hitherto uninfected areas hav ; been detected in all parts of the presidency. Following prompt action by local officers
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Chronic kidney disease awareness among the general population: tool validation and knowledge assessment in a developing country
BMC nephrology
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Chronic kidney disease awareness among the general population: tool validation and knowledge assessment in a developing country Samar Younes1*, Nisreen Mourad1, Jihan Safwan2, Mariam Dabbous2, Mohamad Rahal1, Marah Al Nabulsi1 and Fouad Sakr2 © 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access Abstract Introduction:  Good knowledge and early identification of chronic kidney disease (CKD) can help in preventing dis- ease progression in its early stages and reducing undesired outcomes. The aim of the current study was to assess the level of public knowledge about CKD, determine predictors of better knowledge, and to construct and validate a CKD knowledge scale for public health assessment and research use. Methods:  A community-based cross-sectional study was conducted using an electronic self-administered question- naire. All people living in Lebanon and being 18 years of age and above were considered eligible for recruitment. CKD knowledge was assessed by a 37-item scale that was constructed by principal component analysis and then validated. The score of the CKD knowledge scale was computed from the extracted factors. A multivariable binomial logistic regression model evaluated the sociodemographic and clinical predictors of the knowledge score. Results:  A total of 1308 participants were included. The scale items converged over 9 factors with Eigenvalue greater than 1 and explaining 53.26% of the total variance, and the total scale had a high Cronbach’s alpha of 0.804. All items of the scale significantly correlated with the full scale with correlation coefficients ranging from 0.082 to 0.558. The ROC curve analysis determined an optimal cutoff point of better knowledge at 47.5 with 70.6% sensitivity and 44.2% specificity. The CKD knowledge score had a median of 51.00 (IQR 47.00–55.00). Higher knowledge score was significantly associated with old age (ORa = 1.018, 95% CI 1.006–1.030, P = 0.003),, occupation (ORa = 3.919, 95% CI 2.107–7.288, P <  0.001), and recent renal function assessment (ORa = 2.314, 95% CI 1.532–3.495, P <  0.001). However, a lower knowledge score was significantly associated with lower level of education (ORa = 0.462, 95% CI 0.327–0.653, P <  0.001). Conclusion:  A reliable tool to assess public knowledge and awareness about CKD was developed and validated. The overall knowledge was good, however, important gaps in CKD awareness were detected in some areas and subpopu- lations. Therefore, public health stakeholders need to implement targeted CKD educational activities to minimize the disease burden. Keywords:  Chronic kidney disease, Tool validation, Public awareness, Knowledge assessment, Lebanon *Correspondence: samar.younes@liu.edu.lb 1 School of Pharmacy, Lebanese International University, Bekaa, Lebanon Full list of author information is available at the end of the article Younes et al. BMC Nephrology (2022) 23:266 https://doi.org/10.1186/s12882-022-02889-2 Younes et al. BMC Nephrology (2022) 23:266 https://doi.org/10.1186/s12882-022-02889-2 Introduction consequence and understanding are all parts of CKD awareness among patients [14].i Chronic kidney disease (CKD), which affects more than 10% of the world’s population, has become a global public health crisis in recent decades. CKD is characterized by progressive decline of renal function over three months or more, which is linked to a number of risk factors. Once the kidneys have been damaged, they are unable to filter blood or perform other functions, resulting in a decrease in glomerular filtration rate (GFR) and proteinuria, which can develop to end-stage renal disease (ESRD) or kidney failure. If not treated with dialysis or a kidney transplant, ESRD is deadly and irreversible [1].i A better rate of early identification of those with unde- tected/early CKD or those at risk of developing CKD may be attainable in populations with high levels of knowledge and awareness about CKD [15]. According to research conducted in both developed and develop- ing nations, the public’s understanding of CKD and its risk factors is inadequate [16]. A recent Australian study found that participants had insufficient awareness of the kidneys’ physiological significance, with less than half correctly identifying HTN as a risk factor [16]. A study conducted in Saudi Arabia in 2010 found that only 7.1% of patients with early CKD were aware of their CKD status, and that the study cohort had poor awareness of CKD symptoms [17]. However, another recent study from Saudi Arabia using a non-validated questionnaire found that more than half of the study population cor- rectly identified HTN and DM as CKD risk factors [15]. Another study also found a deficiency of awareness about CKD among Saudis, with respondents from higher edu- cational and economic backgrounds having much more information and those with CKD risk factors also had a better understanding of the condition [18]. Besides, Chow et al. observed a lack of broad public knowledge on CKD and recommended future research in a population of high-risk individuals [19]. y Since the significant rise in the incidence of impor- tant risk factors as diabetes mellitus (DM), hypertension (HTN), a poor diet, little or no physical exercise, and metabolic syndrome, CKD has become a global pub- lic health issue. It claimed the lives of 409,000 people in 1990 and 956,000 people in 2013. CKD as a result of DM was responsible for 46,000 deaths in 1990 and 173,000 in 2013 [2]. © 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Younes et al. BMC Nephrology (2022) 23:266 Younes et al. BMC Nephrology (2022) 23:266 Page 2 of 11 Introduction Kidney illnesses have recently been ranked as the world’s 12th and 17th leading causes of death and dis- ability [3]. Around 10–13% of the general population are affected by CKD, with an estimated global population of over 500 million people [4, 5]. Low GFR was listed as the 12th greatest cause of death at the worldwide level, and the 14th largest cause of Disability-Adjusted Life-Years (DALYs) among 79 risk factors in the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) in 2013 [3, 6]. CKD is a leading cause of death and disability around the world [20]. In Lebanon, there is a paucity of data on public awareness and knowledge of CKD, as well as vali- dated tools to assess public knowledge. This study aimed to assess the level of public knowledge about CKD and to determine predictors of better knowledge. It also aimed to construct and validate a CKD knowledge scale for public health assessment and research use. Kidney failure, cardiovascular disease (CVD), and early death are all elevated by 8 to 10 times in people with CKD [4]. Acute kidney injury, anemia, mineral and bone disorders, fractures, and hospitalizations are among the other complications [7, 8].ii CKD is also associated with a significant financial bur- den, accounting for more than 2–3% of annual health- care expenditures in high-income countries, despite the fact that patients with ESRD account for only 0.03% of the total population, and lower socioeconomic status is linked to a higher risk of ESRD [9]. The burden of CKD in developing countries is substantially greater due to addi- tional hazards associated with poverty, such as infections, hazardous job, inadequate education, and poor maternal health, as well as the additional expense of screening and treatment, which must be paid directly by patients [10, 11]. Data analysis Data were analyzed using IBM Statistical Package for Social Sciences (IBM SPSS) version 26.0. Descriptive statistics were used to evaluate the sociodemographic and clinical characteristics of participants. Continuous variables were reported by their mean (± standard devia- tion), and categorical variables were reported by their frequencies and percentages. The CKD knowledge scale responses were coded with 2 for the correct answer and 1 for wrong answers of each multiple-choice item. Fac- tor analysis with principal component analysis was run to construct and then validate the CKD knowledge scale with varimax rotation. Factors retained in the final scale had Eigen values greater than one. Adequate Kaiser– Meyer–Olkin (KMO) measure of sampling adequacy and Bartlett’s test of sphericity were ensured. KMO and Bartlett’s indicate the suitability of the data for structure detection. The KMO measure of sampling adequacy indi- cates the proportion of variables’ variance that might be caused by underlying factors. Bartlett’s test of sphericity tests whether the correlation matrix is an identity matrix to confirm that the factor model is appropriate. The score of the CKD knowledge scale was computed from the extracted factors. The correlation of each scale com- ponent with the whole scale was determined by Pearson correlation. Cronbach’s alpha measured the full-scale internal consistency and reliability. The scale sensitivity and specificity were determined by receiver operating characteristic (ROC) curve analysis, with optimal cutoff point determined by J-index. The normal distribution of the final knowledge score was assessed by histogram and Shapiro-Wilk test. The score was skewed so it was dichot- omized according to its median. Knowledge scores above the median were considered associated with better CKD knowledge. A multivariable binomial logistic regression model utilizing forward likelihood ratio method evalu- ated the sociodemographic and clinical characteristics of participants as independent variables predicting a bet- ter knowledge score as the dependent variables. Results The scale was constructed by 4 pharmacotherapy spe- cialists after a comprehensive PubMed literature review. The initial scale included 39 items and was reviewed and verified by 2 external nephrologists. The scale items included multiple choice questions about renal physiol- ogy, CKD risk factors, renal assessment, CKD screening, clinical presentation and complications, correlation of kidney disease with cardiovascular and metabolic disor- der, and CKD preventive measures and treatment options (Additional file 1: Annex 1). Study instrument and outcomesh The questionnaire of the study was composed of 2 parts. The first part included general sociodemographic infor- mation relating to age, gender, area of residence, level of education, occupation, marital status, monthly income, and smoking and alcohol use. It also assessed clinical char- acteristics relating to present medical and family histories relating to cardiovascular, metabolic and kidney diseases. The second part was composite of specific questions about the kidneys and renal function to generate a genuine CKD knowledge scale. Knowledge of CKD involved informa- tion about various aspects of the disease that the respond- ents had collected over their lives through experience or education, usually pertaining to the causes of the disease and exacerbating factors, identification of symptoms and complications, and available treatment modalities. As for awareness, it goes beyond knowledge to include perceiving preventative actions, screening and check-ups, therefore, it plays an important role in disease control.h Sample size calculation complete the questionnaire was about 10 to 15 minutes. Data collection was started with a pilot study on 10 par- ticipants to explore clarity, consistency, and time needed to complete the questionnaire. The minimal sample size was calculated using the Cent- ers for Disease Control and Prevention (CDC) Epi Info version 7.2.4 (population surveys). The expected fre- quency was kept at 50% to yield the largest minimal sam- ple size required. Accordingly, a minimal sample of 384 participants was required to allow for adequate power of statistical analysis, and produce a 95% confidence level and an acceptable margin of error of 5%. For the scale validation, a ratio of participants to items should be at least 10:1 according to the rule of thumb [21]. Therefore, a minimal sample size of 370 was required to validate the CKD knowledge scale. Study design and participantsh This was a cross-sectional study that included partici- pants from all over Lebanon. A snowball sampling was used to collect data through an electronic self-admin- istered questionnaire between May 2021 and Decem- ber 2021. The weblink to the questionnaire was shared on Facebook, LinkedIn, and WhatsApp. Involvement of illiterate participants was encouraged to explore their knowledge and awareness about CKD, which could affect their behavior toward the disease prevention. Therefore, family members or/and caregivers were encouraged to interview their illiterate family members and record the responses on behalf of them. All people living in Lebanon and being 18 years of age and above were considered eli- gible for recruitment. The questionnaire included a cover letter to explain the objectives of the study. The time to The public’s awareness and understanding of CKD is a critical aspect in CKD preventive and screening pro- grams’ success, whereby early detection and manage- ment of CKD can help prevent disease progression in its early stages [12]. Despite this, the majority of CKD cases are not detected early [13]. General knowledge of CKD, its risk factors, and individual risk and CKD status Younes et al. BMC Nephrology (2022) 23:266 Younes et al. BMC Nephrology (2022) 23:266 Younes et al. BMC Nephrology (2022) 23:266 Page 3 of 11 Data analysis The study outcomes were to determine the public knowledge and awareness about CKD through a valid and reliable tool, and to determine predictors of better knowledge about CKD. Sociodemographic and clinical characteristics of participants The CKD knowledge score for total correct knowledge had a median of 51.00 (IQR 47.00–55.00) with a mini- mum and maximum values of 38 and 70 respectively. There is a significant positive association between age and area of residence with the knowledge score. Older par- ticipants had significantly higher score (ORa = 1.018, 95% CI 1.006–1.030, P = 0.003). Residents of Bekaa, Mount Lebanon, and North governorates also had significantly higher scores compared to South residents (ORa = 1.623, 95% CI 1.056–2.493, P = 0.027); (ORa = 1.671, 95% CI 1.116–2.501, P = 0.013); and (ORa = 1.691, 95% CI 1.050– 2.722, P = 0.031) respectively.hi A total of 1308 participants were included in the study. The mean age was 37.15 (±16.24), 55.4% were females, 89.9% were Lebanese, and 33% were from Beirut. The majority of participants (61.7%) had a university degree, 49.1% were married, 33.6% were non-healthcare profes- sionals, and most of the participants were non-smokers and non-alcoholic (52.8 and 82.6% respectively). For the monthly income, 49.2% had a family income between 500 to 1500 USD based on the official Lebanese rate of 1 USD = 1515 LBP. Greater than half of participants (61.2%) did not have a current cardiovascular or chronic kidney disease, yet their family history included HTN (57.2%), DM (45.1%), and CKD (9.5%). With respect to renal function screening assessment by a blood or urine test, 33.7% never screened their kidney function and 16.7% don’t know if they ever had the kidneys assessed. The complete sociodemographic and clinical characteris- tics of participants are reported in Table 1. There is a significant association between the level of education and occupation with the knowledge score. A significantly lower knowledge score was associated with secondary school (ORa = 0.462, 95% CI 0.327–0.653, P <  0.001), primary school (ORa = 0.386, 95% CI 0.249– 0.600, P <   0.001), and illiteracy (ORa = 0.248, 95% CI 0.145–0.422, P <   0.001) compared to university educa- tion. A significantly higher score was associated with being a healthcare professional (ORa = 3.919, 95% CI 2.107–7.288, P <  0.001) and a student (ORa = 2.497, 95% CI 1.585–3.934, P <   0.001) compared to unemployed participants. Factor analysish The factor analysis selection variable was Lebanese par- ticipants from the total sample representing the general population including Lebanese and non-Lebanese partic- ipants. After eliminating 5 variables from the initial CKD knowledge scale because they didn’t load well on any fac- tor, the final scale included a total of 34 variables (Addi- tional file 2: Annex 2). No variables of the final scale over correlated with each other (r > 0.9), had low communality (< 0.3), or low factor loading (< 0.3). The KMO measure of sampling adequacy was 0.881 with a significant Bartlett’s test of sphericity (P <  0.001). The scale items converged over 9 factors with Eigenvalue greater than 1 and explain- ing 53.26% of the total variance. The varimax rotated matrix of the scale components is shown in Table 2. Finally, there is a significant positive association between recent renal function assessment and knowl- edge score. Participants with a renal function assess- ment within the last 6 months and 1 year had significantly higher score compared to those who never tested their kidney function (ORa = 2.314, 95% CI 1.532–3.495, P <   0.001) and (ORa = 2.124, 95% CI 1.291–3.496, P = 0.003) respectively. The multivariable logistic regres- sion of CKD knowledge score predictor is reported in Table 4. Correlation and reliabilityh The current study constructed and validated the CKD knowledge scale as a reliable tool to assess public knowl- edge about CKD, and promote public health awareness and research. It also assessed the level of knowledge on CKD and determined predictors of better knowl- edge among the Lebanese population. We found a good overall knowledge about CKD, with a significant better knowledge associated with age, area of residence, level of education, occupation, and a recent assessment of kid- ney function. The scale had a high Cronbach’s alpha of 0.804. All items of the scale significantly correlated with the full scale. The correlation coefficients ranged from 0.082 to 0.558. Table 3 reports the correlation of each item of the CKD knowledge scale with the whole scale. Ethical aspectsh The study was approved by the Ethics and Research Committee of the School of Pharmacy at the Lebanese International University (2021RC-116-LIUSOP). The study carried no harmful risks as it was observational, and anonymity of all participants was warranted as per- sonal identifiers weren’t traced during data collection and analysis. All participants agreed to participate and provided informed consent. Illiterate people had the informed consent read and explained to them by a car- egiver or family member. Informed consent was provided electronically by ticking 3 compulsory options about understanding that participation is voluntarily and con- fidential, and agreeing to participate before being able to proceed to the first section of the questionnaire. Younes et al. BMC Nephrology (2022) 23:266 Page 4 of 11 from the general population. The ROC curve analysis determined an optimal cutoff point of better knowledge at 47.5 with 70.6% sensitivity and 44.2% specificity. The area under the curve was 0.597 (95% CI 0.545–0 .649, P <  0.001). were reported as adjusted odds ratios (ORa) with 95% confidence interval (CI). P values < 0.05 were consid- ered statistically significant with an acceptable margin of error = 5%. Validity measures The nine factors in the scale had a very good internal consistency [25], and the results suggested very good reproducibil- Table 1  Sociodemographic and clinical characteristics of participants Variable Frequency or (Mean ± S.D., range) Percentage Age 37.15 (±16.24, 18–90) Age Group   18–30 649 49.6   31–49 311 23.8   50+ 348 26.6 Gender   Male 583 44.6   Female 725 55.4 Nationality   Lebanese 1176 89.9   Non-Lebanese 132 10.1 Area of residence   Beirut 431 33   Bekaa 189 14.4   Mount Lebanon 239 18.3   North 144 11   South 305 23.3 Marital status   Single 572 43.7   Married 642 49.1   Divorced/Wid- owed/Separated 94 7.2 Education status   Illiterate 97 7.4   Primary school 160 12.2   Secondary school 244 18.7   University 807 61.7 Occupation   Student 380 29.1   Healthcare 138 10.6   Non-healthcare 439 33.6   Unemployed 274 20.9   Retired 77 5.9 Alcoholic   Yes 228 17.4   No 1080 82.6 Smoker   Yes 618 47.2   No 690 52.8 Family income    < 500$ 145 11.1   500–1500$ 644 49.2    > 1500$ 519 39.7 Table 1  (continued) Variable Frequency or (Mean ± S.D., range) Percentage Present medical history   Hypertension 325 24.8   Diabetes mellitus 209 16   Hyperlipidemia 221 16.9   Chronic kidney disease 121 9.3   Cardiac disease 107 8.2   Cerebrovascular disease 26 2   None 801 61.2 Family history   Hypertension 748 57.2   Diabetes mellitus 590 45.1   Hyperlipidemia 397 30.4   Chronic kidney disease 124 9.5   Cardiac disease 387 29.6   Cerebrovascular disease 55 4.2   None 276 21.1 Last renal function assessment/ screening    <  6 months 225 17.2   6–12 months 129 9.9   1–2 years 133 10.2    >  2 years 162 12.4   Never been tested for renal function 441 33.7   Do not know/ remember 218 16.7 Attended any CKD awareness presentation   Yes 164 12.5   No 1144 87.5 S.D Standard deviation, CKD Chronic kidney disease a NSSF National social security fund b COOP Cooperation healthcare fund Table 1  (continued) Variable Frequency or (Mean ± S.D., range) Percentage Present medical history   Hypertension 325 24.8   Diabetes mellitus 209 16   Hyperlipidemia 221 16.9   Chronic kidney disease 121 9.3   Cardiac disease 107 8.2   Cerebrovascular disease 26 2   None 801 61.2 Family history   Hypertension 748 57.2   Diabetes mellitus 590 45.1   Hyperlipidemia 397 30.4   Chronic kidney disease 124 9.5   Cardiac disease 387 29.6   Cerebrovascular disease 55 4.2   None 276 21.1 Last renal function assessment/ screening    <  6 months 225 17.2   6–12 months 129 9.9   1–2 years 133 10.2    >  2 years 162 12.4   Never been tested for renal function 441 33.7   Do not know/ remember 218 16.7 Attended any CKD awareness presentation   Yes 164 12.5   No 1144 87.5 S.D Standard deviation, CKD Chronic kidney disease a NSSF National social security fund b COOP Cooperation healthcare fund Attended any CKD awareness presentation This research was able to develop and validate a novel CKD knowledge scale that intends to use evidence-based literature to assess the public awareness about CKD [16, 22–24]. Validity measures Figure 1 presents the ROC curve of the CKD knowledge scale comparing Lebanese to non-Lebanese participants Page 5 of 11 Younes et al. BMC Nephrology (2022) 23:266 Table 1  Sociodemographic and clinical participants Variable Frequency or (Mean ± S.D., range) Per Age 37.15 (±16.24, 18–90) Age Group   18–30 649 49.6   31–49 311 23.8   50+ 348 26.6 Gender   Male 583 44.6   Female 725 55.4 Nationality   Lebanese 1176 89.9   Non-Lebanese 132 10.1 Area of residence   Beirut 431 33   Bekaa 189 14.4   Mount Lebanon 239 18.3   North 144 11   South 305 23.3 Marital status   Single 572 43.7   Married 642 49.1   Divorced/Wid- owed/Separated 94 7.2 Education status   Illiterate 97 7.4   Primary school 160 12.2   Secondary school 244 18.7   University 807 61.7 Occupation   Student 380 29.1   Healthcare 138 10.6   Non-healthcare 439 33.6   Unemployed 274 20.9   Retired 77 5.9 Alcoholic   Yes 228 17.4   No 1080 82.6 Smoker   Yes 618 47.2   No 690 52.8 Family income    < 500$ 145 11.1   500–1500$ 644 49.2    > 1500$ 519 39.7 Health insurance   ­NSSFa 658 50.3   ­COOPb 92 7   Private 306 23.4   None 318 24.3 Table 1  Sociodemographic and clinical characteristics of participants Variable Frequency or (Mean ± S.D., range) Percentage Age 37.15 (±16.24, 18–90) Age Group   18–30 649 49.6   31–49 311 23.8   50+ 348 26.6 Gender   Male 583 44.6   Female 725 55.4 Nationality   Lebanese 1176 89.9   Non-Lebanese 132 10.1 Area of residence   Beirut 431 33   Bekaa 189 14.4   Mount Lebanon 239 18.3   North 144 11   South 305 23.3 Marital status   Single 572 43.7   Married 642 49.1   Divorced/Wid- owed/Separated 94 7.2 Education status   Illiterate 97 7.4   Primary school 160 12.2   Secondary school 244 18.7   University 807 61.7 Occupation   Student 380 29.1   Healthcare 138 10.6   Non-healthcare 439 33.6   Unemployed 274 20.9   Retired 77 5.9 Alcoholic   Yes 228 17.4   No 1080 82.6 Smoker   Yes 618 47.2   No 690 52.8 Family income    < 500$ 145 11.1   500–1500$ 644 49.2    > 1500$ 519 39.7 Health insurance   ­NSSFa 658 50.3   ­COOPb 92 7   Private 306 23.4 None 318 24.3 This research was able to develop and validate a novel CKD knowledge scale that intends to use evidence-based literature to assess the public awareness about CKD [16, 22–24]. Our findings provided evidence that the scale is valid and reliable in determining the level of knowledge on CKD especially in the Lebanese population. NSAIDs Nonsteroidal anti-inflammatory drugs, CKD Chronic kidney disease Validity measures Our findings provided evidence that the scale is valid and reliable in determining the level of knowledge on CKD especially in the Lebanese population. The nine factors in the scale had a very good internal consistency [25], and the results suggested very good reproducibil- ity as all components correlated with the full scale with high significance. Although further research is still rec- ommended to confirm the reproducibility and validity of the scale as the correlation coefficients of the scale’s items with the full scale were low. Younes et al. BMC Nephrology (2022) 23:266 Page 6 of 11 Table 2  Varimax rotated matrix of CKD knowledge scale Loading factor Communalities Loading factor Item number NSAIDs Nonsteroidal anti-inflammatory drugs, CKD Chronic kidney disease a A Awareness Factor 1   Hypertension aA6.1 0.676 0.504   Keep blood pressure under control aA8.3 0.620 0.505   Help in maintaining blood pressure bK2.5 0.612 0.564   Blood pressure monitoring aA3.4 0.515 0.509   Heart diseases such as heart failure or heart attack aA6.4 0.426 0.435   Affected organs: heart aA5.1 0.451 0.487 Factor 2   Nausea and vomiting bK7.1 0.710 0.545   Loss of appetite bK7.3 0.665 0.549   Keep blood sugar levels under control aA8.2 0.531 0.508   Tiredness/ fatigue bK7.2 0.519 0.444   Diabetes aA6.2 0.469 0.550   Fever bK7.4 0.414 0.524 Factor 3   Pain killers (e.g., NSAIDs) bK6.5 0.561 0.472   Fluid overload bK7.5 0.503 0.482   Limit the intake of juices and soft drinks aA8.1 0.472 0.475   Dialysis bK9.2 0.469 0.426   Family history of CKD aA6.3 0.465 0.383   Drugs bK9.1 0.442 0.380 Factor 4   Obesity aA6.6 0.725 0.649   Excess stress aA6.7 0.684 0.570   Keep body weight under control aA8.4 0.549 0.537 Factor 5   Produce substances that break down fats bK2.6 0.765 0.632   Break down protein in the body b*K2.2 0.728 0.579   Help in keeping the bones healthy bK2.4 0.591 0.556 Factor 6   Affected organs: lungs aA5.2 0.617 0.492   Affected organs: skin aA5.3 0.611 0.498   Affected organs: brain aA5.4 0.603 0.614 Factor 7   Produce urine bK2.1 0.696 0.644   Clean blood/ filter waste products in the blood bK2.3 0.621 0.522   CKD stages bK4 0.465 0.455 Factor 8   Renal assessment via fecal test bK3.3 0.661 0.544   Number of kidneys in a healthy individual bK1 0.625 0.571 Factor 9   Renal assessment via urine test aA3.1 0.747 0.664   Renal assessment via blood test aA3.2 0.654 0.603 Factor 9 b K Knowledge Younes et al. BMC Nephrology (2022) 23:266 Page 7 of 11 Table 3  Correlation of each item of CKD knowledge scale with the whole scale CKD Chronic kidney disease, NSAIDs Nonsteroidal anti-inflammatory drugs Total CKD knowledge scale Correlation P-value 1. Number of kidneys in a healthy individual 0.082 0.003 2.1. Produce urine 0.242 <  0.001 2.2. Break down protein in the body 0.357 <  0.001 2.3. Clean blood/ filter waste products in the blood 0.368 <  0.001 2.4. Help in keeping the bones healthy 0.484 <  0.001 2.5. Help in maintaining blood pressure 0.570 <  0.001 2.6. Produce substances that break down fats 0.300 <  0.001 3.1. Renal assessment via urine test 0.257 <  0.001 3.2. Renal assessment via blood test 0.398 <  0.001 3.3. Renal assessment via fecal test 0.239 <  0.001 3.4. Blood pressure monitoring 0.493 <  0.001 4. CKD stages 0.181 <  0.001 5.1. Affected organs: heart 0.212 <  0.001 5.2. Affected organs: lungs 0.282 <  0.001 5.3. Affected organs: skin 0.316 <  0.001 5.4. Affected organs: brain 0.328 <  0.001 6.1. Hypertension 0.479 <  0.001 6.2. Diabetes 0.558 <  0.001 6.3. Family history of CKD 0.448 <  0.001 6.4. Heart diseases such as heart failure or heart attack 0.541 <  0.001 6.5. Pain killers (e.g., NSAIDs) 0.475 <  0.001 6.6. Obesity 0.552 <  0.001 6.7. Excess stress 0.487 <  0.001 7.1. Nausea and vomiting 0.471 <  0.001 7.2. Tiredness/ fatigue 0.565 <  0.001 7.3. Loss of appetite 0.536 <  0.001 7.4. Fever 0.362 <  0.001 7.5. Fluid overload 0.462 <  0.001 8.1. Limit the intake of juices and soft drinks 0.279 <  0.001 8.2. Keep blood sugar levels under control 0.543 <  0.001 8.3. Keep blood pressure under control 0.533 <  0.001 8.4. Keep body weight under control 0.590 <  0.001 9.1. Drugs 0.356 <  0.001 9.2. Dialysis 0.377 <  0.001 additional tool of evaluation in the form of a valid scale of knowledge assessment. The construct validity was con- firmed by computing the sensitivity and specificity of the scale. Our scale had a very good sensitivity supporting its use for public knowledge assessment. Nonetheless, the specificity of the scale was relatively low. This could be justified by the fact that the scale items were general and aimed at being understood and self-administered by the general population. Factor 9 g We found a good level of knowledge about CKD as determined by the median of the score of the knowl- edge scale. Our results are consistent with other findings that reported good knowledge about CKD. A study con- ducted in Jordan revealed that most of the participants had appropriate knowledge about CKD [28]. Moreover, nearly half of the participants in a study conducted by Tegegne et  al. were well-knowledgeable on the preven- tion and early detection of CKD [29]. Contrary to these findings, in a study of the Australian public’s understand- ing of CKD, half of the participants had knowledge scores of less than the median [16]. Furthermore, Tuot et al. [30], Ng et al. [31], Agustiyowati [32], as well as, Hussain et al. [14] reported low levels of knowledge and awareness of CKD. Our results added to the literature that knowledge scores above the median or the mean could reflect sub- stantial awareness about CKD, as our ROC curve analy- sis determined a lower cutoff point for better knowledge which was below the knowledge score median. The results of our study revealed a significant positive association between age and a better CKD knowledge. Older population appear to be more knowledgeable about CKD. Maturity and more years of life experience could be behind this association. Our findings support the literature that determined better knowledge of CKD among older population. In a study conducted in Saudi Arabia, participants with a greater age showed a substan- tially higher CKD knowledge score [18]. The risk of car- diovascular and renal diseases increases with age and is associated with high rates of morbidity and mortality [33, 34]. Older patients may also be more concerned about their health, which could be the reason to acquire more information about chronic conditions including kidney disease to prevent or control their medical conditions.i Our findings support the literature that reported bet- ter health literacy in urban regions compared to rural areas. These findings are in line with those reported by Asmelash et al. where urban residents were 2.21 times more knowledgeable about CKD when compared to rural residents [35]. Likewise, higher mean knowledge score was observed among urban participants when compared to rural participants in a study conducted in Tanzania [36]. Factor 9 The above results showed better knowl- edge in Bekaa, Mount Lebanon, and North governorates A number of surveys assessed public awareness about CKD and provided validated questionnaires for this assessment. For instance, Gheewala et  al. developed a validated questionnaire to assess public knowledge of CKD in Australia [16]. Furthermore, CKD knowl- edge questionnaires developed by Peng et  al. [26] and Wei et al. [27] showed favorable construct validity, reli- ability, and consistency. The present study provided an Younes et al. BMC Nephrology (2022) 23:266 Page 8 of 11 compared to the South of Lebanon. The reason for this is not fully understood. Though it could be explained by that the South population are less concerned about their health and medical conditions. This is reflected by recent national reports that showed lower immuniza- tion rates and acceptance of COVID-19 vaccination in this region [37]. enough knowledge and practice towards CKD [40]. This is not surprising, as healthcare professionals are expected to be competent with the basics of all medical conditions including nephrology. Moreover, students appear to have better knowledge about CKD. This could be justified by higher digital literacy among this subpopulation and the wider access they have to medi- cal information [41, 42]. The results of the current study are consistent with the normal trend of having a better health literacy with higher level of education [38]. Our results determined that having a university degree is an important pre- dictor of knowledge about CKD. For instance, partici- pants with a higher education level had a higher CKD knowledge score, according to Chow et  al. [23]. Also, in a study conducted by Stanifer et al., results demon- strated that participants who had completed secondary or post-secondary school had higher mean scores than those who had just completed primary school or had no education [36]. In fact, highly educated populations are reportedly more knowledgeable about various med- ical and non-medical conditions [39], and this research confirms that CKD is not an exception. Healthcare providers were also found to have significantly bet- ter knowledge; this was in line with findings in Ethio- pia which showed that care provider professionals had People who are more concerned about their health conditions appear to have a better awareness about CKD. We found a significantly better knowledge in this context among those who screened or assessed their renal func- tion lately within a maximum of 1 year. Factor 9 This subpopula- tion is assumed to have better attitude toward selfcare and awareness, which is associated with routine medi- cal checkups and better knowledge relating to various chronic conditions including CKD [43]. Acknowledgements The authors would like to acknowledge all participants who responded to the study survey. Conclusion In conclusion, the current study developed and validated a reliable tool to assess the general public’s knowledge and awareness about CKD. Findings revealed that Leba- nese knowledge was good in general and was associated with age, area of residence, level of education, occupa- tion, and a recent assessment of kidney function. None- theless, significant gaps in CKD awareness were observed in some areas and subpopulations. Therefore, responsible organizations should make an extra effort to raise com- munity awareness and implement targeted CKD edu- cational activities to improve the early detection and management of CKD. ORa Adjusted odd’s ratio, 95% CI 95% Confidence interval Implications for practiceh p p This study was able to generate and validate a reliable assessment tool for public knowledge and awareness about CKD. Although a good level of knowledge was determined, important gaps around CKD awareness were found among younger, less educated, and unemployed subpopulations. Similar gaps appear to exist in rural Fig. 1  ROC curve of CKD knowledge scale Fig. 1  ROC curve of CKD knowledge scale Younes et al. BMC Nephrology (2022) 23:266 Younes et al. BMC Nephrology (2022) 23:266 Page 9 of 11 Table 4  Multivariable logistic regression of CKD knowledge score predictors ORa Adjusted odd’s ratio, 95% CI 95% Confidence interval Variable ORa P-value 95% CI Age 1.018 0.003 1.006–1.030 Area of residence ▪ Beirut vs. South 0.902 0.537 0.649–1.253 ▪ Bekaa vs. South 1.623 0.027 1.056–2.493 ▪ Mount Lebanon vs. South 1.671 0.013 1.116–2.501 ▪ North vs. South 1.691 0.031 1.050–2.722 Level of education ▪ Illiterate vs. University 0.248 <  0.001 0.145–0.422 ▪ Primary school vs. University 0.386 < 0.001 0.249–0.600 ▪ Secondary school vs. University 0.462 < 0.001 0.327–0.653 Occupation ▪ Healthcare vs. Unemployed 3.919 < 0.001 2.107–7.288 ▪ Non-healthcare vs. Unemployed 1.041 0.820 0.739–1.466 ▪ Retired vs. Unemployed 1.126 0.699 0.618–2.049 ▪ Student vs. Unemployed 2.497 < 0.001 1.585–3.934 Last assessment of renal function ▪ < 6 months vs. Never 2.314 < 0.001 1.532–3.495 ▪ 6–12 months vs. Never 2.124 0.003 1.291–3.496 ▪ 1–2 years vs. Never 1.394 0.156 0.881–2.205 ▪ > 2 years vs. Never 1.422 0.107 0.927–2.181 ▪ Don’t know or remember vs. Never 0.923 0.665 0.642–1.326 design of the study, which cannot determine temporal- ity. Moreover, the sensitivity and specificity of the scale were relatively low, and the study sample included rela- tively young participants. Therefore, additional research is recommended to include older participants to confirm the validity of the scale. Finally, although this novel tool of assessment appears to be valid and reliable, the vali- dation involved exploratory factor analysis only. Further research is suggested in this context to confirm the fac- tor analysis and validity measures of this assessment and research tool. Abbreviations CKD: Chronic kidney disease; GFR: Glomerular filtration rate; ESRD: End-stage renal disease; DM: Diabetes mellitus; HTN: Hypertension; DALYs: Disability- Adjusted Life-Years; GBD: Global Burden of Diseases, Injuries, and Risk Factors Study; CVD: Cardiovascular disease; CDC: Centers for Disease Control and Prevention; SPSS: Statistical Package for Social Sciences; KMO: Kaiser–Meyer– Olkin; ROC: Receiver operating characteristic; ORa: Adjusted odds ratios; CI: Confidence interval; NSAIDs: Nonsteroidal anti-inflammatory drugs. areas and among people who are less concerned about their medical wellbeing. The findings of this research provide insight for public health stakeholders to stipulate more attention, education, and healthcare for those pop- ulations in order to minimize the disease burden. Strengths and limitationsh The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12882-​022-​02889-2. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12882-​022-​02889-2. This study has several strengths. It provides public health researchers and healthcare providers with a reli- able, consistent, and valid tool to assess public awareness about CKD. This tool could be modified and adapted in future research to assess public awareness about addi- tional medical conditions. The sample size was consider- ably large and thus allowed for a high power of statistical analysis. On the other hand, the limitations of the study include a possible selection bias as a result of the snow- ball sampling. However, the risk of this bias is minimized as the sample included participants from districts all over Lebanon. Furthermore, a possible information bias that could be related to self-administration of the question- naire cannot be precluded. Though it is believed that this kind of bias is also minimized since the questionnaire was in simple lay language and didn’t include any medical jar- gons. Another limitation is related to the cross-sectional Additional file 1: Annex 1. The study questionnaire including the initial CKD knowledge scale. Additional file 1: Annex 1. The study questionnaire including the initial CKD knowledge scale. Additional file 2: Annex 2. The final validated CKD knowledge scale. Authors’ contributions SY conceptualized and designed the study. SY, FS, JS, MD, and NM wrote the first draft of the manuscript; SY, FS, JS, MD, MA and NM acquired the data and designed the methodology; FS carried out the analysis; SY, FS, and JS interpreted the results, MR was responsible for project administra- tion and supervision. All authors read and approved the final version of the manuscript. References 21. Boateng GO, Neilands TB, Frongillo EA, Melgar-Quiñonez HR, Young SL. Best practices for developing and validating scales for health, social, and behavioral research: A primer. Front Public Health. 2018;6:149. 1. CKD Evaluation and Management – KDIGO [Internet]. [cited 2022 Mar 16]. Available from: https://​kdigo.​org/​guide​lines/​ckd-​evalu​ation-​and-​ manag​ement/. 1. CKD Evaluation and Management – KDIGO [Internet]. [cited 2022 Mar 16]. Available from: https://​kdigo.​org/​guide​lines/​ckd-​evalu​ation-​and-​ manag​ement/. 22. AlSogair AAA, Albalawi AMA, Ahmed M, Alharbi AAH, Almansour ADM. Awareness of chronic kidney disease in hail region. Egypt J Hosp Med. 2018;71(7):3634–40. 2. Damtie S, Biadgo B, Baynes HW, Ambachew S, Melak T, Asmelash D, et al. Chronic kidney disease and associated risk factors assessment among diabetes mellitus patients at A tertiary hospital, Northwest Ethiopia. Ethiop J Health Sci. 2018;28(6):691–700. 2. Damtie S, Biadgo B, Baynes HW, Ambachew S, Melak T, Asmelash D, et al. 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Availability of data and materials 11. Norton JM, Moxey-Mims MM, Eggers PW, Narva AS, Star RA, Kimmel PL, et al. Social determinants of racial disparities in CKD. J Am Soc Nephrol. 2016;27(9):2576–95. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 12. Fauziyati A. Global challenge of early detection and Management of Chronic Kidney Disease. J Kedokt Dan Kesehat Indones. 2017;8(1):1–2. Competing interests The authors declare that they have no competing interests. 18. Alobaidi S. Knowledge of chronic kidney disease among the population of Saudi Arabia evaluated using a validated questionnaire: A cross-sec- tional study. Patient Prefer Adherence. 2021;15:1281–8. Funding None. Page 10 of 11 Page 10 of 11 Younes et al. BMC Nephrology (2022) 23:266 Younes et al. BMC Nephrology Author details 1 1 School of Pharmacy, Lebanese International University, Bekaa, Lebanon. 2 School of Pharmacy, Lebanese International University, Beirut, Lebanon. 19. Chow WL, Joshi VD, Tin AS, van der Erf S, Lim JFY, Swah TS, et al. Limited knowledge of chronic kidney disease among primary care patients – a cross-sectional survey. BMC Nephrol. 2012;13(1):54. Received: 25 April 2022 Accepted: 14 July 2022 Received: 25 April 2022 Accepted: 14 July 2022 20. Bikbov B, Purcell CA, Levey AS, Smith M, Abdoli A, Abebe M, et al. Global, regional, and national burden of chronic kidney disease, 1990–2017: a systematic analysis for the global burden of disease study 2017. Lancet. 2020;395(10225):709–33. Declarations 13. Johnson DW, Atai E, Chan M, Phoon RK, Scott C, Toussaint ND, et al. KHA- CARI guideline: early chronic kidney disease: detection, prevention and management. Nephrology. 2013;18(5):340–50. Ethics approval and consent to participate All methods were performed in accordance with the Declaration of Helsinki and have been approved by the Ethics and Research Committee of the School of Pharmacy at the Lebanese International University (2021RC-116-LIUSOP). The study carried no harmful risks as it was observational, and anonymity of all participants was warranted as personal identifiers weren’t traced during data collection and analysis. All participants agreed to participate and provided informed consent. Illiterate people had the informed consent read and explained to them by a caregiver or family member. 14. Hussain S, Habib A, Najmi AK. Limited knowledge of chronic kidney disease among type 2 diabetes mellitus patients in India. Int J Environ Res Public Health. 2019;16(8):1443. 15. Ahmed IAB, Alharbi SH, Alateeq FA, Aloriney AM, Alharbi AAH, AlSogair ARA, et al. Knowledge and awareness towards chronic kidney disease risk factors in Saudi Arabia. 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Kidney Med. 2019;1(2):43–50. 10. Kumela Goro K, Desalegn Wolide A, Kerga Dibaba F, Gashe Fufa F, Wakjira Garedow A, Edilu Tufa B, et al. Patient awareness, prevalence, and risk fac- tors of chronic kidney disease among diabetes mellitus and hypertensive patients at Jimma University medical center, Ethiopia. Biomed Res Int. 2019;2019:e2383508. 31. Ng CY, Lee ZS, Goh KS. Cross-sectional study on knowledge of chronic kidney disease among medical outpatient clinic patients. Med J Malaysia. 2016;71(3):99–104. 32. Agustiyowati THR. Knowledge and Attitude Toward Chronic Kidney Disease Among Pre-Dialysis Patients in Indonesia. Int J Caring Sci Knowl Attitude Chronic Kidney Dis Pre-Dial Patients Indones. 2020;13(1):283–7. Page 11 of 11 Younes et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 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https://www.biorxiv.org/content/biorxiv/early/2022/11/01/2022.11.01.514664.full.pdf
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Within-patient and global evolutionary dynamics of<i>Klebsiella pneumoniae</i>ST17
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. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 partment of Medical Microbiology, Stavanger University Hospital, Stavanger, Norway 2 Department of Biological Sciences, Faculty of Mathematics and Natural Sciences, University of Bergen, Bergen, Norway 2 Department of Biological Sciences, Faculty of Mathematics and Natural Sciences, University of Bergen, Bergen, Norway 3 Department of Infectious Diseases, Central Clinical School, Monash University, Melbourne, Australia 3 Department of Infectious Diseases, Central Clinical School, Monash University, Melbourne, Australia 4 Research Section Food Safety and Animal Health, Department of Animal Health and Food Safety, Norwegian Veterinary Institute, Oslo, Norway search Section Food Safety and Animal Health, Department of Animal Health and Food Safet i V t i I tit t O l N 4 Research Section Food Safety and Animal Health, Department of Animal Health and Food Safety, 4 Research Section Food Safety and Animal Health, Department of Animal Health and Food 4 Research Section Food Safety and Animal Health, Department of Animal Health and Food Safety, Norwegian Veterinary Institute Oslo Norway Norwegian Veterinary Institute, Oslo, Norway 5 Department of Paediatrics, Stavanger University Hospital, Stavanger, Norway 6 Faculty of Health Sciences, University of Stavanger, Stavanger, Norway 7 Department of Medical Biology, Faculty of Health Sciences, UiT – The Arctic University of Norway, Tromsø, Norway 7 Department of Medical Biology, Faculty of Health Sciences, UiT – The Arctic University of Norway, Tromsø, Norway 8 Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway 8 Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway 9 Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom 9 Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom 10 Department of Clinical Science, Faculty of Medicine, University of Bergen, Bergen, Norway 10 Department of Clinical Science, Faculty of Medicine, University of Bergen, Bergen, Norway Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 Marit A. K. Hetland 1,2*, Jane Hawkey 3, Eva Bernhoff 1, Ragna-Johanne Bakksjø 1, Håkon Kaspersen 4, Siren Irene Rettedal 5,6, Arnfinn Sundsfjord 7,8, Kathryn E. Holt 3,9, Iren H. Löhr 1,10 Marit A. K. Hetland 1,2*, Jane Hawkey 3, Eva Bernhoff 1, Ragna-Johanne Bakksjø 1, Håkon Kaspersen 4, Siren Irene Rettedal 5,6, Arnfinn Sundsfjord 7,8, Kathryn E. Holt 3,9, Iren H. Löhr 1,10 Marit A. K. Hetland , Jane Hawkey , Eva Bernhoff , Ragna Johanne Bakksjø , Håkon Kaspersen 4, Siren Irene Rettedal 5,6, Arnfinn Sundsfjord 7,8, Kathryn E. Holt 3,9, Iren H. Löhr 1,10 1 Department of Medical Microbiology, Stavanger University Hospital, Stavanger, Norway 2 Department of Biological Sciences, Faculty of Mathematics and Natural Sciences, University of Bergen, Bergen, Norway 3 Department of Infectious Diseases, Central Clinical School, Monash University, Melbourne, Australia 4 Research Section Food Safety and Animal Health, Department of Animal Health and Food Safety, Norwegian Veterinary Institute, Oslo, Norway 5 Department of Paediatrics, Stavanger University Hospital, Stavanger, Norway 6 Faculty of Health Sciences, University of Stavanger, Stavanger, Norway 7 Department of Medical Biology, Faculty of Health Sciences, UiT – The Arctic University of Norway, Tromsø, Norway 8 Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway 9 Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom 10 Department of Clinical Science, Faculty of Medicine, University of Bergen, Bergen, Norway * Corresponding author: Marit A. K. Hetland, marit.hetland@uib.no Keywords: Klebsiella pneumoniae; ST17; in vivo evolution; infection; colonisation; global dynamics Repositories: BioProject PRJEB36392, GenBank accession numbers: CP075591-CP075594. * Corresponding author: Marit A. K. Hetland, marit.hetland@uib.no * Corresponding author: Marit A. K. Hetland, marit.hetland@uib.no Keywords: Klebsiella pneumoniae; ST17; in vivo evolution; infection; colonisation; global dynamics Keywords: Klebsiella pneumoniae; ST17; in vivo evolution; infection; colonisation; global dynamics Repositories: BioProject PRJEB36392, GenBank accession numbers: CP075591-CP075594. Repositories: BioProject PRJEB36392, GenBank accession numbers: CP075591-CP075594. Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 1 Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 1 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint Abstract Here, we investigated the within-host evolution of ST17 in 45 of those children during long-term colonisation and compared the outbreak with 254 global strains. Ninety-two outbreak-related isolates were whole-genome sequenced. They had capsule locus KL25, O locus O5 and carried yersiniabactin. During within-host colonisation ST17 remained stable with few single nucleotide polymorphisms, no acquisition of antimicrobial resistance (AMR) or virulence determinants, and persistent carriage of a blaCTX-M-15-encoding IncFII(K) IncFIB(K) plasmid (pKp2177_1). The global collection included ST17 from 1993-2020 from 34 countries, that were from human infection (41.3%), colonisation (39.3%) and respiratory specimens (7.3%), from animals (9.3%), and from the environment (2.7%). We estimate that ST17 emerged mid-to-late 19th century (1859, 95% HPD 1763-1939) and diversified through recombinations of the K and O loci to form several sublineages, with various AMR genes, virulence loci and plasmids. There was limited evidence of persistence of AMR genes in any of these lineages. A globally disseminated sublineage with KL25/O5 accounted for 52.7% of the genomes. It included a monophyletic subclade that emerged in the mid-1980s which comprised the Stavanger NICU outbreak Klebsiella pneumoniae sequence type (ST) 17 is a global problem clone that causes multidrug-resistant (MDR) hospital infections worldwide. In 2008-2009, an outbreak of MDR ST17 occurred at a neonatal intensive care unit (NICU) in Stavanger, Norway. Fifty-seven children were colonised. We observed intestinal persistence of ST17 in all of the children for up to two years after hospital discharge. Here, we investigated the within-host evolution of ST17 in 45 of those children during long-term colonisation and compared the outbreak with 254 global strains. Ninety-two outbreak-related isolates were whole-genome sequenced. They had capsule locus KL25, O locus O5 and carried yersiniabactin. During within-host colonisation ST17 remained stable with few single nucleotide polymorphisms, no acquisition of antimicrobial resistance (AMR) or virulence determinants, and persistent carriage of a blaCTX-M-15-encoding IncFII(K) IncFIB(K) plasmid (pKp2177_1). The global collection included ST17 from 1993-2020 from 34 countries, that were from human infection (41.3%), colonisation (39.3%) and respiratory specimens (7.3%), from animals (9.3%), and from the environment (2.7%). We estimate that ST17 emerged mid-to-late 19th century (1859, 95% HPD 1763-1939) and diversified through recombinations of the K and O loci to form several sublineages, with various AMR genes, virulence loci and plasmids. There was limited evidence of persistence of AMR genes in any of these lineages. A globally disseminated sublineage with KL25/O5 accounted for 52.7% of the genomes. Abstract Klebsiella pneumoniae sequence type (ST) 17 is a global problem clone that causes multidrug-resistant (MDR) hospital infections worldwide. In 2008-2009, an outbreak of MDR ST17 occurred at a neonatal intensive care unit (NICU) in Stavanger, Norway. Fifty-seven children were colonised. We observed intestinal persistence of ST17 in all of the children for up to two years after hospital discharge. Here, we investigated the within-host evolution of ST17 in 45 of those children during long-term colonisation and compared the outbreak with 254 global strains. Ninety-two outbreak-related isolates were whole-genome sequenced. They had capsule locus KL25, O locus O5 and carried yersiniabactin. During within-host colonisation ST17 remained stable with few single nucleotide polymorphisms, no acquisition of antimicrobial resistance (AMR) or virulence determinants, and persistent carriage of a blaCTX-M-15-encoding IncFII(K) IncFIB(K) plasmid (pKp2177_1). The global collection included ST17 from 1993-2020 from 34 countries, that were from human infection (41.3%), colonisation (39.3%) and respiratory specimens (7.3%), from animals (9.3%), and from the environment (2.7%). We estimate that ST17 emerged mid-to-late 19th century (1859, 95% HPD 1763-1939) and diversified through recombinations of the K and O loci to form several sublineages, with various AMR genes, virulence loci and plasmids. There was limited evidence of persistence of AMR genes in any of these lineages. A globally disseminated sublineage with KL25/O5 accounted for 52.7% of the genomes. It included a monophyletic subclade that emerged in the mid-1980s, which comprised the Stavanger NICU outbreak and 10 genomes from three other countries, which all carried pKp2177_1. The plasmid was also observed in a KL155/OL101 subclade from the 2000s. Three clonal expansions of ST17 were identified, all were healthcare-associated and carried either yersiniabactin and/or pKp2177_1. To conclude, ST17 is globally disseminated and associated with opportunistic hospital-acquired infections. It contributes to the burden of global MDR infections, but many diverse lineages persist without acquired AMR. We hypothesise that non-human sources and human colonisation may play a crucial role for severe infections in vulnerable patients, such as preterm neonates. Klebsiella pneumoniae sequence type (ST) 17 is a global problem clone that causes multidrug-resistant (MDR) hospital infections worldwide. In 2008-2009, an outbreak of MDR ST17 occurred at a neonatal intensive care unit (NICU) in Stavanger, Norway. Fifty-seven children were colonised. We observed intestinal persistence of ST17 in all of the children for up to two years after hospital discharge. Impact statement Klebsiella pneumoniae is an opportunistic pathogen that frequently causes hospital- associated multidrug-resistant (MDR) infections. Infections with K. pneumoniae strains that are resistant to third generation cephalosporins and/or carbapenems are considered a major public health threat, as there are limited treatment options available. Some MDR K. pneumoniae clones, including ST307 and ST258, are global problem clones because they disproportionately contribute to the burden of MDR infections and are common causes of such infections and/or outbreaks in hospitals around the world. Here we describe another such clone, ST17, which caused an MDR outbreak in our neonatal intensive care unit, affecting 57 children. We found that this clone underwent minor within-host evolution during two years of long-term gastrointestinal colonisation after hospital discharge. We then investigated the evolutionary history of ST17 globally, as it had not previously been studied. When we compared ST17 isolates from around the world with the isolates from our hospital, we discovered that whilst ST17 with antimicrobial resistance has contributed to outbreaks in other neonatal care units, it also frequently causes infections or colonises humans and non- human sources without any antimicrobial resistance. Klebsiella pneumoniae is an opportunistic pathogen that frequently causes hospital- associated multidrug-resistant (MDR) infections. Infections with K. pneumoniae strains that are resistant to third generation cephalosporins and/or carbapenems are considered a major public health threat, as there are limited treatment options available. Some MDR K. Abstract It included a monophyletic subclade that emerged in the mid-1980s, which comprised the Stavanger NICU outbreak and 10 genomes from three other countries, which all carried pKp2177_1. The plasmid was also observed in a KL155/OL101 subclade from the 2000s. Three clonal expansions of ST17 were identified, all were healthcare-associated and carried either yersiniabactin and/or pKp2177_1. To conclude, ST17 is globally disseminated and associated with opportunistic hospital-acquired infections. It contributes to the burden of global MDR infections, but many diverse lineages persist without acquired AMR. We hypothesise that non-human sources and human colonisation may play a crucial role for severe infections in vulnerable patients, such as preterm neonates. Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 2 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint Introduction The Gram-negative bacteria Klebsiella pneumoniae are a common cause of infections in both healthcare and community settings (1, 2). Notably, they are a major pathogen responsible for bacterial outbreaks in neonatal intensive care units (NICUs) (3-5). Several sequence types (STs) of K. pneumoniae are associated with multidrug-resistance (MDR) (6). Some clones carry genes encoding extended-spectrum beta-lactamases (ESBLs) or carbapenemases and co-linked resistance determinants, resulting in severely limited treatment options (5). Some MDR clones remain localised, e.g. within a healthcare or community setting, whilst others have spread across continents and become global problem clones (5). One such clone is K. pneumoniae ST17, which has been detected in several countries, often with an MDR phenotype causing difficult-to-treat hospital infections (5, 7-9). ST17 has also been found colonising healthy humans in the community setting (10-12), in animals (13, 14) and in the environment (15-17). From November 2008 to April 2009 an outbreak caused by ESBL-producing (blaCTX-M-15) ST17 occurred in a NICU at Stavanger University Hospital, Norway (8). The outbreak was first discovered when ESBL-producing K. pneumoniae was recovered from three neonates: one clinical sample from conjunctiva and two routine screening samples. Subsequent screening revealed that 92% (22/24) of the patients in the ward were colonised with ESBL- producing K. pneumoniae. The index isolate of the outbreak (Kp2177) was isolated from breast milk from one of the mothers. During the outbreak, a total of 57 children were colonised with ST17. Only one of them developed a bloodstream infection (BSI), which was successfully treated with meropenem. A follow-up study revealed intestinal persistence of the ST17 strain in all 57 children after hospital discharge, all carrying a blaCTX-M-15-encoding IncFII(K) IncFIB(K) plasmid, for up to two years (median 12 months) (18, 19). Here, we first characterised the genomic diversity and in vivo evolution of ST17 within and between 45 NICU patients in Stavanger, to study the evolution of this strain during long-term gut colonisation. As the blaCTX-M-15 ST17 strain persisted for up to two years in these children, we wanted to examine how the in vivo evolution compared to the evolutionary dynamics of ST17 on a global scale. Whilst ST17 has been observed in several countries and settings, specific analyses to determine its global dynamics have not been reported previously. Data summary The genome sequences generated in this study have been deposited at the European Nucleotide Archive under BioProject PRJEB36392. The BioSample accession numbers and associated metadata for the genomes are available in Table S1. The completed annotated genome assembly of Kp2177 is available in GenBank under accession numbers CP075591 (chromosome), CP075592 (pKp2177_1), CP075593 (pKp2177_2) and CP075594 (pKp2177_3). The global dataset of 300 ST17 genomes is available for interactive viewing in Microreact at https://microreact.org/project/kpst17. Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 3 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 4 Collection and sequencing of the Stavanger NICU outbreak isolates During the outbreak in the Stavanger NICU, 57 children were colonised. They were examined for intestinal persistence over a two-year period (19), and for 45 of the children, we had two ST17 isolates available for genomic comparison in this study. For each child, one sample was isolated during the NICU stay (between January and March 2009) and another after hospital discharge (between June 2009 and November 2010). The duration of colonisation between the two isolates from each child ranged 3-21 months (median 11). All of the colonising isolates were from faecal swabs except one nasal swab isolate. We performed whole-genome sequencing using Illumina MiSeq (n=46) or HiSeq (n=46) technology to produce 300 bp or 150 bp paired-end short-read sequences of in total 92 isolates: the 45 NICU and 45 follow-up isolates, the one BSI isolate and the index (breast milk) isolate Kp2177. BioSample accessions are given in Table S1. To generate a reference genome for the outbreak, Kp2177 was also sequenced with the Oxford Nanopore Technologies (ONT) platform, and hybrid assembly was performed to resolve the complete genome sequence (deposited in GenBank under accessions CP075591-94; details in Supplementary Methods). Introduction We therefore carried out a literature and genome search to assess the global presence of ST17, followed by core genome evolutionary analyses of 300 ST17 strains from around the world, that were isolated from human infections and colonisation, animals, plants and water. al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 4 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 5 Genome assembly and genotyping All short-read Illumina sequences were filtered with TrimGalore v0.6.6 All short-read Illumina sequences were filtered with TrimGalore v0.6.6 (https://github.com/FelixKrueger/TrimGalore) to exclude low-quality reads and adapter- contamination before de novo assembly was performed with Unicycler v0.4.8 (24), using SPAdes v3.13.0 (25) and Pilon v1.23 (26), with default settings. Detection of antimicrobial resistance (AMR) and virulence genes was based on analysis of genome assemblies using Kleborate v2.2.0 (20), which uses a manually curated version of CARD v3.1.13 for acquired AMR determinants. We also used SRST2 v0.2.0 (27) to query the unassembled short-reads directly, using the same database. If a gene was missing in the Kleborate analysis of assemblies but found with SRST2 analysis of reads, we considered it present in the genome. Capsule (K) and lipopolysaccharide (LPS) O-antigen serotype predictions were performed with Kaptive v2.0.0; as low-confidence calls are most likely due to assembly- quality, we reported the best matching K and O loci regardless of confidence call, except for those called with low-confidence as KL107 which were designated unknown (28). Plasmid replicons were detected using Abricate v1.0.1 (https://github.com/tseemann/abricate) with the PlasmidFinder database v2021-01-13 (https://bitbucket.org/genomicepidemiology/plasmidfinder_db). Global dataset of ST17 genomes A literature search querying PubMed for titles or abstracts containing the word “ST17” with and without “Klebsiella pneumoniae” resulted in 36 peer-reviewed articles that contributed novel data on K. pneumoniae ST17 (as of November 2021), but only 29 ST17 isolates were whole-genome sequenced and available for download. We therefore also included ST17 genomes from other sources: a recent study analysed 13,156 publicly available K. pneumoniae genomes and identified their STs (20). Those that were confirmed ST17 and had <500 assembled contigs, known year of collection and sample source (n=149) were included in our global dataset. Additionally, we included 64 ST17 genomes from the recent SpARK study (17) and 41 ST17 genomes from Norwegian surveillance studies that were not linked to the Stavanger NICU outbreak (11, 21-23). For analyses of global dynamics, we also included 46 of the Stavanger NICU outbreak genomes, resulting in a global dataset of 300 genomes (BioSample accessions are given in Table S2). al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 5 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint Phylogenetic inference and molecular dating of the global ST17 collectio We performed ML and Bayesian phylogenetic analyses of 300 genomes to compare the Stavanger NICU outbreak (n=44 follow-up faecal, 1 blood, 1 breast milk) with ST17 from other geographical locations and collection years (n=254), and to estimate the evolutionary rate of the clone. We generated a core chromosomal SNP alignment of the genomes against Kp2177 with RedDog as described in the Supplementary Methods. Gubbins v3.1.6 (34) was used to identify and filter recombinant SNPs from the alignment, which was then passed to RaxML v8.2.12 (31) to infer an ML phylogeny. We investigated the relationship between the root-to-tip distance in the ML tree and the years of isolation using linear regression in TempEST v1.5.3 (35). To estimate the evolutionary rate and dated phylogeny, we performed Bayesian phylodynamic analysis with BEAST2 v2.6.5 (36) on a subset of 145 of the 300 genomes, which were selected to capture the diversity across the clades in the full ML tree as detailed in the Supplementary Methods. The temporal signal of the clone was confirmed by performing date-randomisation tests (Figure S1). Between and within-host comparison of the Stavanger NICU outbreak genomes To identify single nucleotide polymorphisms (SNPs) between and within the outbreak genomes, core genome alignments were produced with RedDog v1beta.11 (https://github.com/katholt/RedDog) by aligning the raw reads of the 92 outbreak genomes against Kp2177. Within-host SNPs were defined as SNPs between the NICU and follow-up genome of a child, from the overall core genome alignment. Bowtie2 v2.3.5.1 (29) was used for sequence alignment and SAMTools v1.9 (30) to identify SNPs. Further details are given in the Supplementary Methods. RAxML v8.2.12 (31) was used to infer a core genome maximum likelihood (ML) phylogeny from the chromosomal alignment of 113 SNPs. To estimate a substitution rate (SNPs/year) for the outbreak, we divided the number of SNPs against Kp2177 from each genome by the difference in isolation time. To identify genes that had been gained or lost between the NICU and follow-up genomes, we characterised their pangenome with Panaroo v1.2.7 (32), using annotations from Prokka v1.14.6 (33) as input. As all the genomes belonged to the same outbreak strain and we wanted to identify any changes in gene content, we ran Panaroo using sensitive mode with the protein family identity set to 90%. et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 6 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint Statistical analyses All statistical analyses were performed with R version 4.0.2 (2020-06-22) (37). Paired data were analysed with a two-tailed Wilcoxon paired signed rank test. Comparisons of groups were analysed with Wilcoxon signed rank test and binomial data were compared with Chi square test. P-values <0.05 were considered statistically significant. Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 7 The Stavanger NICU outbreak The index isolate (Kp2177) of the Stavanger NICU outbreak, which was isolated from breast milk, belonged to ST17, had capsule locus KL25 and O locus O5, and consisted of a 5.38 Mbp chromosome and three plasmids (pKp2177_1, 182 Kbp, IncFII(K) and IncFIB(K); pKp2177_2, 83 Kbp, IncR and IncFIA(H1); and pKp2177_3, 3 Kbp, no plasmid replicon marker detected). A total of 5,303 genes were annotated in the genome. pKp2177_1 harboured the ESBL gene blaCTX-M-15, the beta-lactamase gene blaTEM1-D and the aminoglycoside resistance gene aac(3)-Iia. pKp2177_2 carried strAB, dfrA14, sul2, and cat2 encoding resistance towards streptomycin, trimethoprim, sulphonamides, and phenicols, respectively. Of known virulence factors, yersiniabactin (ybt 16) was chromosomally encoded on an ICEKp12 mobile genetic element. No other siderophores or hypermucoviscosity-encoding genes were present. et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 7 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint All of the colonising NICU (n=45) and subsequent follow-up isolates (n=45) belonged to the same ST17 strain, with KL25/O5 and ≤8 SNPs compared to the index Kp2177 isolate. The three Kp2177 plasmids were conserved in all 45 NICU genomes, with high sequence coverage (98.3%-100%, Table S1). The AMR and virulence genes in Kp2177 were also present in all of the NICU genomes, except for one that did not harbour cat2 (however the follow-up genome for this child did). There was more variation in the follow-up genomes, which were collected up to 21 months (median 11) after initial colonisation (Figure 1). The blaCTX-M-15-encoding plasmid pKp2177_1 was persistent in all of the strains with minimal variation (≤1 SNP and 68.8%–100% sequence coverage, median 100%). The pKp2177_2 and pKp2177_3 plasmids varied in coverage and AMR-gene content and were missing in 16% (n=7) and 11% (n=5) of the 45 follow-up genomes. The Stavanger NICU outbreak In six of the follow-up genomes we detected plasmid replicon markers that were not present in Kp2177 or in the NICU genomes. However, none of the 90 colonising genomes encoded additional AMR or virulence determinants not present in Kp2177. Some children were given antibiotics during their NICU stay or after hospital discharge (Table S3). No significant difference in the number of AMR genes was observed between the children that had received antibiotics (n=34) and those that did not (n=11) (p=0.92). Only one child developed a systemic infection (KN_0144A) (8). The BSI genome also belonged to the Kp2177 strain and was highly similar (1 SNP, wza-G178A), with no acquisition of genes that were unique to this genome. The follow-up genome for this patient (KN_0144A-L), which was isolated from gut colonisation 14 months after the BSI occurred, had the most SNPs of any in the outbreak (n=10). None of them were present in other outbreak genomes (Figure 1A and Table S4), nor in the global ST17 dataset (see below). Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 8 Diversity and evolution within and between hosts It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint We observed few genome changes within-hosts during up to 21 months of colonisation. Within-host SNPs were detected in 84.4% (38/45) NICU and follow-up pairs (Table S3). The number of SNPs in each child ranged from 0-10 (median 2), of which up to 6 were nonsynonymous SNPs (nsSNPs) and the rest synonymous or intergenic. Most of the SNPs (91.7%, n=110/120) occurred only once, in one genome each. However, in eight genes, >1 SNP was observed, in different genomes (Table S4). Notably, two genes had several SNPs: barA and uvrY, that make up the BarA-UvrY two-component system (TCS) (38). In uvrY, there were two SNPs leading to different amino acid changes in the same codon (G76W, G76R). In barA, four nsSNPs were present, two resulting in premature stop codons (Q260*, R305H, V311G and Q466*). These six mutations were variably present in nine genomes from children that were colonised >6 months (median 1.2 years). Of these mutations, only uvrY-G76R was also present in the global ST17 dataset (see results section below), in one genome (KP_NORM_URN_2013_95870), but an additional 12 nsSNPs in these two genes were present across the global phylogeny in 19 genomes. Among the 91 colonising genomes there were 5,742 unique genes. Of those, 76.5% (n=4,393) were core, i.e. present in all of the genomes. The accessory genes were either very common (60.3% of the 1,349 accessory genes were present in >90% of the genomes) or rare (32.2% were present in <5% of genomes). Overall, there was a significant within-host increase in the number of genes in the follow-up (median 5,279; range 5,081-5,428) compared to the NICU genomes (median 5,253; 5,175-5,306) (p=0.005, Figure S2). Within each child, between 6 and 311 (median 71) gene gain or loss events occurred during the colonisation period. In five of the 45 children, more than 150 genes had been gained during colonisation; none of them encoded known AMR or virulence determinants or plasmid replicon markers. There were also two children in which over 150 genes had been lost. Diversity and evolution within and between hosts To assess the diversity and in vivo evolution of the outbreak strain over a 21-month period, we performed a core genome phylogenetic analysis using Kp2177 as the reference genome. In total, 120 unique SNPs were identified, 113 of which were in positions in the chromosome, 3 in pKp2177_1 and 4 in pKp2177_2 (full list of SNPs and their consequences are given in Table S4). The 45 NICU genomes differed from Kp2177 by 0-3 SNPs (median 1). In contrast, the 45 follow-up genomes differed from the index by 0-8 SNPs (median 2) and showed significantly more evolutionary divergence from Kp2177 (p<0.0001). Based on the chromosomal alignment and the time between Kp2177 and the isolate collection dates, we calculated a substitution rate for the outbreak of 2.60 SNPs/year (95% CI 1.96–3.24), or 4.84x10-7 SNPs/year/site (95% CI 3.64x10-7–6.02x10-7). Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 8 Figure 1. Phylogeny of 92 Klebsiella pneumoniae ST17 outbreak isolates and their gene content. A) Midpoint-rooted maximum likelihood phylogeny of the 45 NICU (orange tip) and 45 follow-up (purple) isolates from colonisation and the BSI isolate (red) against the outbreak index isolate Kp2177 (green). B) Presence (colour) or absence (white) of AMR genes as listed in the columns (blocks are coloured by drug class). C) Presence (colour) or absence of the Kp2177 plasmids and D) of acquired plasmid replicon markers. Figure 1. Phylogeny of 92 Klebsiella pneumoniae ST17 outbreak isolates and their gene content. A) Midpoint-rooted maximum likelihood phylogeny of the 45 NICU (orange tip) and 45 follow-up (purple) isolates from colonisation and the BSI isolate (red) against the outbreak index isolate Kp2177 (green). B) Presence (colour) or absence (white) of AMR genes as listed in the columns (blocks are coloured by drug class). C) Presence (colour) or absence of the Kp2177 plasmids and D) of acquired plasmid replicon markers. et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 9 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. ST17 is globally disseminated and highly diverse To understand the ST17 variation in the Norwegian children in the context of the global diversity of this strain, we included 300 ST17 genomes for comparative and phylogenetic analyses. The genomes were globally distributed, including isolates from 34 countries in 12 United Nations defined geographical regions over a 28-year period (1993–2020) (Figure 2 and Table S2). The majority were from humans (88%, n=264/300): infection (n=124, most from blood [n=79] or urine [n=43]), colonisation (n=118, most from faeces [n=109, the Stavanger NICU outbreak accounted for 44 of these]), and respiratory sites with unknown infection status (n=22). The genomes from non-human sources (12%, n=36/300) came from animals (n=26, from dogs, broilers, a chicken, cows, a fly, pigs and turkeys), Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 10 Figure 2. Geographical distribution of Klebsiella pneumoniae ST17 genomes, their K and O loci, and reports of ST17. A) World map highlighting countries of collection for ST17 genomes that were available for download (coloured by world region as in Figure 3) and countries with isolates reported but not included in the global dataset (coloured dark grey). The pie charts show the distribution of K and O loci per world region (n=number of genomes), highlighting loci with ≥10 genomes. B) Number of ST17 genomes by collection year (bars, coloured by KL/OL) and cumulative number (red line), and cumulative number of reports of ST17 in PubMed abstracts or titles (black line) as of November 2021. Figure 2. Geographical distribution of Klebsiella pneumoniae ST17 genomes, their Figure 2. Geographical distribution of Klebsiella pneumoniae ST17 genomes, their K and O loci, and reports of ST17. A) World map highlighting countries of collection for ST17 genomes that were available for download (coloured by world region as in Figure 3) and countries with isolates reported but not included in the global dataset (coloured dark grey). The pie charts show the distribution of K and O loci per world region (n=number of genomes), highlighting loci with ≥10 genomes. B) Number of ST17 genomes by collection year (bars, coloured by KL/OL) and cumulative number (red line), and cumulative number of reports of ST17 in PubMed abstracts or titles (black line) as of November 2021. Figure 2. Geographical distribution of Klebsiella pneumoniae ST17 genomes, their K and O loci, and reports of ST17. ST17 is globally disseminated and highly diverse A) World map highlighting countries of collection for ST17 genomes that were available for download (coloured by world region as in Figure 3) and countries with isolates reported but not included in the global dataset (coloured dark grey). The pie charts show the distribution of K and O loci per world region (n=number of genomes), highlighting loci with ≥10 genomes. B) Number of ST17 genomes by collection year (bars, coloured by KL/OL) and cumulative number (red line), and cumulative number of reports of ST17 in PubMed abstracts or titles (black line) as of November 2021. et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 11 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint marine bivalves (n=2), different sources of water (n=7) and a sugar cane (n=1). The animal genomes were sampled from intestinal isolates, except one from a chicken meat isolate. A maximum likelihood phylogeny of the 300 genomes was created (Figure 3, also available for interactive viewing at https://microreact.org/project/kpst17). Due to the size and diversity of the dataset, 145 representative sequences were selected to estimate the evolutionary rate of ST17 using Bayesian modelling in BEAST2 (Figure S3) (see details in Supplementary Methods). The Bayesian analysis of 11,345 SNPs in 145 genomes indicated that ST17 emerged in 1859 (95% HPD 1763-1939) with an estimated evolutionary rate of 4.04× 10−7 substitutions/site/year (95% HPD 2.30× 10−7–5.79× 10−7), equivalent to 2.17 SNPs per year. Since its emergence, the clone has undergone several recombination events (the ratio of nucleotide substitutions introduced by recombination vs point mutation was estimated as r/m=3.27), which were centred in a hotspot surrounding the K and O loci (Figure 4A). These recombinations have led to a diversification of ST17 strains by creating distinct sublineages with different combinations of K and O loci. ST17 is globally disseminated and highly diverse In total, there were 8 O and 27 K loci (including unknown KLs), and 34 combinations of these, among the 300 ST17 genomes (Figure 4B). Over half (52.7%, n=158/300) of the genomes belonged to a clade that had KL25/O5 (except for two genomes with KL2/O2v1, one KL166/O5 and five unknown KL25/O5) that emerged in 1943 (95% HPD 1905-1972). This clade was found in 19 countries across 11 world regions (all except Southern Asia), and in all sample types. The genomes in the remaining sublineages of ST17 were found in 29 countries (all world regions except Eastern Europe) and had a much larger variety of K and O loci (32 combinations), with KL23/O1/O2v2 (n=29), KL155/OL101 (n=19) and KL122/O1/O2v2 (n=18) being the most prevalent (Figure 2). KL23/O1/O2v2 and KL122/O1/O2v2 were intermingled with genomes carrying other combinations of K/O loci, whilst KL155/OL101 formed a monophyletic clade. The dominant KL25/O5 clade was also distinct from the rest of the tree by high prevalence of yersiniabactin: 84.2% (133/158) of the genomes in this clade carried a yersiniabactin locus, compared to 7.0% (10/142) of the genomes in the rest of the tree. The majority of the yersiniabactin loci were ybt 15, located on ICEKp11, (n=63, complete or truncated) and ybt 16, on ICEKp12, (n=52, complete or truncated), which formed distinct subclades in the KL25/O5 sublineage (see Figure 3). Other yersiniabactin loci (n=10) were sporadically present across the tree, in 28 genomes, including in these subclades. There were few hypervirulence-associated genes, but aerobactin (iuc 1 and 3) or salmochelin (iro 3) with incomplete hypermucoviscosity rmp genes were present in three genomes. t al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 12 Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 13 Figure 3. Global phylogeny of 300 Klebsiella pneumoniae ST17 genomes. A) Maximum likelihood tree with tips coloured by world region of collection. Additionally, the Stavanger NICU outbreak genomes are coloured red and other genomes from Norway green. The three clonal expansions that were observed in ST17 are labelled. B) Sample type and loci as indicated in the column names. The most prevalent loci are ndicated in the inset legend. C) Presence (colour) or absence (white) of genes encoding resistance to the listed antimicrobial resistance (AMR) drug classes (blocks are coloured by drug class). Lighter colour in the ESBL column indicates blaCTX-M-15. ST17 is globally disseminated and highly diverse D) Presence (colour) or absence (white) of the Kp2177 plasmids. Several plasmid replicon markers were present across ST17, these are shown in Figure S4. More details about the metadata and genotypes are available for interactive viewing at https://microreact.org/project/kpst17. * Acquired genes and mutations. Gl b l h l f 300 Kl b i ll i ST17 A) M i lik lih d t ith ti l d b ld Figure 3. Global phylogeny of 300 Klebsiella pneumoniae ST17 genomes. A) Maximum likelihood tree with tips coloured by world region of collection. Additionally, the Stavanger NICU outbreak genomes are coloured red and other genomes from Norway green. The three clonal expansions that were observed in ST17 are labelled. B) Sample type and loci as indicated in the column names. The most prevalent loci are indicated in the inset legend. C) Presence (colour) or absence (white) of genes encoding resistance to the listed antimicrobial resistance (AMR) drug classes (blocks are coloured by drug class). Lighter colour in the ESBL column indicates blaCTX-M-15. D) Presence (colour) or absence (white) of the Kp2177 plasmids. Several plasmid replicon markers were present across ST17, these are shown in Figure S4. More details about the metadata and genotypes are available for interactive viewing at https://microreact.org/project/kpst17. * Acquired genes and mutations. Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint Figure 4. Recombinations and K/O loci diversity. A) Recombination counts per base calculated over non-overlapping windows of 1000 base pairs. There is a peak in the recombination count, which surrounds the K and O loci. The red arrow indicates the gene galF, which is the 5’ K locus gene. B) Combinations of K and O loci found among the 300 genomes. ST17 is globally disseminated and highly diverse Bubbles indicate the number of genomes and are coloured by the K/O Figure 4. Recombinations and K/O loci diversity. A) Recombination counts per base calculated over non-overlapping windows of 1000 base pairs. There is a peak in the recombination count, which surrounds the K and O loci. The red arrow indicates the gene galF, which is the 5’ K locus gene. B) Combinations of K and O loci found among the 300 genomes. Bubbles indicate the number of genomes and are coloured by the K/O bi ti i Fi 2 d 3 Figure 4. Recombinations and K/O loci diversity. A) Recombination counts per base calculated over non-overlapping windows of 1000 base pairs. There is a peak in the recombination count, which surrounds the K and O loci. The red arrow indicates the gene galF, which is the 5’ K locus gene. B) Combinations of K and O loci found among the 300 genomes. Bubbles indicate the number of genomes and are coloured by the K/O combinations as in Figures 2 and 3. t al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 14 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint Several different AMR determinants (n=81) and plasmid replicon markers (n=44) were detected in the ST17 genomes (Table S2 and Figure S4). In the overall collection, the number of AMR genes ranged from 0 to 19 (median 3), with 42.7% (128/300) genomes having no AMR genes present at all and 48.3% (145/300) being MDR (carried resistance genes to ≥1 antimicrobial agent in >3 antimicrobial classes). We observed no differences in the number of AMR determinants or MDR genomes between the samples from humans (infection, respiratory and colonisation). However, significantly fewer AMR genes were detected in the non-human isolate genomes (non-human: median 0, range 0-11 vs human: median 4.5, range 0-19, p<0.0001). ST17 is globally disseminated and highly diverse Of the AMR-encoding genomes, 127 carried ESBL genes; blaCTX-M-15 (n=92), blaCTX-M-14 (n=20), blaCTX-M-27 (n=1), blaCTX-M-63 (n=1), blaSHV-12 (n=13), blaSHV-2 (n=3), blaSHV-5 (n=2), blaSHV-7 (n=1), blaSHV-24 (n=1), and 25 carried carbapenemase- encoding genes; blaKPC-2 (n=10), blaNDM-1 (n=6), blaKPC-3 (n=4), blaOXA-48 (n=3), blaNDM-7 (n=1), blaOXA-181 (n=1) and blaVIM-1 (n=1). The AMR genes were found across many sublineages, but there was limited evidence of AMR-persistence within subclades. Clonal expansions of ST17 were hospital-associated and carried yersiniabactin or a blaCTX-M-15-encoding plasmid Only three clonal expansions (≥15 genomes) were observed across the global dataset, and all were hospital-associated (see Figure 3). One of these included the Stavanger NICU outbreak, which formed a monophyletic clade. Its closest neighbours were a cluster of four identical NICU blood isolates from Kilifi, Kenya (2009; 47 SNPs from Kp2177), five blood isolates from Nigeria (2009; 40 SNPs, 2015-2016: 143-831 SNPs) and one from the United Kingdom (UK) (2011; 38 SNPs) (Figure S5). The most recent common ancestor (MRCA) for this cluster was 1984 (95% HPD 1967-1998). These genomes carried the blaCTX-M-15- encoding pKp2177_1 plasmid that was persistent in the Stavanger NICU outbreak (≤1 SNP and 100% sequence coverage), and were highly similar, except for four genomes from Nigeria; two that had KL2/O2v1 instead of KL25/O5, and two that carried ybt 13 (on ICEKp2) rather than ybt 16 (on ICEKp12). The blaCTX-M-15 pKp2177_1 plasmid was present (≤2 SNPs and ≥95% sequence coverage, median 100%) in one other clonal expansion of ST17, in a cluster of 17 genomes, which all had KL155/OL101, of which only one genome encoded yersiniabactin (ybt 9 on ICEKp3) (see Figure 3). These were invasive isolates from Kilifi, Kenya in 2011 (n=15, 1 SNP between them), Nigeria in 2015 (n=1, 89 SNPs from the ones from Kenya) and Ghana (n=1, 90 SNPs) in 2015, with an MRCA of 2000 (95% HPD 1990-2007). Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 15 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint The last of the three clonal expansions consisted of 16 isolates from gut colonisation in a hospital in Padua, Italy during 2018 (17) (see Figure 3). These had KL25/O5 and carried ybt 15 (on ICEKp11) but no AMR genes (except one with aac(6’)-Ib-Suzhou). In 2017-2018, 48 other ST17s were isolated from different sources in Padua, Italy. Clonal expansions of ST17 were hospital-associated and carried yersiniabactin or a blaCTX-M-15-encoding plasmid These were not related to the clonal expansion in the local hospital, but were found across the entire phylogeny, carrying a range of KL/OLs, and with varying presence of yersiniabactin and AMR genes. The same was the case for the Norwegian ST17 genomes that were not part of the Stavanger NICU outbreak (n=43). To see if the pKp2177_1 plasmid had spread in Norway between STs, we screened 3,212 non-ST17 K. pneumoniae genomes for the presence of the blaCTX-M-15 pKp2177_1 plasmid (see Supplementary Methods). These covered 1,076 STs from multiple human and non- human sources in Norway between 2001 and 2020. We found only one genome that carried this plasmid, with 100% sequence coverage; a clinical ST307 urine isolate from 2013, previously described in (39). Discussion In this study we investigated the global dynamics of K. pneumoniae ST17 and the in vivo evolution within and between children that were colonised following an outbreak in an NICU in Stavanger, Norway in 2008–2009. During up to 21 months of colonisation, few changes (0-10 SNPs) occurred in the core genome, and the majority were present in only one genome each. However, there was one SNP that was shared among several follow-up genomes and it was also found in one genome in the global dataset. This was in the gene uvrY (G76R). UvrY is a response regulator that is part of a TCS with BarA, a sensor kinase, that has been described in uropathogenic Escherichia coli (38). Across the outbreak and global ST17, several independent nsSNPs were seen in these two genes. Pernestig et al. (38) found that mutations in either of those genes can affect survival in long-term competition cultures; when using media with gluconeogenic carbon sources, E. coli with knockout mutations in barA or uvrY had a clear growth advantage over the wild type, whereas when using media with carbon sources feeding into the glycolysis, the wild type had the growth advantage. Another study found that homologues of these genes in other bacteria led to decreased virulence in infection models (40, 41). It is possible that the mutations in these genes in ST17, if rendering the genes non-functional, gave an advantage to long-term colonisation in the gut, which is a source of gluconeogenic carbon. Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 16 t al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 16 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. Discussion ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint The Stavanger NICU outbreak strain persistently carried a blaCTX-M-15-encoding IncFII(K) IncFIB(K) plasmid (pKp2177_1) for up to two years, but the presence of other plasmids varied. Whilst some genes were gained during the follow-up period, no additional known virulence or antibiotic resistance encoding determinants were acquired. This could be a result of the children acquiring a more diverse microbiome once they left the hospital, enabling horizontal gene transfer to or from the ST17 strains. The outbreak and the global ST17 had overlapping evolutionary rates, but unlike the relatively stable genomes within the Stavanger NICU outbreak, ST17 exists globally with many sublineages, harbouring various AMR determinants and plasmid replicons, frequently carrying yersiniabactin and in a few cases also the hypervirulence-associated aerobactin and salmochelin loci. The first detected ST17 was isolated from a pig brain in the UK in 1993 (14), and the earliest ESBL-producing isolate was a blaCTX-M-15-carrying clinical isolate from Canada in 2002 (42). However, the Bayesian phylogeny analysis suggested that ST17 emerged much earlier than this, in the mid-to-late 19th century. ST17 is older, and has a slower evolutionary rate (4.04× 10−7 substitutions/site/year), compared to other global problem MDR lineages that have been estimated, such as ST307 (1.18 × 10−6 substitutions/site/year, MRCA 1994), ST258 (1.03×10−6, 1995) and ST147-KL64 (1.03×10−6, 1994) (39, 43, 44). Since its emergence, recombination has driven diversification of the K and O biosynthesis loci in ST17, which has led to the formation of several sublineages. This is similar to that seen in other MDR clones, including ST258 and ST147 (45). The MDR clones ST258, ST307 and ST147 frequently encode ESBL or carbapenemase genes (5). Unlike these, ST17 was not strongly associated with any specific AMR genes. In fact, less than half of the ST17 genomes carried resistance to third generation cephalosporins and/or carbapenems. On the other hand, 48.3% (145/300) of ST17 genomes were MDR, carrying various AMR genes, including carbapenemases in 25 genomes and ESBL in 127, of which 92 carried blaCTX-M-15. MDR strains were found in many sublineages, but there was limited evidence of persistence of any AMR genes within these, nor within specific countries or collection years, with the possible exception of the blaCTX-M-15-encoding pKp2177_1 plasmid, which was present in two clonal expansions. One of the sublineages, KL25/O5, which emerged in the mid-1940s, contained over half the ST17 genomes and was globally disseminated. Discussion The Stavanger NICU outbreak was located on this sublineage, in a cluster with 10 genomes from Nigeria, Kenya and the UK. They all carried the blaCTX-M-15-harbouring pKp2177_1 plasmid, with an estimated MRCA in 1984. The plasmid was also seen in another sublineage, KL155/OL101, with 17 genomes from Kenya, et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 17 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint Ghana and Nigeria (MRCA 2000). The isolates from the two sublineages in Kenya were from 2009 and 2011, and both were from the same NICU in Kilifi County Hospital (46). In that hospital, ST17 with/without ESBL phenotypes were observed in blood culture isolates almost every year since it first emerged (46), indicating undetected transmission of this strain in the hospital or region, and that the pKp2177_1 plasmid is circulating between ST17 strains. Ghana and Nigeria (MRCA 2000). The isolates from the two sublineages in Kenya were from 2009 and 2011, and both were from the same NICU in Kilifi County Hospital (46). In that hospital, ST17 with/without ESBL phenotypes were observed in blood culture isolates almost every year since it first emerged (46), indicating undetected transmission of this strain in the hospital or region, and that the pKp2177_1 plasmid is circulating between ST17 strains. Given the MRCAs of these pKp2177_1 plasmid-encoding clusters and the larger diversity of the pKp2177_1-encoding genomes in Nigeria, it is probable that the plasmid was acquired by an ancestor in Nigeria in the mid-1980s, where it subsequently diversified and transmitted to the other countries, and that the plasmid later transferred into or between these two ST17 subclades, either in Nigeria or Kenya. In a previous study, only minor fitness costs were associated with carriage of pKp2177_1 in the Stavanger NICU outbreak, indicating that the plasmid was well adapted to its bacterial host (18). Discussion It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint It has been estimated that around half of healthcare-associated infections caused by K. pneumoniae arise from opportunistic colonising strains infecting immunocompromised and vulnerable patients, such as neonates (1, 47, 48). Consistent with this, the ST17 NICU outbreak in Stavanger mainly caused intestinal colonisation and only one systemic infection occurred, after colonisation. In Kilifi, Kenya, only invasive infections with ST17 were reported, however colonisation was not screened for and it is likely that it existed without being detected. The ability of ST17 to cause infection may be related to opportunity, such as host factors and environment, rather than its own intrinsic properties. ST17 isolates have predominantly been reported in studies aiming to characterise Gram-negative bacteria or K. pneumoniae isolated from given populations (often ESBL- or carbapenemase-producing), as minor STs (i.e. incidental findings), and not because they were causing disease outbreaks (Table S5). For example, a study of AMR Gram-negative bacteria in low- and middle-income countries found that K. pneumoniae was the main cause of neonatal sepsis (49). In that study, ST17 accounted for 3.1% (8/258) of the K. pneumoniae isolates. Some outbreak reports do exist, and notably they mainly concern NICU outbreaks, e.g. in Norway (8), Kenya (46) and China (7, 9). Taken together, this indicates that ST17 strains typically colonise a host before opportunistically causing infections, rather than causing clonal expansions of infectious outbreaks within wards, although there are exceptions in highly vulnerable host populations such as those found in NICUs. In conclusion, ST17 is a globally disseminated clone that frequently colonises both humans and non-human sources, and opportunistically causes infections, often in neonates. During within-host colonisation for up to 21 months, ST17 in the Stavanger NICU outbreak remained comparatively stable, with few SNPs and no acquisition of AMR or virulence determinants. Globally, ST17 is highly diverse and harbours a range of AMR genes, virulence loci and plasmids. It contributes to the global burden of MDR infections but is also frequently found without AMR. The ESBL-encoding pKp2177_1 plasmid or yersiniabactin may offer some advantage in clonal expansions of ST17 strains in hospitals, but more data would be needed to assess this. Discussion Further, even though the plasmid carried a complete conjugation machinery, we observed no evidence of plasmid transfer to E. coli in vitro (18) or to other bacterial hosts in vivo during the two years of colonisation (19). Additionally, only one of 3,212 non-ST17 genomes from Norway carried pKp2177_1, further supporting that pKp2177_1 is well adapted to ST17. Additionally, only one of 3,212 non-ST17 genomes from Norway carried pKp2177_1, further supporting that pKp2177_1 is well adapted to ST17. We included two large collections of ST17 from Norway (n=89) and Padua, Italy (n=69) (17). The genomes were spread across the ST17 sublineages and were found in several host species and sample types. This shows that the diversified descendants of the ancestral ST17 co-circulate both within and between ecological niches, even in highly localised geographic regions. Further, it suggests that there have been multiple independent importations of ST17 to these locations, and that the same patterns would be observed for other countries given larger sample sizes, which was also the case for ST307 (39). The NICU outbreaks in the collection indicate that this highly flexible and widely disseminated clone can rapidly colonise neonates in hospital settings. This may be associated with the presence of the blaCTX-M-15 pKp2177_1 plasmid and/or yersiniabactin, although more data would be needed to confirm this: The 2009 NICU outbreaks in Stavanger, Norway and Kilifi, Kenya carried the pKp2177_1 plasmid and ybt 16 on ICEKp12; the 2011 NICU outbreak in Kilifi carried the plasmid but no yersiniabactin locus; and in Padua, Italy, the genomes did not carry the plasmid, but had ybt 15 on ICEKp11. In addition to these large hospital transmissions, some small local clusters (≤7 genomes) occurred. These were mainly reported from studies investigating the movement of strains in close communities, e.g. circulation of ST17 between animals (22) or between animals and people in the community (13), where the same strain was present in animals and humans, indicating that animals may be reservoirs for ST17. et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 18 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. Funding This study was supported by the Western Norway Regional Health Authority (grant number F-12508 to MAKH) and was part of the KLEB-GAP project (project number TMS2019TMT03) funded by the Trond Mohn Foundation (https://mohnfoundation.no/amr- prosjekter/). Author contributions MAKH, JH, KEH, AS and IHL conceptualised the study. SR collected samples and data from the Stavanger NICU outbreak. EB and RJB performed whole-genome sequencing. HK contributed bacterial genomes from animals. MAKH performed the genomic analyses. MAKH wrote the first draft of the manuscript. All authors contributed to data interpretation, reviewed and edited the manuscript, and have read and agreed to the published version of the manuscript. Discussion Given the movement of the pKp2177_1 plasmid, the presence of similar ST17 strains in animals and humans, and the deep-branching global phylogeny, we hypothesise that non-human sources and human colonisation play a crucial role for severe infections in vulnerable patients, such as in preterm neonates. t al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 19 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514664 doi: bioRxiv preprint Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements We wish to thank Olav Natås and Knut Øymar for their support during the NICU outbreak and follow-up study, and Umaer Naseer for his contribution and expertice related to previous experimental plasmid studies. We thank Edward Feil for providing then unpublished genomes from the SpARK One Health consortium project, and others who have made genomes and metadata from their studies publicly available in online repositories. We also wish to thank Benjamin Silvester, Zoe Dyson, Kelly Wyres, Margaret Lam and Ryan Wick for valuable discussions. 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Peirano G, Sang JH, Pitondo-Silva A, Laupland KB, Pitout JD. Molecular epidemiology of extended-spectrum-β-lactamase-producing Klebsiella pneumoniae over a 10 year period in Calgary, Canada. J Antimicrob Chemother. 2012;67(5):1114-1120. doi:10.1093/jac/dks026 43. Bowers JR, Kitchel B, Driebe EM, et al. Genomic Analysis of the Emergence and Rapid Global Dissemination of the Clonal Group 258 Klebsiella pneumoniae Pandemic. PLoS One. 2015;10(7):e0133727. Published 2015 Jul 21. doi:10.1371/journal.pone.0133727 44. Rodrigues C, Desai S, Passet V, Gajjar D, Brisse S. Genomic evolution of the globally disseminated multidrug-resistant Klebsiella pneumoniae clonal group 147. Microb Genom. 2022;8(1):000737. doi:10.1099/mgen.0.000737 45. Wyres KL, Wick RR, Judd LM, et al. Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae. PLoS Genet. 2019;15(4):e1008114. Published 2019 Apr 15. doi:10.1371/journal.pgen.1008114 45. Wyres KL, Wick RR, Judd LM, et al. Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae. PLoS Genet. 2019;15(4):e1008114. Published 2019 Apr 15. doi:10.1371/journal.pgen.1008114 46. Henson SP, Boinett CJ, Ellington MJ, et al. Molecular epidemiology of Klebsiella pneumoniae invasive infections over a decade at Kilifi County Hospital in Kenya. Int J Med Microbiol. 2017;307(7):422-429. doi:10.1016/j.ijmm.2017.07.006 46. Henson SP, Boinett CJ, Ellington MJ, et al. References Molecular epidemiology of Klebsiella pneumoniae invasive infections over a decade at Kilifi County Hospital in Kenya. Int J Med Microbiol. 2017;307(7):422-429. doi:10.1016/j.ijmm.2017.07.006 47. Gorrie CL, Mirceta M, Wick RR, et al. Gastrointestinal Carriage Is a Major Reservoir of Klebsiella pneumoniae Infection in Intensive Care Patients. Clin Infect Dis. 2017;65(2):208-215. doi:10.1093/cid/cix270 47. Gorrie CL, Mirceta M, Wick RR, et al. Gastrointestinal Carriage Is a Major Reservoir of Klebsiella pneumoniae Infection in Intensive Care Patients. Clin Infect Dis. 2017;65(2):208-215. doi:10.1093/cid/cix270 48. Thuy DB, Campbell J, Thuy CT, et al. Colonization with Staphylococcus aureus and Klebsiella pneumoniae causes infections in a Vietnamese intensive care unit. Microb Genom. 2021;7(2):000514. doi:10.1099/mgen.0.000514 48. Thuy DB, Campbell J, Thuy CT, et al. Colonization with Staphylococcus aureus and Klebsiella pneumoniae causes infections in a Vietnamese intensive care unit. Microb Genom. 2021;7(2):000514. doi:10.1099/mgen.0.000514 49. Sands K, Carvalho MJ, Portal E, et al. Characterization of antimicrobial-resistant Gram- negative bacteria that cause neonatal sepsis in seven low- and middle-income countries. Nat Microbiol. 2021;6(4):512-523. doi:10.1038/s41564-021-00870-7 Hetland et al. | Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17 | Page 24
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Improving smoking cessation after myocardial infarction by systematically implementing evidence-based treatment methods: a prospective observational cohort study
European heart journal
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Improving Smoking Cessation After Myocardial Infarction by Systematically Implementing Evidence-Based Treatment Methods: A Retrospective Observational Cohort Study Margret Leosdottir  (  Margret.Leosdottir@med.lu.se ) Margret Leosdottir  (  Margret Lund University Sanne Wärjerstam  Skåne University Hospital Halldora Michelsen  Helsingborgs lasarett Mona Schlyter  Skåne University Hospital Emma Hag  Ryhov Hospital Jönköping John Wallert  Karolinska Institute Matz Larsson  Örebro University Hospital Research Article Keywords: Smoking cessation, myocardial infarction, cardiac rehabilitation, varenicline, nicotine replacement therapy, implementation. Posted Date: September 27th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-823380/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at European Heart Journal on October 1st, 2021. See the published version at https://doi.org/10.1093/eurheartj/ehab724.1409. Version of Record: A version of this preprint was published at European Heart Journal on October 1st, 2021. See the published version at https://doi.org/10.1093/eurheartj/ehab724.1409. Page 1/15 Page 1/15 Abstract We compared the odds of smoking cessation at 2-months post-myocardial infarction (MI) before and after implementing routines, optimizing the use of evidence-based methods for smoking cessation, with start during admission. The following routines were implemented at six hospitals in Sweden: cardiac rehabilitation nurses offering smokers short consultation during admission, optimizing nicotine replacement therapy, increasing prescription of varenicline, and contacting patients by telephone during the 1st week post-discharge. Using logistic regression, odds for smoking cessation at 2-months post-MI before (n = 188) and after (n = 195) routine implementation were compared. Secondary outcomes included adherence to implemented routines. After implementation, a larger proportion of patients (65% vs 54%) were abstinent at 2-months (OR 1.60 [1.04–2.48], p = 0.034). Including only those counselled during admission, 74% were abstinent (OR 2.50 [1.42–4.41], p = 0.002). After implementation patients were more often counselled during admission (50% vs 6%, p < 0.001), prescribed varenicline (23% vs 7%, p < 0.001), and contacted by telephone post-discharge (18% vs 2%, p < 0.001). Being prescribed varenicline had the strongest association with smoking abstinence at 2-months (adjusted OR 4.09 [1.68- 10.00], p = 0.002). In conclusion, readily available methods for aiding smoking cessation can be implemented effectively in routine practice, with beneficial effects on smoking cessation for the high-risk group of smoking MI patients. Introduction Smoking is one of the primary risk factors for coronary heart disease (CHD) 1. For smokers who suffer a myocardial infarction (MI), ceasing to smoke after the MI is the most effective of all preventive measures. Similarly, tobacco abstinence is associated with a lowered risk of reinfarction by 30–40% 2,3 and death by 45% 4. Supporting patients to quit smoking after an MI, and to remain abstinent long-term, are therefore primary goals of cardiac rehabilitation (CR). The Swedish quality registry SWEDEHEART (The Swedish Web-system for Enhancement and Development of Evidence-based care in Heart disease Evaluated According to Recommended Therapies) registers data from an unselected MI-patient population, with nationwide coverage and one-year follow- up data available for > 80% of those < 80 years of age (www.swedeheart.se). According to the most recent SWEDEHEART annual report, the proportion of active smokers among patients with MI in Sweden was 23%, which also has decreased successively over the last decade alongside a general population reduction in daily smoking 5,6. The proportion of smokers who reported being abstinent at 2-months post- MI was 57% and 56% at one-year post-MI, which is somewhat higher than the European average of ~ 45% 6,7. However, no visible progress in smoking abstinence rates among Swedish patients with MI has been observed for more than ten years (Fig. 1) 6. Current CR guidelines advocate professional support, with smoking cessation programmes initiated already during hospital admission 8,9. Drug interventions are also strongly recommended, using nicotine Page 2/15 Page 2/15 replacement therapy (NRT), varenicline and/or bupropion 8,9. Varenicline is the most effective medical treatment to support smoking cessation 10. Recent studies have confirmed its safety not only in patients with stable CHD but also in patients with acute coronary syndromes, including MI 11–13. In the fifth and most recent European Action on Secondary and Primary Prevention by Intervention to Reduce Events (EUROASPIRE) survey, most currently smoking patients with MI were offered personal advice by a healthcare professional to stop smoking but NRT was prescribed to only 7% of smokers and varenicline to 2%, with no meaningful change between EUROASPIRE III and V 7,14. Consequently, there is clearly a considerable room for improvement with respect to medical treatment for active smokers with MI. Study design A retrospective observational multicentre cohort study. Methods The aim of this study was to compare the odds of smoking cessation at 2-months post-MI before versus after implementing a set of pre-specified routines for optimization of evidence-based treatment methods for smoking cessation, with start during admission. Setting and participants As a joint venture for clinical quality improvement, structured routines for early smoking cessation counselling and treatment optimization were implemented at six hospitals in Sweden in 2018-2019. Two are university hospitals. The largest hospital is situated in Malmö, the third largest city in Sweden. Two are of medium size (Örebro and Jönköping) and three are smaller urban or rural hospitals (Trelleborg, Eksjö and Värnamo). The three largest hospitals have acute percutaneous coronary intervention facility, and all have a coronary intensive care unit and an outpatient cardiac rehabilitation (CR) centre. The CR team in Malmö was the first to start implementing the new working routines. The Malmö team thereafter visited the other hospitals to introduce the CR and coronary intensive care personnel to the new routines and support implementation. The participating CR centres report to the SWEDEHEART registry 6 and provide comprehensive CR to post- MI patients. The “usual care” CR programme starts with an individual patient assessment with a nurse at 2-3 weeks after discharge, followed by two registry-based nurse visits at 6-10 weeks (2-month visit) and 11-13 months (1-year visit). Central components of the programme include control and treatment of cardiovascular risk factors, interactive patient education, psychosocial management, smoking cessation counselling (individual or group therapy) if needed, individual patient assessment with a physiotherapist, and supervised centre-based exercise training. The new routines The newly adapted routines included the following: Page 3/15 Page 3/15 A CR nurse should identify all actively smoking MI-patients while still hospitalized for their MI and offer a short consultation with the patient during admission. A new information pamphlet for patients was designed, which included information on the benefits of smoking cessation, tips for avoiding relapse after discharge, plan for follow-up and contact information for the CR centre and generally available anti-smoking aid (telephone and online support). The CR nurse should give advice to the coronary intensive care nurses and physicians on optimal dosage of NRT during admission and encourage prescription at discharge (even though NRT is available over the counter in Sweden), as an attempt to increase adherence. The CR nurse should identify eligible patients for treatment with varenicline, and alert the responsible physician, who in turn would initiate treatment if deemed suitable. The CR nurse should identify eligible patients for treatment with varenicline, and alert the responsible physician, who in turn would initiate treatment if deemed suitable. Varenicline treatment should preferably be started before discharge. Varenicline treatment should preferably be started before discharge. The patient should be contacted by telephone, by the same nurse that consulted the patient during admission, during the first week after discharge. Thereafter, usual care CR commenced. If the patient opted out from varenicline treatment during admission and was still smoking at the telephone contact or 1st CR follow-up visits, treatment should be offered again. If the patient opted out from varenicline treatment during admission and was still smoking at the telephone contact or 1st CR follow-up visits, treatment should be offered again. Finally, centres were encouraged to strive for continuity in nurse-patient care, meaning the same CR nurse should care for the patient from the first contact during admission throughout CR follow-up. As such, the CR nurse was the key professional in implementing the new routines. CR nurses at all participating centres had tobacco counselling qualifications prior to the start of the project. If requested, a brief refreshment course and coaching guide was provided by a tobacco counsellor. At some centres, the routines were predominantly new, and at others only smaller amendments or refreshing of routines were needed (see Supplementary Table 1). Availability of data and materials The SWEDEHEART registry data that support the findings of this study are available from Uppsala Clinical Research Center (UCR) in Sweden but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Third-party data usage is not allowed, irrespective of whether the data contain potentially identifying or sensitive data or not. Instead, given ethical study approval from the Swedish Ethical Review Authority, access to SWEDEHEART data supporting the present findings can be applied for from UCR. Further information can be found on the UCR (www.ucr.uu.se/en/) and Swedish Ethical Review Authority (etikprovningsmyndigheten.se) websites. The study protocol has not been previously published but is available from the corresponding author on reasonable request. Ethics approval and consent to participate The Swedish Ethical Review Authority approved the study (2019-06177). As it involved evaluating outcomes after implementing evidence-based and guideline-recommended clinical routines in daily practice, the need for informed consent was waived. As for SWEDEHEART data, a nurse or physician verbally informs all eligible patients at hospital admission of their registration and opt-out rights. All patients have the right to deny registration and retain the right to be removed from the registry at any time. Data collection and outcomes Smoking cessation at 2-months post-MI for currently smoking patients aged 18-74 years admitted to the coronary intensive care units at the participating hospitals with a type-1 MI, before (reference period) and after (implementation period) routine implementation, were compared. The implementation and reference periods were of equivalent length at each participating hospital. For the three hospitals with the longest implementation periods (8-14 months), the end of the reference period was set one month prior to start of the implementation period. The remaining three hospitals had shorter implementation periods (4 months), and for which the same period the year prior to the implementation year, was used to minimize seasonal bias. The start and length of the study periods per hospital is listed in Supplementary Table 2. Baseline data and smoking status at 2-months was retrieved from the SWEDEHEART registry. Current smoking was self-reported and defined as daily smoking during the last month. Adherence to the pre-set routines was retrieved from patient journal records at each participating hospital. According to the original study protocol, primary outcome was smoking abstinence at the 1-year follow- up visit. Since the Covid-19 pandemic started at the time the last patients included in the implementation Page 4/15 Page 4/15 period had their 2-month follow-up - seriously debilitating planned follow-up routines at all CR centres - primary outcome was changed to smoking abstinence at 2-months post-MI. Secondary outcomes included (i) adherence to the newly implemented routines and (ii) prognostic value of each of the new routines for smoking abstinence at 2-months. Continuity in nurse-patient care was defined as 2/3 or more of same nurse-patient contact (physical visits during admission and follow-up and telephone contacts during follow-up). Sample size calculations In the randomized controlled double-blind Evaluation of Varenicline in Smoking Cessation for Patients Post-Acute Coronary Syndrome (EVITA) trial, patients with acute coronary syndrome and who were smokers at baseline were randomized to receive either varenicline or placebo before discharge 11. In the intervention arm, 58% of patients were smoke-free at first follow-up (12 weeks), compared to 36% in the placebo arm (21.3% difference, p<0.0001). In the current study, at the CR centres implementing the new routines, 59% of current smokers at the time of MI were abstinent at the first SWEDEHEART follow-up (6- 10 weeks) in 2017 15. Based on EVITA results, we concluded that a 15%-point change improvement in smoking cessation rates was a reasonable target for the current study. A somewhat lower improvement rate compared to EVITA was expected because the proportion of smokers abstinent at 2-months is already quite high and not all patients were expected to be identified and thus potentially benefitting from the new routines. With a power of 80% and a two-sided significance level of 0.05 we calculated that we need to include 150 patients in each group. Page 5/15 Page 5/15 Statistical analysis Baseline patient characteristics are presented as medians (q1, q3) and percentages. Adherence to the implemented routines are described using percentages and comparisons made using chi-square test. The primary and secondary outcomes were analyzed using logistic regression. The primary outcome analysis was first done for all current smokers admitted during the predefined implementation period (intention to treat) and thereafter including only current smokers counselled during admission (per protocol). For the secondary outcomes, each routine´s association with smoking cessation at 2-months for patients from the implementation period only were evaluated. Finally, all routines were included as independent variables simultaneously. For all regression analyses, crude models were fitted, whereafter models were adjusted for age, gender, size of center (small, medium, large), length of hospital stay, and whether the patient was discharged during the weekend (Saturday-Sunday), Mondays or during holidays. Mondays were included as patients discharged on Mondays often had returned home before the nurse had a chance to consult the patients. Results Implementation of the new routines started in Malmö in October 2018 with the last hospitals (Eksjö and Värnamo) joining in September 2019 (see Additional file 3). The time frame for patient inclusion ended early January 2020, when 195 current smokers had been admitted for MI at the participating hospitals (Figure 2). During the pre-specified reference period, 188 active smokers were admitted. A somewhat higher number of patients were included in the analysis than indicated by the sample size calculations, to compensate for attrition. Baseline characteristics are shown in Table 1. Median age was 60 (range 33-74) years in both groups. The proportion of men was somewhat higher in the reference group (77% vs 71%, p=0.1). Primary outcome In crude models, being (i) counselled during admission, (ii) contacted during the 1st week after discharge, (iii) prescribed varenicline at discharge or during follow-up, and (iv) nurse-patient care continuity were each positively associated with 2-month smoking cessation. Adjusting for covariates, contact during the 1st week after discharge and prescription of varenicline remained statistically significant. Entering all routines simultaneously into a single model, being prescribed varenicline was the only routine independently associated with smoking cessation at 2- months (adjusted OR 4.09 [1.68-10.00], p=0.002). Being a retrospective study, no harms, or unintended effects on account of the current study were noted or documented. Primary outcome The total number of contacts (by telephone or visits) during the 2-month follow-up period was higher during the intervention period (2.8 vs 2.3 contacts, p<0.001) and more patients had continuity in their nurse- patient care (64% vs 51%, p=0.010). During the implementation period, comparing patients counselled during admission vs those not counselled, 33% vs 14% received a varenicline prescription at discharge or during follow-up (p=0.002), 30% vs 17% received a NRT prescription at discharge or during follow-up (p=0.033), and 36% vs 0% were contacted by telephone during the first week after discharge (p<0.001). Patients not counselled during admission more often had short hospital stays (3 days or less, 51% vs 32%, p=0.001) and were more often discharged during weekends/Mondays/holidays (33% vs 15%, p=0.004). During the implementation period, patients were more often counselled during admission (50% vs 6%, p<0.001), prescribed varenicline at discharge (14% vs 2%, p<0.001) and during follow-up (up until and including the 2-month follow-up visit: 12% vs 5%, p=0.011; at discharge or during follow-up combined: 23% vs 7%, p<0.001). Patients slightly more often received an NRT prescription at discharge (21% vs 14%, p=0.051) but less often during follow-up (5% vs 12%, p=0.017; at discharge or during follow-up combined: 23% vs 16%, p=0.093). During the intervention period, patients were more often contacted by telephone during the first post discharge week, compared to during the reference period (18% vs 2%, p<0.001). The total number of contacts (by telephone or visits) during the 2-month follow-up period was higher during the intervention period (2.8 vs 2.3 contacts, p<0.001) and more patients had continuity in their nurse- patient care (64% vs 51%, p=0.010). During the implementation period, comparing patients counselled during admission vs those not counselled, 33% vs 14% received a varenicline prescription at discharge or during follow-up (p=0.002), 30% vs 17% received a NRT prescription at discharge or during follow-up (p=0.033), and 36% vs 0% were contacted by telephone during the first week after discharge (p<0.001). Patients not counselled during admission more often had short hospital stays (3 days or less, 51% vs 32%, p=0.001) and were more often discharged during weekends/Mondays/holidays (33% vs 15%, p=0.004). Results from the regression analysis are shown in Table 2. Primary outcome In total, 159 out of the 188 (85%) and 179 out of the 195 (92%) active smokers at baseline during the reference and implementation periods, respectively, attended a 2-month follow-up. Including all these patients, irrespective of whether they had been subject to any of the new routines or not (intention to treat), significantly more patients were abstinent from smoking at the 2-month follow-up after the new routines had been implemented: 64% compared to 54% during the reference period (10% difference, crude OR 1.60 [1.04-2.48], p=0.034; adjusted OR 1.60 [1.03-2.52], p=0.037;) (Figure 3). Comparing only patients who were counselled by a CR nurse during admission (50% of the patients during the implementation period – per protocol), 73% were abstinent at the 2-month follow-up, compared to 54% during the reference period (19% difference, crude OR 2.50 [1.42-4.41], p=0.002; adjusted OR 2.68 [1.46-4.91], p=0.002). In total, 159 out of the 188 (85%) and 179 out of the 195 (92%) active smokers at baseline during the reference and implementation periods, respectively, attended a 2-month follow-up. Including all these patients, irrespective of whether they had been subject to any of the new routines or not (intention to treat), significantly more patients were abstinent from smoking at the 2-month follow-up after the new routines had been implemented: 64% compared to 54% during the reference period (10% difference, crude OR 1.60 [1.04-2.48], p=0.034; adjusted OR 1.60 [1.03-2.52], p=0.037;) (Figure 3). Comparing only patients who were counselled by a CR nurse during admission (50% of the patients during the implementation period – per protocol), 73% were abstinent at the 2-month follow-up, compared to 54% during the reference period (19% difference, crude OR 2.50 [1.42-4.41], p=0.002; adjusted OR 2.68 [1.46-4.91], p=0.002). Secondary outcomes Page 6/15 During the implementation period, patients were more often counselled during admission (50% vs 6%, p<0.001), prescribed varenicline at discharge (14% vs 2%, p<0.001) and during follow-up (up until and including the 2-month follow-up visit: 12% vs 5%, p=0.011; at discharge or during follow-up combined: 23% vs 7%, p<0.001). Patients slightly more often received an NRT prescription at discharge (21% vs 14%, p=0.051) but less often during follow-up (5% vs 12%, p=0.017; at discharge or during follow-up combined: 23% vs 16%, p=0.093). During the intervention period, patients were more often contacted by telephone during the first post discharge week, compared to during the reference period (18% vs 2%, p<0.001). Discussion More time and resources for counselling in future studies and clinical praxis is most likely warranted, also since clinical studies show a positive relationship between number of counselling sessions and abstinence rate 18,19. To achieve optimal effect, smoking cessation programmes should preferably include wide-ranging medical information, behavioral aspects, community-oriented methods and suitable pharmaco-therapeutic treatments 8,18,20. Moreover, it has been proposed that telephone counseling and text messaging is effective for outpatients after a MI 21. At all six hospitals participating in the project, various forms of smoking cessation support were already in-place before project initiation. Thus, the implemented model was largely an implementation that strengthened and structured existing support. Focus was on assessing smoking status early, offering all present smokers brief verbal advice complemented with written material, and pharmacological treatment optimization. Implementation was performed without increasing personnel resources – and although it´s difficult to estimate the exact cost for society – there is little doubt that a nurse-managed smoking cessation program after acute MI is cost-effective. In a study by Quist-Paulsen, cost of a nurse-led smoking intervention program compared favourably to other treatment modalities for CHD patients, being approximately 1/25 the cost of statins and angiotensin-converting enzyme inhibitors 16. In another study by Krumholz et al that evaluated the cost-effectiveness of a similar nurse-led intervention, that included three hours of extra counselling time with a nurse, was cost-effective at an estimated $220/year of life saved 17. In the present study we estimate that the extra counselling time (patient identification, p y g (p counselling during admission and more telephone contacts during follow-up) to be approximately 1-2 hours per patient. More time and resources for counselling in future studies and clinical praxis is most likely warranted, also since clinical studies show a positive relationship between number of counselling sessions and abstinence rate 18,19. To achieve optimal effect, smoking cessation programmes should preferably include wide-ranging medical information, behavioral aspects, community-oriented methods and suitable pharmaco-therapeutic treatments 8,18,20. Moreover, it has been proposed that telephone counseling and text messaging is effective for outpatients after a MI 21. Out of all current smokers at baseline less than 10% were concomitantly using wet snuff during the reference and implementation periods, respectively. Use of wet snuff is common in Sweden, with 18% of men and 5% of women using wet snuff daily 5 and using wet snuff as means to quit smoking cigarettes is quite common. Discussion The present study showed that implementing evidence-based, low cost- and readily available methods for smoking cessation can have a significant impact on smoking habits after an MI. Implementation resulted in a higher proportion of abstinent patients at 2-month follow up; 64% vs 54% during the reference period, and including only patients counselled during admission smoking abstinence was 73% vs 54%. To stop smoking following onset of CHD or after undergoing revascularization results in a considerably lower risk of death. Information and support for smoking cessation should be delivered routinely to all patients with a diagnosis of CHD 3,9. This risk reduction is evident across strata by sex, age, index cardiac event, country, and year of study initiation 3. Page 7/15 Page 7/15 Page 7/15 According to SWEDEHEART data, being abstinent from smoking at 2-months after MI is associated with a 50% reduction in 10-year mortality risk (Figure 4) 6. The population of patients as well as the structure of follow-up in SWEDEHEART is practically equivalent to the present study´s control period population. Hence, more abstinent patients 2-months after MI should be associated with a significant improvement regarding both long-term life expectancy. At all six hospitals participating in the project, various forms of smoking cessation support were already in-place before project initiation. Thus, the implemented model was largely an implementation that strengthened and structured existing support. Focus was on assessing smoking status early, offering all present smokers brief verbal advice complemented with written material, and pharmacological treatment optimization. Implementation was performed without increasing personnel resources – and although it´s difficult to estimate the exact cost for society – there is little doubt that a nurse-managed smoking cessation program after acute MI is cost-effective. In a study by Quist-Paulsen, cost of a nurse-led smoking intervention program compared favourably to other treatment modalities for CHD patients, being approximately 1/25 the cost of statins and angiotensin-converting enzyme inhibitors 16. In another study by Krumholz et al that evaluated the cost-effectiveness of a similar nurse-led intervention, that included three hours of extra counselling time with a nurse, was cost-effective at an estimated $220/year of life saved 17. In the present study we estimate that the extra counselling time (patient identification, counselling during admission and more telephone contacts during follow-up) to be approximately 1-2 hours per patient. Discussion The small number of current wet snuff users in our study, however, limited the possibilities for meaningful sub-analyses of this group. As the CR nurses at the participating hospitals only work office hours Monday to Friday, lack of time to identify and subsequently counsel patients during admission was probably the major barrier to offering counselling during admission to more patients, but only 50% of the patients included in the current analysis received counselling. This in turn affected the CR nurses´ possibility to improve treatment during admission and at discharge. Also, if the patients were not identified during admission, they were not contacted by telephone during the first week after discharge. Instead, first contact was at the initial CR assessment visit (2-3 weeks post discharge), as it was during the reference period and which is the general rule in Sweden. The importance of identifying patients early is supported in our data by the large Page 8/15 Page 8/15 difference in odds for smoking cessation when only including patients who received counselling (per protocol, OR 2.50 [1.42-4.41], p=0.002) vs all patients treated during the implementation period (intention to treat, OR 1.60 [1.04-2.48], p=0.034). Given the potential benefits in gained lives and cost-effectiveness of a relatively simple intervention, increasing CR nurse resources to facilitate counselling even on weekends and during odd hours should be a priority. Sweden has a low prevalence of daily smokers around 7% yet the proportion of MI patients that smoke is 23% 5,6. Many European countries have significantly higher prevalence of smoking among MI patients, including Spain (41%), Turkey (42%), Serbia (46%) and Cyprus (57%) 22. Accordingly, simple interventions such as the one evaluated in the current study, in countries with a high proportion of active smokers, could have major effects on the general prognosis post-MI. We encourage healthcare authorities to facilitate implementation of structured multi-component smoking cessation methods, which should be offered to every smoker who suffers an MI. The study has several strengths and limitation. The study had a relatively large sample size (383 patients) and several hospitals, both university hospitals and rural hospitals, were involved. All currently smoking MI patients admitted at the participating hospitals were included, increasing study representativeness. Missing data was also minimal. On the other hand, smoking status was assessed by self-report and no biochemical validation was performed to verify abstinence. Nevertheless, self-reporting is considered accurate in most smoking cessation studies 23. Discussion The duration and intensity of smoking (i.e., n packages smoke per time unit, and years of smoking) is not available via SWEDEHEART or medical records. Some of the implemented routines were already in place at the participating hospitals before start of the project, also during the reference period. This likely reduced the difference between implementation and control periods. Although the association of each component of the new routines was assessed in the secondary analysis collinearity is plausible. The short follow-up of 2 months is a limitation. A longer follow-up of 6 or 12 months would have been preferred, as abstinence at 12 months is a good predictor for long-term abstinence 24. However, as previously stated the Covid19 pandemic made a longer follow-up period unfeasible. Other factors than the implemented routines, such as public health interventions or general attitudes in society might have changed during the periods and influenced the results. However, major confounding effects of this sort seem unlikely since SWEDEHEART data has shown similar results regarding smoking cessation after MI for ten years prior to and during the study period 6. In conclusion, our results from a real-life clinical setting show that implementing evidence-based and readily available methods for smoking cessation can have a significant positive impact on smoking cessation post-MI. The result was evident even though far from all patients were subject to all implemented routines. The most plausible barrier to implementing the new routines was lack of time for the CR nurses. Strengthening CR nurse resources for this important task should thus be a priority. Abbreviations Page 9/15 Author´s contributions M.Le. and M.La. designed the study. M.Le., S.W., M.S., E.H. and M.Le. were involved in implementing the new routines at the participating hospitals. M.Le., S.W., H.Ö.M. and E.H. contributed to data collection. M.Le. and J.W .were responsible for data analysis. M.Le. and M.La. were responsible for drafting the manuscript. All authors reviewed the manuscript at several stages in the writing process. Page 9/15 EUROASPIRE - European Action on Secondary and Primary Prevention by Intervention to Reduce Events - EVITA - Evaluation of Varenicline in - Smoking Cessation for Patients Post-Acute Coronary Syndrome - Kg - Kilograms STEMI - ST-elevation myocardial infarction - SWEDEHEART - The Swedish Web-system for Enhancement and Development of Evidence-based care in Heart disease Evaluated According to Recommended Therapies SWEDEHEART - The Swedish Web-system for Enhancement and Development of Evidence-based care in Heart disease Evaluated According to Recommended Therapies TnT - Troponin T TnT - Troponin T The authors have no competing financial or non-financial interests to declare. he authors have no competing financial or non-financial interests to declare Page 10/15 Additional information Competing interests’ statement. Competing interests’ statement. The authors have no competing financial or non-financial interests to declare. di Page 10/15 The study was funded by the Swedish Heart and Lung Association and Pfizer. The funding sources had no role in the design of the study or writing of the manuscript. References 1. Teo, K. K. et al. Tobacco use and risk of myocardial infarction in 52 countries in the INTERHEART study: a case-control study. Lancet 368, 647-658, doi:10.1016/S0140-6736(06)69249-0 (2006). 2. Chow, C. K. et al. Association of diet, exercise, and smoking modification with risk of early cardiovascular events after acute coronary syndromes. Circulation 121, 750-758, doi:10.1161/CIRCULATIONAHA.109.891523 (2010). 3. Critchley, J. A. & Capewell, S. Mortality risk reduction associated with smoking cessation in patients with coronary heart disease: a systematic review. JAMA 290, 86-97, doi:10.1001/jama.290.1.86 (2003). 4. United States Public Health Service Office of the Surgeon General. Smoking Cessation: A Report of the Surgeon General. Rockville, MD: Office of the Surgeon General, U.S. Department of Health and Human Services (2020). 5. Use of cigarettes, wet snuff and e-cigarettes (Swedish). Public Health Agency of Sweden. 2020. https://www.folkhalsomyndigheten.se/livsvillkor-levnadsvanor/andts/utveckling-inom-andts- anvandning-och-ohalsa/bruk/tobak-och-liknande-produkter/vuxnas-bruk-av-cigaretter-snus-och-e- cigaretter/. Accessed 8 June 2021. 6. Vasko, P. et al. SWEDEHEART Annual report 2020. (Uppsala Clinical Research Center (UCR), Uppsala, Sweden, 2021). 7. Kotseva, K. et al. Lifestyle and impact on cardiovascular risk factor control in coronary patients across 27 countries: Results from the European Society of Cardiology ESC-EORP EUROASPIRE V registry. Eur J Prev Cardiol 26, 824-835, doi:10.1177/2047487318825350 (2019). 7. Kotseva, K. et al. Lifestyle and impact on cardiovascular risk factor control in coronary patients across 27 countries: Results from the European Society of Cardiology ESC-EORP EUROASPIRE V registry. Eur J Prev Cardiol 26, 824-835, doi:10.1177/2047487318825350 (2019). 8. Ambrosetti, M. et al. Secondary prevention through comprehensive cardiovascular rehabilitation: From knowledge to implementation. 2020 update. A position paper from the Secondary Prevention and Rehabilitation Section of the European Association of Preventive Cardiology. Eur J Prev Cardiol, 2047487320913379, doi:10.1177/2047487320913379 (2020). 9. Piepoli, M. F. et al. 2016 European Guidelines on cardiovascular disease prevention in clinical practice: The Sixth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of 10 societies and by invited experts)Developed with the special contribution of the European Association for Cardiovascular Prevention & Rehabilitation (EACPR). Eur Heart J 37, 2315-2381, doi:10.1093/eurheartj/ehw106 (2016). 9. Piepoli, M. F. et al. References 2016 European Guidelines on cardiovascular disease prevention in clinical practice: The Sixth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of 10 societies and by invited experts)Developed with the special contribution of the European Association for Cardiovascular Prevention & Rehabilitation (EACPR). Eur Heart J 37, 2315-2381, doi:10.1093/eurheartj/ehw106 (2016). 10. Cahill, K., Stevens, S., Perera, R. & Lancaster, T. Pharmacological interventions for smoking cessation: an overview and network meta-analysis. Cochrane Database Syst Rev, CD009329, doi:10.1002/14651858.CD009329.pub2 (2013). 10. Cahill, K., Stevens, S., Perera, R. & Lancaster, T. Pharmacological interventions for smoking cessation: an overview and network meta-analysis. Cochrane Database Syst Rev, CD009329, doi:10.1002/14651858.CD009329.pub2 (2013). Page 11/15 11. Eisenberg, M. J. et al. Varenicline for Smoking Cessation in Hospitalized Patients With Acute Coronary Syndrome. Circulation 133, 21-30, doi:10.1161/CIRCULATIONAHA.115.019634 (2016). 12. Sterling, L. H., Windle, S. B., Filion, K. B., Touma, L. & Eisenberg, M. J. Varenicline and Adverse Cardiovascular Events: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. J Am Heart Assoc 5, doi:10.1161/JAHA.115.002849 (2016). 13. Windle, S. B. et al. Smoking abstinence 1 year after acute coronary syndrome: follow-up from a randomized controlled trial of varenicline in patients admitted to hospital. CMAJ 190, E347-E354, doi:10.1503/cmaj.170377 (2018). 14. Prugger, C. et al. Passive smoking and smoking cessation among patients with coronary heart disease across Europe: results from the EUROASPIRE III survey. Eur Heart J 35, 590-598, doi:10.1093/eurheartj/eht538 (2014). 15. Jernberg, T. et al. SWEDEHEART Annual report 2017. (Uppsala Clinical Research Center (UCR), Uppsala, 2018). 16. Quist-Paulsen, P., Lydersen, S., Bakke, P. S. & Gallefoss, F. Cost effectiveness of a smoking cessation program in patients admitted for coronary heart disease. Eur J Cardiovasc Prev Rehabil 13, 274-280, doi:10.1097/01.hjr.0000192742.81231.91 (2006). 17. Krumholz, H. M., Cohen, B. J., Tsevat, J., Pasternak, R. C. & Weinstein, M. C. Cost-effectiveness of a smoking cessation program after myocardial infarction. J Am Coll Cardiol 22, 1697-1702, doi:10.1016/0735-1097(93)90598-u (1993). 18. A clinical practice guideline for treating tobacco use and dependence: A US Public Health Service report. The Tobacco Use and Dependence Clinical Practice Guideline Panel, Staff, and Consortium Representatives. JAMA 283, 3244-3254 (2000). 19. Olenik, A. & Mospan, C. M. Smoking cessation: Identifying readiness to quit and designing a plan. JAAPA 30, 13-19, doi:10.1097/01.JAA.0000520530.80388.2f (2017). 20. West, R., McNeill, A. & Raw, M. Smoking cessation guidelines for health professionals: an update. Health Education Authority. Figures Figure 1 Annual trends in proportion of patients with MI in Sweden, currently smoking at the time of the index event, who were abstinent at one-year follow-up (self-report). Adapted with permission from SWEDEHEART annual reports 2008-2020. References Thorax 55, 987-999, doi:10.1136/thorax.55.12.987 (2000). 21. Kong, J. H. & Ha, Y. [Effects of a smoking cessation program including telephone counseling and text messaging using stages of change for outpatients after a myocardial infarction]. J Korean Acad Nurs 43, 557-567, doi:10.4040/jkan.2013.43.4.557 (2013). 21. Kong, J. H. & Ha, Y. [Effects of a smoking cessation program including telephone counseling and text messaging using stages of change for outpatients after a myocardial infarction]. J Korean Acad Nurs 43, 557-567, doi:10.4040/jkan.2013.43.4.557 (2013). 22. Snaterse, M. et al. Smoking cessation in European patients with coronary heart disease. Results from the EUROASPIRE IV survey: A registry from the European Society of Cardiology. Int J Cardiol 258, 1-6, doi:10.1016/j.ijcard.2018.01.064 (2018). 22. Snaterse, M. et al. Smoking cessation in European patients with coronary heart disease. Results from the EUROASPIRE IV survey: A registry from the European Society of Cardiology. Int J Cardiol 258, 1-6, doi:10.1016/j.ijcard.2018.01.064 (2018). 23. SRNT Subcommittee on Biochemical Verification. Biochemical verification of tobacco use and cessation. Nicotine Tob Res 4, 149-159, doi:10.1080/14622200210123581 (2002). 23. SRNT Subcommittee on Biochemical Verification. Biochemical verification of tobacco use and cessation. Nicotine Tob Res 4, 149-159, doi:10.1080/14622200210123581 (2002). 24. Nohlert, E., Ohrvik, J., Tegelberg, A., Tillgren, P. & Helgason, A. R. Long-term follow-up of a high- and a low-intensity smoking cessation intervention in a dental setting--a randomized trial. BMC Public Health 13, 592, doi:10.1186/1471-2458-13-592 (2013). Page 12/15 Page 12/15 Figures Figure 1 Annual trends in proportion of patients with MI in Sweden, currently smoking at the time of the index event, who were abstinent at one-year follow-up (self-report). Adapted with permission from SWEDEHEART annual reports 2008-2020. Figures Figure 1 Annual trends in proportion of patients with MI in Sweden, currently smoking at the time of the index event, who were abstinent at one-year follow-up (self-report). Adapted with permission from SWEDEHEART annual reports 2008-2020. Figures Figure 1 Annual trends in proportion of patients with MI in Sweden, currently smoking at the time of the index event, who were abstinent at one-year follow-up (self-report). Adapted with permission from SWEDEHEART annual reports 2008-2020. Page 13/15 Page 13/15 Figure 2 Flow chart for patients in the study. MI, myocardial infarction. Flow chart for patients in the study. MI, myocardial infarction. Figure 3 Figure 4 Crude mortality rate in patients who were smokers at the time of the MI, relative to their smoking status at the 2-month follow-up. The graph includes all patients who attended their 2-month follow-up in 2005- 2020 (approximately 100 000 patients). Reprinted with permission from SWEDEHEART. Crude mortality rate in patients who were smokers at the time of the MI, relative to their smoking status at the 2-month follow-up. The graph includes all patients who attended their 2-month follow-up in 2005- 2020 (approximately 100 000 patients). Reprinted with permission from SWEDEHEART. Figure 3 The proportion of abstinent patients and the crude odds of smoking abstinence at 2-month follow-up during the reference period (far left) and implementation period, for all patients (center) and for those counselled during admission only (far right). OR, odds ratio. Page 14/15 Page 14/15 SupplementaryDataset.pdf Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryDataset.pdf Page 15/15
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A cross-sectional study to evaluate factors related to condom use with commercial sexual partners in workers from Ecuadorian companies
BMC public health
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Abstract Background: Unprotected intercourse with sex workers is one of the major risk factors for HIV infection. Consistent condom use is a prerequisite to lower the incidence of HIV. Methods: We assessed the prevalence of condom use and its determinants among company workers engaged with commercial sexual partners in Ecuador. The study was based on a random sample of 115 companies and 1,732 workers stratified by province and working sector and utilized the “Behavioral Surveillance Surveys – Adult questionnaire” developed by Family Health International. Results: Of the 1,561 sexually active workers, 311 (19.9 %) reported having intercourse with sex workers. Among them 25.9 % did not use a condom at the last sexual intercourse. As for condom use frequency over the last 12 months, 29/208 (13.9 %) reported never, 23 (11.1 %) sometimes, 24 (11.5 %) almost every time and 132 (63.5 %) every time. Factors adversely affecting condom use frequency over the last 12 months were female gender (OR = 4.56, 95 % CI: 1.45-14.4), older age (OR = 1.07, 95 % CI: 1.03-1.10), low educational level (OR = 4.69, 95 % CI: 1.95-11.3) and married workers living with spouse (OR = 7.66, 95 % CI: 3.08-19.1). By contrast, factors such as age at first sexual intercourse, job category, HIV transmission and prevention measure knowledge, single workers, previous exposure to HIV intervention programs and having a casual sexual partner were not affecting condom use frequency. When considering condom use during the last sexual intercourse or during the past 12 months with commercial sexual partners, results were similar. Conclusions: Workers with low education, older age, female gender and those married living with hould be targeted for specific educational interventions. the major risk factors for HIV infection [1, 2]. According to the WHO, a noteworthy decrease in HIV incidence worldwide has been associated with increased condom use [2]. UNAIDS reported that the most important factors to control the spread of the infection were sexual behavior changes; these can be most effectively prompted by improving basic knowledge about HIV/AIDS transmission pathways and prevention measures and by stressing the importance of condom use with risky sexual partners [3]. Sexual workers have been less receptive to condom use because clients generally refuse them; therefore they have come to accept unprotected sexual intercourses to avoid losing their clients [4]. © 2015 Cabezas et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: mariadelcarmencg@yahoo.com 1Public Health Department, University of Liège, Liège, Belgium 2Medical School, Pontifical Catholic University, Quito, Ecuador Full list of author information is available at the end of the article Cabezas et al. BMC Public Health (2015) 15:856 DOI 10.1186/s12889-015-2184-5 Abstract Providing easy access to condoms, promoting education and HIV testing programs, however, have evidenced a positive impact by decreasing HIV prevalence in sexual workers [5, 6]. Background Human immune deficiency virus (HIV) infection is a major health problem worldwide. According to the United Nations Programme on HIV/AIDS (UNAIDS) 2014 Fact Sheet, more than 36.9 million people live with HIV around the world and 2.1 million were newly diagnosed with HIV during 2013 [1]. In Latin America, new HIV infections have continued to grow over the years; incidence figures reported for year 2013 are displayed in Fig. 1. Unprotected sexual intercourse with high risk sexual partners, which is still a common practice especially in countries most affected by the epidemic, represents one of * Correspondence: mariadelcarmencg@yahoo.com 1Public Health Department, University of Liège, Liège, Belgium 2Medical School, Pontifical Catholic University, Quito, Ecuador Full list of author information is available at the end of the article Cabezas et al. BMC Public Health (2015) 15:856 Page 2 of 10 Fig. 1 New HIV infections in Latin America, 2013. (Source: UNAIDS estimates, 2013) A meta-analysis related to condom use in Latinos and Latin Americans in the United States found that, along with other factors such as education level, gender, and US/Latin American nationality, condom use after an edu- cational intervention was positively related to the know- ledge regarding prevention and transmission of HIV [7]. A study in Cotonou (Benin) showed that commercial sex intercourse contributed up to 93 % (84-98 %) of all cu- mulative sexual infections. Further, the prevalence decline of HIV in sexual workers may have prevented 62 % (52– 71 %) of new HIV infections among sexual workers be- tween 1993 and 2008 and 33 % (20–46 %) in the overall population. These results emphasize the need of promot- ing safe sexual intercourses with commercial sexual part- ners [1]. Study design The present study is part of a larger three-phase project (2010-2013) to improve knowledge, attitudes and sexual behavior of workers in Ecuador. It was based on a random sample of Ecuadorian companies stratified by provinces (Pichincha, Guayas and Azuay) and working sectors encompassing the greatest number of workers (commerce, manufacturing and real estate). The number of companies recorded in the National Database of Companies (Super- intendencia de Compañías de Ecuador) was 34,917 and the corresponding number of workers was 311,050. In the database, workers are also classified into job categories (executive, administrative, manual labor and other) [10]. In each stratum, companies were selected randomly and in proportion to their actual number in the database. Ecuador is a middle-income country facing health issues related to low education, poverty, gender dis- parities, race discrimination and religious limitations related to condom use. The Ecuadorian manual labor worker population has been among the groups most affected by HIV infection. In addition, only 1 % of HIV cases in Ecuador are not related to sexual activ- ities [8]. A previous study of our group demonstrated a clear lack of knowledge of HIV transmission and prevention measures among a representative sample of Ecuadorian workers [9]. The present work used the same study material to estimate the prevalence of condom use and to identify factors related to condom use in Ecuadorian workers with commercial sexual partners (COSP). Table 1 Distribution of sampled companies and workers according to province and working sector Company Worker N = 115 N = 1732 Number % Number % Province Pichincha 43 37.1 736 42.5 Guayas 67 58.6 900 52.0 Azuay 5 4.3 96 5.5 Working sector Commerce 46 40.0 624 36.0 Manufacturing 13 11.3 590 34.1 Real Estate 56 48.7 518 29.9 Table 1 Distribution of sampled companies and workers according to province and working sector Cabezas et al. BMC Public Health (2015) 15:856 Page 3 of 10 Cabezas et al. BMC Public Health (2015) 15:856 Workers were then selected proportionally to the actual number of workers in each working sector and category of job. This was achieved by selecting groups of approxi- mately 14, 45 and 10 workers from each company of commerce, manufacturing and real estate, working sec- tors, respectively and keeping a fair representation of job categories. Subject characteristics The socio-demographic variables collected for each par- ticipant included; gender, age (years), age at first sexual intercourse (years), education level (primary, secondary, higher), living-in partner status (married living with spouse, married living with other sexual partner, married not living with a sexual partner, single living with a sexual partner, and single not living with a sexual partner), job category and previous exposure to an educational inter- vention program about HIV/AIDS (yes, no) as well as HIV/AIDS transmission and prevention measures know- ledge (poor, good). Data collected on the subject’s sexual activity included sexual relations over the past 12 months (yes, no) and in last sexual intercourse (vaginal or anal sex) with regular, commercial, and casual sexual partners. In each case, the estimated number of partners was noted along with condom use (yes, no) in the last sexual inter- course and frequency of condom use in the last 12 months (every time, almost every time, sometimes, never). Fre- quency of condom use in the last year, “consistent con- dom use” was defined as condom use during every sexual intercourse with a commercial sexual partner. “Inconsist- ent condom use” was defined as sometimes or almost every Questionnaire and study conduct Questionnaire and study conduct The instrument used in this study was the “Behavioral Surveillance Surveys – Adult questionnaire” developed by Family Health International in 2000. All subsequent ver- sions of this questionnaire developed after 2000 maintained the same basic concepts and included modifications mainly based on local additions rather than significantly modifying the established questions. This questionnaire includes 4 questions about sexual history with commercial sexual partners (partners with whom they had sex in exchange for money) and casual sexual partners (non-regular, non- commercial sexual partners, not including current spouse or live-in sexual partners) [11]. Before implementing the survey, all questions were validated for language and com- prehension of questions by a pilot study in Ecuador. Com- mercial sexual partner and casual sexual partner definitions were thoroughly explained to participants. Questionnaires were self-administered with facilitator help and the average duration was 30 to 45 min. A surveyor manual was elaborated with the help of a soci- ologist to avoid bias in the survey process. The study was conducted by 10 surveyors (4 in Pichincha, 4 in Guayas and 2 in Azuay), 2 call centers and 1 study coordinator. The surveyors went through a two-day training session during which they were provided with information regard- ing the questionnaire and the project, and their questions were answered. The call centers staff received one-day training. Validated informed consent forms explaining the study procedures, including protocols for subject confi- dentiality, were signed by each participant. Electronic data records were kept anonymous and secured. Study design The actual selection of workers was as follows: for each company, the number of workers was known but no lists of names were used to guarantee anonymous selection. The company was contacted by mail or phone to request its participation in the study, and a formal invi- tation letter with information about the study was sent to the General Manager and Human Resources Manager. In case of refusal, another company was drawn from the database. When a company agreed to participate, workers were invited to attend a free information meeting on HIV/ AIDS, where the study and its objectives were explained. After signing the informed consent form to confirm their participation in the study, participants were asked to fill in the “Family Health International” questionnaire. All ques- tionnaires were collected by the investigators, kept an- onymous and sorted by job category. In order to maintain the sample size initially planned for the company and the proportions of job categories, questionnaires in excess were discarded randomly. This procedure was applied in all 115 companies, yielding a total number of 1,732 workers. A detailed description of the selection procedure can be found in a previous publication [9]. In total, 115 companies (refusal rate: 12 %) and 1,732 workers consti- tuted the study material (Table 1). time condom use during sexual intercourse with sexual commercial partner. “Complete lack of condom use” was defined as never use condoms during every sexual inter- course with a commercial sexual partner. Ethics The study was approved and supervised by The Institu- tional Review Board (IRB) of Universidad San Francisco de Quito in Ecuador. Statistical analysis A power calculation showed that with a sample size of 1,500 workers, the prevalence of knowledge, sexual be- havior, and educational needs towards HIV/AIDS could be estimated with a statistical precision of at least 3 %. Expecting a 15 % loss of data during the study conduct, a sample of 1,725 was initially required. Description of the study participants p y p p Table 2 displays the characteristics of the 1,732 workers in- cluded in the study. There were 1,128 (65.1 %) males and 604 (34.9 %) females. The mean age of participants was 31.8 ± 9.0 years and 1,374 (82.9 %) participants were under 40 years of age. Most participants (n = 929, 58.6 %) were married. The education level was primary for 10.6 %, sec- ondary for 32.7 % and higher for 56.7 % of the participants. As for the job category, a majority of the participants (50.1 %) was manual workers. Catholic religion was re- ported by 1,396 (80.6 %) out of 1,732 of subjects. Previous exposure to an HIV/AIDS intervention program was high: 1,103 (67.9 %) participants had been exposed and 522 (32.1 %) had not. Poor knowledge about HIV/AIDS trans- mission was found in 49.1 % (95 % CI: 46.6-51.6) of subjects and incorrect knowledge about preventive measures was found in 32.9 % (95 % CI: 30.6-35.2) of respondents [9]. Quantitative variables were summarized by mean and standard deviation (SD), whereas for qualitative variables the number and percentage of subjects in each category were given. The association between each risk factor and condom use was assessed by logistic regression. Odds ratios (OR) and associated 95 % confidence intervals (95 % CI) were adjusted for the stratifying variables (province and Table 2 Characteristics of study participants (n = 1732 Ecuadorian workers) Variable Category Number % Gender Male 1128 65.1 Female 604 34.9 Age (years)a <40 1374 82.9 >40 284 17.1 Education Level Primary 177 10.6 Secondary 546 32.7 Higher 947 56.7 Marital Status Married 929 58.6 Single 655 41.4 Job Category Executive 89 5.1 Administrative 428 24.7 Manual Labor 868 50.1 Other 347 20.1 Previous Exposure to HIV Intervention Programs Yes 1103 67.9 No 522 32.1 HIV Transmission Knowledge (N = 1543) Good 786 50.9 Poor 757 49.1 HIV Prevention Measures Knowledge (N = 1546) Good 1037 67.1 Poor 509 32.9 aMean age ± SD: 31.8 ± 9.0 years Table 2 Characteristics of study participants (n = 1732 Ecuadorian workers) Measurement methods The number of commercial sexual partners and condom use in the last sexual intercourse and during the last 12 months was recorded. Sexual behavior with commer- cial sexual partners was assessed by questions Q501, Q503, Q506 and Q601 of the questionnaire:  Q501: Had sexual intercourse with a commercial sexual partner (partners with whom you had sex in exchange for money) in the last 12 months.  Q503: The last time you had sex with this commercial partner, did you and your partner use a condom?  Q506: With what frequency did you and all of your commercial sexual partner(s) use a condom during the past 12 months?  Q601: Had sexual intercourse with casual sexual partners in the last 12 months. “Casual sexual Page 4 of 10 Cabezas et al. BMC Public Health (2015) 15:856 working sector). All risks factors, potential confounders and stratifying variables were then combined into a multi- variate logistic regression analysis. Likewise, ordinal logistic regression was used to assess the association of frequency of condom use (every time, almost every time, sometimes, never) in the last 12 months with the risk factors and adjusting for stratification. The strength of association be- tween the outcome and the risk factors was assessed by the area under curve (AUC) expressed in percent; the closer AUC to 100 %, the stronger the association. Calculations were always done on the maximum number of data avail- able, missing data were neither replaced nor imputed. Results were considered statistically significant at the 5 % critical level (P < 0.05). All calculations were done using the SAS statistical package (version 9.2 for Windows). partners” (sexual partners that you are not married to and have never lived with and did not pay (“non-regular” partners) – Do not include current spouse(s) or live-in sexual partner). Subjects with missing answers were not included in the statistical analysis. Sexual behavior Among the 1,732 study subjects, 1,561 (90.7 %) reported to be sexually active. In sexually active subjects, only 5 (0.32 %) reported condom cost as expensive. When asked about the required time to get a condom, 816 (52.3 %) reported a mean time of less than 15 min, 64 (4.1 %) less than 1 h; 66 (4.2 %) more than 1 h and 615 (39.4 %) didn’t know the required time. Among the 1,561 sexually active participants, 311 (19.9 %) had sexual relations with COSP. Of them, only 116 (37.3 %) accepted to be tested for HIV. The comparison of subjects with and without a commercial sexual partner is given in Table 3. Having a commercial sexual partner was significantly associated with; male gender, young age at first sexual intercourse, manual labor job category, low educa- tion level, poor HIV transmission and prevention measures knowledge, lack of previous exposure to HIV prevention programs and having a casual sexual partner. No significant difference was found for age and living-in status. Page 5 of 10 Cabezas et al. BMC Public Health (2015) 15:856 Page 5 of 10 Condom use Among the 239 subjects with a commercial sexual part- ner who provided information about condom use during the last sexual intercourse, 177 (74.1 %) indicated they used a condom and 62 (25.9 %) did not. As for the 208 subjects with a commercial sexual partner who reported about the frequency of condom use during the last 12 months, the distribution was as follows: every time (63.5 %), almost every time (11.5 %), sometimes (11.1 %) and never (13.9 %). Sexual behavior Thus, consistent condom use amounted 63.5 % (95 % CI: 57.0-70.0), inconsistent condom use 22.6 % (95 % CI: 16.9-28.3) and complete lack of condom Table 3 Comparison of sexually active subjects with and without commercial sexual partners (COSP) COSP Variable Category N Yes No P-value* Number (%) 1561 311 (19.9) 1250 (80.1) Gender 1561 <.0001 Male 284 (26.5) 788 (73.5) Female 27 (5.5) 462 (94.5) Age (years) Mean ± SD 1561 32.1 ± 9.7 31.8 ± 8.5 0.67 Age at first sexual intercourse (years) Mean ± SD 1561 15.6 ± 2.5 17.4 ± 3.6 <.0001 Level of education 1504 <.0001 Primary 57 (37.3) 96 (62.7) Secondary 124 (24.7) 378 (75.3) Higher 112 (13.2) 737 (86.8) Living in status 1434 0.16 A* 138 (17.6) 647 (82.4) B* 13 (22.8) 44 (77.2) C* 17 (26.6) 47 (73.4) D* 36 (17.5) 170 (82.5) E* 70 (21.7) 252 (78.3) Job category 1561 <.0001 Executive 13 (15.7) 70 (84.3) Administrative 45 (11.7) 340 (88.3) Manual Labor 199 (25.5) 583 (74.5) Other 54 (17.4) 257 (82.6) Previous exposure to HIV Prevention Programs 1469 0.0014 Yes 162 (16.2) 836 (83.8) No 116 (24.6) 355 (75.4) HIV Transmission knowledge 1392 <.0001 Good 96 (13.5) 616 (86.5) Poor 154 (22.7) 526 (77.3) HIV Prevention measure knowledge 1394 0.031 Good 149 (15.7) 800 (84.3) Poor 93 (20.9) 352 (79.1) Casual Sexual Partner 1561 <.0001 Yes 199 (40.7) 290 (59.3) No 112 (10.5) 960 (89.5) A* Married, living with spouse B* Married, living with other sexual partner C* Married, not living with a sexual partner D* Not married, living with a sexual partner E* Not married, not living with a sexual partner *Univariate logistic regression but adjusted for province and working sector Table 3 Comparison of sexually active subjects with and without commercial sexual partners (COSP) Table 3 Comparison of sexually active subjects with and without commercial sexual partners (COSP) COSP of sexually active subjects with and without commercial sexual partners (COSP) about the frequency of condom use during the last 12 months, the distribution was as follows: every time (63.5 %), almost every time (11.5 %), sometimes (11.1 %) and never (13.9 %). Thus, consistent condom use amounted 63.5 % (95 % CI: 57.0-70.0), inconsistent condom use 22.6 % (95 % CI: 16.9-28.3) and complete lack of condom Condom use Among the 239 subjects with a commercial sexual part- ner who provided information about condom use during the last sexual intercourse, 177 (74.1 %) indicated they used a condom and 62 (25.9 %) did not. As for the 208 subjects with a commercial sexual partner who reported Cabezas et al. BMC Public Health (2015) 15:856 Page 6 of 10 Table 4 Relationship between and frequency of condom use during last 12 months and condom use of last sexual intercourse with commercial sexual partners Last sexual intercourse condom use Frequency of condom use last 12 months Every time Almost every time Sometimes Never Total N (%) N (%) N (%) N (%) N (%) Yes 122 (95.3) 18 (94.7) 9 (36.0) 1 (4.0) 150 (76.9) No 6 (4.7) 1 (5.3) 14 (64.0) 24 (96.0) 45 (23.1) Total 128 (100) 19 (100) 23 (100) 25 (100) 195 (100) P < 0.0001 Table 4 Relationship between and frequency of condom use during last 12 months and condom use of last sexual intercourse with i l l t and adjusting for stratification factors, only female gender, low level of education, non-single living-in status and to a lesser extent age were significant. The strength of associ- ation between frequency of condom use and risk factors assessed by the AUC was equal to 66.9 %. use 13.9 (95 % CI: 9.19-18.6). When cross-classifying condom use and frequency of condom use (see Table 4), a highly significant association was found between the two responses (P < 0.0001). Unprotected last sexual intercourse with a commercial sexual partner was significantly associated with; female gender (OR = 7.16, 95 % CI: 2.34-21.9), older age (OR = 1.06, 95 % CI: 1.02-1.10), low level of education (OR = 3.91, 95 % CI: 1.53-9.98), and married workers living spouse (OR = 6.82, 95 % CI: 2.48-18.7) or living with other sexual partner (OR = 16.5, 95 % CI: 3.71-73.4) as com- pared to single living subjects. Factors, such as age at the first sexual intercourse, job category, previous exposure to HIV prevention programs, HIV transmission and preven- tion measures knowledge, and having a casual sexual part- ner were not related to condom use during the last sexual intercourse with commercial sexual partners (see Table 5). Discussion Th The present study focused on sexually active workers who declared having sexual intercourse with COSP. In terms of HIV/AIDS, COSP is one of the highest risk groups of propagating the epidemic. Subjects with a COSP reported low socio-economic conditions, poor HIV transmission and prevention measures knowledge and an early age of sexual initiation. We found that 25.6 % of subjects did not use condom in their last sexual intercourse with a COSP. When looking at the last 12 months, complete lack of condom use was 13.9 % while inconsistent condom use amounted 22.6 %. Interestingly, the present study showed that the risk factors characterizing subjects having sexual intercourse with a COSP were not necessarily the same as those re- lated to condom use in such individuals. Indeed, charac- teristics such as male gender, younger age at first sexual intercourse, manual labor, non-exposure to previous HIV prevention programs, lack of HIV transmission and pre- vention knowledge, and having a casual sexual partner described the typical pattern of “COSP subjects”. By con- trast, factors like female gender (despite the small number of women in the high-risk group), older age, and married but living with another sexual partner, were affecting condom use in a negative way. The only factor common to the two outcomes was low educational level. Condom use When combining all variables into a multivariate logistic regression analysis and adjusting for stratification factors, only female gender, young age, living-in status with spouse or other sexual partner and to a lesser extent low educa- tion remained significant. The strength of association be- tween condom use and risk factors amounted 84.7 %. Frequency of condom use A study of prisoners of both genders in Ecuador in 2008 reported that 35.5 % of subjects had more than Table 5 Factors related to condom use during the last sexual intercourse with a commercial sexual partner Condom use at last sexual intercourse Variable Category N No Yes OR (95 % CI)* P-value* P-value** Number (%) 239 62 (25.9) 177 (74.1) Gender 239 0.0006 0.0005 Male 51 (22.9) 172 (77.1) 1.0 Female 11 (68.7) 5 (31.3) 7.16 (2.34 – 21.9) Age (years) Mean ± SD 223 34.8 ± 10.1 30.1 ± 8.1 1.06 (1.02 – 1.10) 0.0014 0.079 Age of first sexual intercourse (years) Mean ± SD 224 15.8 ± 2.7 15.5 ± 2.4 1.05 (0.93 – 1.18) 0.45 0.63 Level of education 229 0.016 0.051 Primary 16 (38.1) 26 (61.9) 3.91 (1.53 – 9.98) Secondary 25 (24.5) 77 (75.5) 1.71 (0.81 – 3.63) Higher 15 (17.6) 70 (82.4) 1.0 Living in status 212 0.0005 0.042 A* 37 (36.6) 64 (63.4) 6.82 (2.48 – 18.7) B* 7 (58.3) 5 (41.7) 16.5 (3.71 – 73.4) C* 4 (30.8) 9 (69.2) 4.34 (0.96 – 19.6) D* 4 (16.0) 21 (84.0) 2.21 (0.54 – 9.19) E* 5 (8.2) 56 (91.8) 1.0 Job category 239 0.10 0.96 Executive 3 (37.5) 5 (62.5) 1.0 Administrative 5 (14.3) 30 (85.7) 0.25 (0.04 – 1.45) Manual Labor 47 (30.3) 108 (69.7) 0.67 (0.15 – 3.00) Other 7 (17.1) 34 (82.9) 0.31 (0.06 – 1.67) Previous exposure to HIV prevention programs 227 0.57 0.83 Yes 32 (24.1) 101 (75.9) 1.0 No 26 (27.7) 68 (72.3) 0.84 (0.45 – 1.57) HIV transmission knowledge 200 0.86 0.52 Good 18 (22.8) 61 (77.2) 1.0 Poor 30 (24.8) 91 (75.2) 1.06 (0.51 – 2.19) HIV prevention measures knowledge 201 0.18 0.82 Good 29 (22.7) 99 (77.3) 1.0 Poor 23 (31.5) 50 (68.5) 1.55 (0.81 – 2.97) Casual sexual partner 239 0.93 0.75 Yes 38 (25.8) 109 (74.2) 1.03 (0.56 – 1.88) No 24 (26.1) 68 (73.9) 1.0 A* Married, living with spouse B* Married, living with other sexual partner C* Married, not living with a sexual partner D* Not married, living with a sexual partner E* Not married, not living with a sexual partner *Univariate logistic regression but adjusted for province and working sector **Multivariate logistic regression adjusted for province and working sector (N = 187) – AUC = 84.7 % ours but truck drivers are known to be particularly at risk by travelling across large areas all year long [13]. Frequency of condom use When assessing the association between frequency of con- dom use with commercial sexual partners in the last 12 months with the various risk factors, results were simi- lar to those obtained for condom use during the last sex- ual intercourse. Specifically, females were at higher risk than males (OR = 4.56, 95 % CI: 1.45-14.4). The risk of in- consistent condom use increased with age (OR = 1.07, 95 % CI: 1.03-1.10), low educational level (OR = 4.69, 95 % CI: 1.95-11.3) and with living-in status in the sense that, in all subject categories, the odds ratios (range: 4.48 to 7.66) were markedly higher than in the single living-in individ- uals. Factors such as age at first sexual intercourse, job category, HIV transmission and prevention knowledge, previous exposure to HIV prevention programs and hav- ing a casual sexual partner were not related to condom use frequency during the last 12 months with commercial sexual partners (see Table 6). When combining all vari- ables into a multivariate ordinal logistic regression analysis The figures obtained respectively for lack of condom use in the last sexual intercourse and for inconsistent condom use in the last 12 months with commercial sexual partners were comparatively high to those found in other studies. In an Indian study, only 19 % of subjects used a condom during intercourse with their non-regular sexual partners compared with 63.5 % in our study [12]. Another study in Bolivia on truck drivers reported that 31 % of them never used condoms while 43 % used them within the last month. The latter percentages are somewhat worse than Cabezas et al. BMC Public Health (2015) 15:856 Page 7 of 10 Page 7 of 10 ours but truck drivers are known to be particularly at risk by travelling across large areas all year long [13]. In Ecuador, only few studies have focused on the num- ber and type of sexual partners of the sexually active population. A survey of university students in 2007 re- ported that, among the 219 sexually active, 44.8 % of males and 5.4 % females had more than one sexual partner at that time. The study however did not specify the type of sexual partners. Compared with the present study, males from that study had more sexual partners [14]. Frequency of condom use males and 5.4 % females had more than one sexual partner at that time. The study however did not specify the type of sexual partners. Compared with the present study, males from that study had more sexual partners [14]. In Ecuador, only few studies have focused on the num- ber and type of sexual partners of the sexually active population. A survey of university students in 2007 re- ported that, among the 219 sexually active, 44.8 % of A study of prisoners of both genders in Ecuador in 2008 reported that 35.5 % of subjects had more than Cabezas et al. BMC Public Health (2015) 15:856 Page 8 of 10 Page 8 of 10 one sexual partner in the last 12 months, a percentage similar to that of the present study (34.5 %) [14]. The 74.1 % of participants reported condom use with their last COSP. Frequency of condom use Another study in men who have sex with Table 6 Factors related to frequency of condom use with commercial sexual partners in the last 12 months Frequency of condom use Variable Category N Every time Almost every time Sometimes Never OR (95 % CI)* P-value* P-value** Number (%) 208 132 (63.5) 24 (11.5) 23 (11.1) 29 (13.9) Gender 208 0.0095 0.0041 Male 129 (65.5) 24 (12.2) 19 (9.6) 25 (12.7) 1.0 Female 3 (27.3) 0 (0.0) 4 (36.4) 4 (36.4) 4.56 (1.45 – 14.4) Age (years) Mean ± SD 208 29.0 ± 7.8 30.5 ± 8.3 36.1 ± 12.2 36.3 ± 10.5 1.07 (1.03 – 1.10) 0.0001 0.053 Age at first sexual intercourse (years) Mean ± SD 208 15.3 ± 2.4 15.3 ± 1.7 15.9 ± 2.8 15.7 ± 2.6 1.09 (0.96 – 1.22) 0.17 0.15 Level of education 199 0.0009 0.0055 Primary 16 (41.0) 5 (12.8) 7 (18.0) 11 (28.2) 4.69 (1.95 – 11.3) Secondary 58 (67.4) 12 (14.0) 10 (11.6) 6 (7.0) 1.31 (0.63 – 2.72) Higher 56 (75.7) 4 (5.4) 5 (6.8) 9 (12.1) 1.0 Living in Status 182 0.0006 0.021 A* 42 (50.0) 11 (13.1) 14 (16.7) 17 (20.2) 7.66 (3.08 – 19.1) B* 3 (50.0) 2 (33.3) 0 (0.0) 1 (16.7) 4.48 (0.77 – 26.0) C* 9 (56.3) 1 (6.2) 0 (0.0) 6 (37.5) 7.13 (2.07 – 24.6) D* 12 (57.1) 3 (14.3) 2 (9.5) 4 (19.1) 5.81 (1.79 – 18.9) E* 48 (87.2) 3 (5.5) 3 (5.5) 1 (1.8) 1.0 Job category 208 0.44 0.34 Executive 5 (71.4) 0 (0.0) 0 (0.0) 2 (28.6) 1.0 Administrative 22 (75.8) 1 (3.5) 1 (3.5) 5 (17.2) 0.64 (0.11 – 3.70) Manual Labor 80 (59.7) 17 (12.7) 17 (12.7) 20 (14.9) 1.23 (0.25 – 6.06) Other 25 (65.8) 6 (15.8) 5 (13.1) 2 (5.3) 0.85 (0.15 – 4.62) Previous exposure to HIV Prevention Programs 197 0.97 0.86 Yes 72 (63.2) 18 (15.8) 13 (11.4) 11 (9.7) 1.0 No 55 (66.3) 6 (7.2) 7 (8.4) 15 (18.1) 0.99 (0.54 – 1.82) HIV Transmission knowledge 180 0.19 0.47 Good 46 (64.8) 6 (8.5) 8 (11.2) 11 (15.5) 1.0 Poor 74 (67.9) 16 (14.7) 7 (6.4) 12 (11.0) 0.63 (0.32 – 1.27) HIV Prevention knowledge 175 0.50 0.34 Good 74 (67.3) 14 (12.7) 12 (10.9) 10 (9.1) 1.0 Poor 42 (64.6) 5 (7.7) 5 (7.7) 13 (20.0) 1.23 (0.66 – 2.33) Casual Sexual Partner 208 0.15 0.78 Yes 80 (59.3) 20 (14.8) 16 (11.8) 19 (14.1) 1.56 (0.84 – 2.88) No 52 (71.2) 4 (5.5) 7 (9.6) 10 (13.7) 1.0 A* Married, living with spouse B* Married, living with other sexual partner C* Married, not living with a sexual partner D* Not married, living with a sexual partner E* Not married, not living with a sexual partner *Univariate logistic regression but adjusted for province and working sector **Multivariate logistic regression adjusted for province and working sector (N = 165) – AUC = 66.9 % ed to frequency of condom use with commercial sexual partners in the last 12 months 74.1 % of participants reported condom use with their last COSP. Frequency of condom use Another study in men who have sex with men in Ecuador showed that 55 % had unprotected penetrative sex with their last three sexual partners [15]. one sexual partner in the last 12 months, a percentage similar to that of the present study (34.5 %) [14]. The study of commercial sexual partners in Ecuador in 2009 reported that 97.2 % of them used a condom with their last client. By contrast, in the present study, Cabezas et al. BMC Public Health (2015) 15:856 Page 9 of 10 Page 9 of 10 by the HIV epidemic in Ecuador. In this context, wider delivery of effective behavior change strategies to promote protected sexual intercourse with risky sexual partners is central to reversing the global HIV epidemic. Human be- havior will remain critical as new prevention strategies are unlikely to be 100 % effective in preventing transmission [24]. Therefore, the HIV/AIDS epidemic remains a serious health challenge worldwide; in countries where the epi- demic continues to expand as it is the case for Ecuador, there is an urgent need to pursue the current effective strategies to promote safer behaviors [1]. In this per- spective, an educational video specifically dedicated to companies was developed to promote HIV-related know- ledge, attitudes and prevention for company workers [25]. Unfortunately, as alluded to in a recent study [17], corre- lates of efficacy for condom use remain largely unstudied because behavioural change theories presume that self- efficacy predicts condom use. In another study among brewery workers in Nigeria with similar socio-demographical profile as those from the present study reported a consistent condom use in 12 %, which contrasts with the present study that found condom use to be higher [16]. A study on young male workers in Mexico has shown that psychosocial and psychosexual factors may influence the self-efficacy for condom use among male clients with sexual workers [17]. Thus, to efficiently promote consistent condom use, the present findings underline the need to understand the workers vulnerability, their sexual practices, the experi- enced barriers to condom use, and their knowledge and related attitudes towards HIV [18, 19]. This study has a number of limitations. The database of the “Superintendencia de Compañías de Ecuador” does not contain all existing companies in the country; there- fore, informal companies have not been accounted for. Authors' contributions MC i d h d i Authors contributions MC carried out the design, implementation, and acquisition of data in the study and drafted the manuscript. MF has been involved in revising it critically for important intellectual content. AA participated in the design of the study, has been involved in revising it critically for important intellectual content and supervised the statistical analysis. TB has been involved in the implementation of the study and revising it critically for important intellectual content. ND performed the statistical analyses and contributed to data interpretation. DB has been involved in the implementation of the study and revising it critically for important intellectual content. All authors read and approved the final manuscript. In Latin America there are only a few studies targeting the working populations. Studies in healthcare workers with a focus on HIV/AIDS prevention and transmission know- ledge have shown a poor knowledge of HIV transmission/ prevention. However, they did not provide information on factors related to condom use [21–23]. Thus, the results of this study should trigger other initiatives in the domain. Frequency of condom use It is clear that the material and financial resources of the companies and their working sector (commerce, manufac- turing or real estate) influence the educational level of their employees. Therefore, the proportion of workers with a low education level in the sample is most likely underestimated. The study has been limited to three prov- inces; Pichincha, Guayas and Azuay. Although they are the most important provinces, the other provinces may exhibit differences regarding risk factors associated with lack of condom use. Another limitation is the lack of in- formation regarding the gender of COSP and their sexual behavior. The study defined commercial sexual partners with whom they had sex in exchange for money not for other goods. Therefore, it is likely that there were male and female sexual workers. In addition, important vari- ables such urban and rural location, ethnicity and nation- ality and transgender of participants were not registered. Conclusions h The present study demonstrates a major lack of con- dom use among workers having sexual intercourse with commercial sexual partners in Ecuador. Having a com- mercial sexual partner is influenced by factors such as young age at the first sexual intercourse, male gender, low education level, poor HIV transmission and prevention knowledge, lack of previous exposure to HIV prevention programs and having a casual sexual partner. Condom use with COSP was negatively influenced by factors such as old age, female gender, low education level and married status of living with a spouse or other sexual partner. Workers with low education level, older age, female gender and married should be targeted for specific interventions emphasizing the importance of consistent condom use with commercial sexual partners. The study stresses the need for more evaluations and educational intervention programs to reduce HIV/AIDS transmission in companies in Ecuador. To the best of our knowledge, the present study is the first to look at factors associated with condom use (or lack thereof) in occupational settings in Ecuador. It is worth mentioning that in 2003 UNAIDS already pre- pared a document for the business and industrial sectors emphasizing the importance of implementing prevention programs in occupational settings [20]. The WHO recom- mended that companies implement HIV/AIDS policies and prevention programs because it is a favorable invest- ment to avoid discrimination and prevent HIV/AIDS in occupational settings. Competing interest h h d l Competing interest The authors declare to have no competing interests. p g The authors declare to have no competing interests. Maria de Carmen Cabezas, MD, PhD., researcher at the School of Medicine of Pontifical Catholic University, Quito, Ecuador; her expertise is in HIV prevention programs. Cabezas et al. BMC Public Health (2015) 15:856 Marco Fornasini, MD, PhD in epidemiology is a full time professor and researcher at Universidad de las Americas, Quito, Ecuador; his expertise is in chronic diseases and infectious diseases. 16. Sunmola AM. Factors associated with consistent condom use by employees in the brewery industry in Nigeria. J Soc Aspects of HIV/AIDS Res Alliance. 2004;1(1):27–34. Nadia Dardenne, MSc, is a biostatistician at the School of Public Health, University of Liège, Liège, Belgium. 17. Volkmann T, Wagner KD, Strathdee SA, Semple SJ, Ompad DC, Chavarin CV, et al. Correlates of self-efficacy for condom use among male clients of female sex workers in Tijuana, Mexico. Arch Sex Behav. 2014;43(4):719–27. Nadia Dardenne, MSc, is a biostatistician at the School of Public Health, University of Liège, Liège, Belgium. David Barmettler, MSc in Public Health, is coordinator in Accreditation Canada Ecuador. David Barmettler, MSc in Public Health, is coordinator in Accreditation Canada Ecuador. David Barmettler, MSc in Public Health, is coordinator in Accreditation Canada Ecuador. 18. Ojo O, Verbeek J, Rasanen K, Heikkinen J, Isotalo L, Mngoma N, et al. Interventions to reduce risky sexual behaviour for preventing HIV infection in workers in occupational settings (Review). Cochraine Library. 2011;12:1–68. Teresa Borja, PhD in Psychology is a full time professor and researcher at the Universidad San Francisco de Quito, Ecuador; her expertise is in sexology and HIV. Teresa Borja, PhD in Psychology is a full time professor and researcher at the Universidad San Francisco de Quito, Ecuador; her expertise is in sexology and HIV. g y 19. Sunmola A. Sexual practices, barriers to condom use and its consistent use among long distance truck drivers in Nigeria. AIDS Care. 2005;17(2):208–21. Adelin Albert, PhD, is professor emeritus of medical informatics and biostatistics, School of Public Health, University of Liège, Liège, Belgium. 20. UNAIDS. HIV It’s your business, vol. 1. Geneva: UNAIDS; 2003. 21. Cabieses B, Ferrer L, Villarroel L, Cianelli R, Sarah Miner S, Bernales M. HIV/ AIDS knowledge and occupational risk in primary care health workers from Chile. In: Investigación y Educación en Enfermería, vol. 29. Medellin: Investigación y Educación en Enfermería; 2011. p. 212–21. Acknowledgements Thi d This study was supported by a mixed (Belgium–Ecuador) grant (MC) from the Belgian Development Agency (CTB-BTC) and UNAIDS Ecuador. 22. Torales G. Knowledge of Occupational Transmission of HIV and 22. Torales G. Knowledge of Occupational Transmission of HIV and Biosafety Standards IN Healthcare. Revista Nacional (Itagua). 2010;2(2):21–9. 22. Torales G. Knowledge of Occupational Transmission of HIV and Biosafety Standards IN Healthcare. Revista Nacional (Itagua). 2010;2(2):21–9. 23. Guzmán I, Vega E, Rodríguez M. Assessment of knowledge about occupational transmission of HIV/AIDS in nursing staff in a tertiary level hospital. Medwave Revista Biomédica Revisada por Pares. 2008;8(7):559. Author details 1 1Public Health Department, University of Liège, Liège, Belgium. 2Medical School, Pontifical Catholic University, Quito, Ecuador. 3Translational Research Center, Universidad de las Américas (UDLA), Quito, Ecuador. 4Accreditation Canada International, Ottawa, Canada. 5Psychology School, Universidad San Francisco de Quito (USFQ), Cumbaya, Ecuador. School, Pontifical Catholic University, Quito, Ecuador. Translational Research Center, Universidad de las Américas (UDLA), Quito, Ecuador. 4Accreditation Canada International, Ottawa, Canada. 5Psychology School, Universidad San Francisco de Quito (USFQ), Cumbaya, Ecuador. 24. Prevention Working Group: Behavior Change and HIVPrevention: (Re)Considerations for the 21st Century. 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DECLARACION DE COMPROMISO SOBRE VIH-SIDA, UNGASS. Informe del Gobierno del Ecuador Relativo al Período 2006-2007. In: Library W, editor. UNGASS 2008 Ecuador. 1st ed. Geneva: UNAIDS; 2008. p. 1–88. 9. Cabezas M, Fornasini M, Dardenne N, Borja T, Albert A. A cross-sectional study to assess the knowledge about HIV/AIDS transmission and prevention measures in enterprise workers in Ecuador. In: BMC Public Health. vol. 13. London: BMC Public Health; 2013;139. 10. Consulta de Compañias Activas [http://www.supercias.gov.ec/consultas/ inicio.html] 10. Authors' information d b Maria de Carmen Cabezas, MD, PhD., researcher at the School of Medicine of Pontifical Catholic University, Quito, Ecuador; her expertise is in HIV prevention programs. National reports have persistently indicated that manual labor workers have been one of the most affected groups Page 10 of 10 Cabezas et al. BMC Public Health (2015) 15:856 Cabezas et al. BMC Public Health (2015) 15:856 Page 10 of 10 References Consulta de Compañias Activas [http://www.supercias.gov.ec/consultas/ inicio.html] 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: 11. FHI: Behavioural Survellance Survey: Guidelines for Repeated Behavioral Surveys in Populations at Risk of HIV, vol. 1. Arlington: Family Health International; 2000. 12. Sougat R, Bhaskar V, Bhandari S, Kodange C. Impact of a behaviour change intervention model for promotion of condoms amongst young adult males in an occupational setting. Med J Armed Forces India. 2012;68:316–21. • Convenient online submission 13. Sorensen W, Peter B, Anderson R, VILCHES J. Assessment of condom use among Bolivian truck drivers through the lens of social cognitive theory. Health Promot Int. 2007;22(1):37–41. 14. Ministerio de Salud Pública del Ecuador and ONUSIDA. Informe Nacional Sobre los Progresos Realizados en la Aplicación del UNGASS- Ecuador. Enero de 2008- Diciembre de 2009, vol. 1. Geneva: UNAIDS; 2010. 15. Gutiérrez J, Molina D, Morrison K, Samuels F, Bertozzi S. Correlates of condom use in a sample of MSM in Ecuador. BMC Public Health. 2006;6:152. 15. Gutiérrez J, Molina D, Morrison K, Samuels F, Bertozzi S. Correlates of condom use in a sample of MSM in Ecuador. BMC Public Health. 2006;6:152.
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Pathos or logos? How governance legitimacy perception influences individual privacy trade-offs during COVID-19 pandemic
Humanities & social sciences communications
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1 College of Arts and Media, Tongji University, Shanghai, China. 2 School of Media and Communication, Shanghai Jiao Tong University, Shanghai, China. 3 School of Journalism and Communication, Shanghai University, Shanghai, China. ✉email: li.yungeng@sjtu.edu.cn HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2024) 11:451 | https://doi.org/10.1057/s41599-024-02969-8 Introduction T the context of crisis management, such as the COVID-19 pan- demic, governance legitimacy is vital for the effective functioning of governmental systems (Christensen et al., 2016). The voluntary compliance of the public with government rules and guidelines is essential for an optimal response to the pandemic (Martela et al., 2021). During the COVID-19 period, the perceived legitimacy of health authorities plays a significant role in shaping individuals’ belief that following their guidelines is the appropriate and response course of action (Gerber et al., 2021). Therefore, legiti- macy can be seen as a measure of the government’s ability to elicit compliance with public health orders and directives (Khemani, 2020). T he COVID-19 pandemic has underscored the tension between safeguarding individual privacy and ensuring effective public health governance. Governments rely heavily on access to private information for implementing mea- sures against the virus (Li et al., 2022; Ram and Gray, 2020; Yuan, 2021), raising concerns about privacy breaches and cyber violence (Huang, 2020; Lian et al., 2022; Liang, 2020; Parker et al., 2020). The introduction of the General Data Protection Regulation (GDPR) by the European Union defines the inclusion of personal data such as IP address, geolocation data, and biometric data (Vanberg, 2021). Furthermore, the potential for misuse of col- lected information has led to criticisms that invasive technologies like digital contact tracing can encroach upon privacy rights, data protection, and other fundamental civil and human rights (Huang, 2020; Singer and Sang-Hun, 2020). T h In the context of the pandemic, the government’s collection of users’ personal privacy information demands a stronger and more stable foundation of government legitimacy. This is necessitated by the highly sensitive nature of the data involved, the unique circumstances of data collection during the COVID-19 outbreak, and the potential adverse consequences of data breaches. Users’ willingness to provide this information for effective pandemic management hinges on their perception of the governments’ legitimacy. Consequently, this paper proposes the following research hypotheses: g g g Despite these concerns, individuals have sacrificed their per- sonal privacy for the greater cause of pandemic management. This collective willingness to disclose personal information can be attributed to two approaches. One explanatory approach focuses on the functional dimension, which emphasizes the practical benefits individuals derive from using digital contact tracing technologies. Introduction T For instance, studies have identified that positive attitudes towards contact-tracing apps are influenced by per- ceived effectiveness (von Wyl et al., 2021; Zabel et al., 2023) and ease of use (Dowthwaite et al., 2022). Individuals also weigh risks, such as privacy violations, against benefits, such as protection against COVID-19 infection (Hassandoust et al., 2021). Another explanatory approach pertains to the values dimension. Indivi- duals’ trust in the government and health authorities has also played a significant role in their decision to voluntarily disclose personal information. Trust serves as a crucial factor in shaping individuals’ confidence in the government’s ability to handle the crisis. Research has indicated that higher levels of trust in the government, big tech companies, or large hospitality venues increase individuals’ likelihood of using COVID-19 digital con- tact tracing (Dowthwaite et al., 2022). Moreover, individuals perceive contact tracing as contributing to the “greater good” (“Public Attitudes towards COVID-19 Contact Tracing Apps,” 2021) and feel a moral obligation based on social norms (Zabel et al., 2023) and social influence (Hassandoust et al., 2021). H1a: Cognitive legitimacy has a positive influence on privacy disclosure. H1b: Moral legitimacy has a positive influence on privacy disclosure. All for one, or one for all? How altruism mediates the gov- ernance legitimacy and privacy disclosure. Altruistic behavior refers to prosocial acts driven by a genuine intention to benefit others without seeking personal rewards (Feigin et al., 2014). In the context of the COVID-19 pandemic, sharing personal infor- mation for pandemic management, while vital, can risk data leaks and social stigmatization, challenging altruistic motives. As the costs and risks of privacy concessions rise, especially in interac- tions with non-acquaintances, altruistic motivations become less effctive. Therefore, we introduce perceived legitimacy as a vital factor in sustaining altruistic behavior,fostering the belief that adhering to government guidelines during COVID-19 govern- ance is the right course of action (Gerber et al., 2021). While the functional explanatory path sheds light on indivi- duals’ disclosure behaviors during the COVID-19 pandemic from an individual pragmatic perspective, the values explanatory path complements this understanding by examining how external factors influence disclosure behaviors. However, the values pathway primarily focuses on how individuals’ perceptions are influenced by their evaluations and perceptions of others or institutions, particularly in relation to the use of technology tracking techniques, driven by trust in political institutions and social influence from others who have also disclosed information. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-024-02969-8 ARTICLE Introduction T This interpretation overlooks the possibility of individuals eval- uating the governance legitimacy of the event itself and how this evaluation impacts their perceptions and attitudes. Thus, this paper aims to address this gap in the literature by exploring the role of governance legitimacy evaluation in shaping individuals’ perceptions and attitudes towards privacy disclosure during crisis like the COVID-19 pandemic. g During the COVID-19 pandemic, governments globally have sought the public’ support and adherence to various pandemic governance measures. These measures include voluntary com- pliance with self-isolation protocols, participation in nucleic acid testing, and self-health monitoring (Calvo et al., 2020). Rituals and symbols plays a significant role in constructing narratives that contribute to successful pandemic responses (Jing, 2021). For instance, slogans such as “Fighting the pandemic is everyone’s responsibility” effectively communicate the idea that each individual has a duty and obligation to actively engage in pandemic governance. By framing the pandemic response as a symbol of social solidarity, the government reframes individual behaviors, such as providing health information and wearing masks, as acts of altruism rather than self-protection (K. K. Cheng et al., 2022). This framing highlights the importance of collective responsibility and motivates the public to actively participate in pandemic preventive behaviors (Walker, 2022). Pathos or logos? How governance legitimacy perception influences individual privacy trade-offs during COVID-19 pandemic Xiaoxiao Meng 1, Yungeng Li 2✉& Qijun He3 The COVID-19 pandemic has brought attention to the delicate balance between individual privacy concerns and the governance of public health emergencies. Governments are leveraging a wide range of digital methodologies to acquire individual-level data for purposes such as contact tracing, isolation protocols, and surveillance, all aimed at effectively miti- gating the deleterious consequences of the epidemic. However, the surrender of individual health information depends on individuals’ perception of the legitimacy of governance. In this research, our objective is to examine how individuals’ perceptions of the legitimacy of gov- ernance impact their decisions regarding privacy disclosure. This study stands out by dis- secting cognitive and moral legitimacy of governance, uniquely examining their influence on individuals’ altruistic privacy disclosure during a crisis. Unlike previous research, our approach offers a more nuanced understanding of the interplay between governance legitimacy and privacy concessions. From July 15th to August 14th, 2022, amid the 2022 lockdown in Shanghai, China, this study utilizes surveys with established measurement scales, alongside structural equation modeling (SEM), to explore the relationship between individuals’ per- ceptions of government legitimacy in managing the pandemic and their willingness to compromise health information. The study distinguishes between moral legitimacy (pathos) and cognitive legitimacy (logos). The results find that both cognitive and moral legitimacy positively influence altruism, thus enhancing the efficacy of voluntary disclosure of personal health information to government agencies for pandemic governance. However, it is note- worthy that education level moderates the impact of these two dimensions of legitimacy on altruism. This research provides empirical evidence to enhance our understanding of how different dimensions of citizens’ perceptions of governance legitimacy in crisis situations shape their attitudes and behaviors towards privacy trade-offs. 1 1 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2024) 11:451 | https://doi.org/10.1057/s41599-024-02969-8 Literature review h l This notion of moral privacy suggests that privacy is not solely an individual’s possession but can be relinquished and governed in certain circumstances, particularly when it conflicts with the interests of others and the broader public values. Building upon these insights, this paper aims to investigate the following research hypotheses: H2a: Altruism mediates the relationship between cognitive legitimacy and privacy disclosure. H2b: Altruism mediates the relationship between moral legitimacy and privacy disclosure. Divergent altruism? How education moderates the impact of governance legitimacy on altruism. Education level can poten- tially moderate the relationship between legitimacy and altruism for several reasons. Firstly, education equips individuals with critical thinking skills and the ability to evaluate and comprehend the legitimacy of governmental’ actions (Matthes and Marquart, 2013; Cao, 2008). Higher levels of education may enable indivi- duals to make more informed judgments regarding the govern- ment’s initiatives and perceive them as more legitimate. This enhanced perception of legitimacy can positively influence their willingness to disclose personal information. Secondly, education plays a pivotal role in shaping individuals’ moral values and altruistic tendencies (White, 2016). Higher levels of education are often associated with increased empathy, social awareness, and a sense of responsibility towards the well-being of others. Indivi- duals with higher education may possess stronger altruistic motivations and a greater inclination to contribute to the col- lective good. This may extend to sacrificing privacy for the sake of public health, as they understand the potential benefits and importance of sharing personal information for effective crisis management. Measures. The Cronbach alpha coefficient is used to measure the internal consistency of related scales. The Cronbach’s alpha coefficients of privacy disclosure, cognitive legitimacy, moral legitimacy, and altruism are 0.83, 0.90, 0.92, and 0.71, respec- tively. The specific values are explicitly provided in the sub- sequent sections detailing the measurement of each variable. A higher alpha coefficient indicates better consistency among different items in the scale, meaning greater reliability when measuring the same concept across various items. In this paper, when calculating the alpha coefficient, we used scores from all items within a concept measurement scale. Mean (M) repre- sents the average value, while the standard deviation (SD) is a single numerical value that summarizes the variability within a dataset. Furthermore, education empowers individuals with knowledge about the potential benefits and risks associated with privacy disclosure. Literature review h l Literature review Pathos or logos? How perceived governance legitimacy influ- ence privacy disclosure. Legitimacy encompasses the public’s willingness to perceive the actions of authorities as valid and appropriate within a socially constructed systems of norms, values, beliefs, and definitions (Lipset, 1959; Suchman, 1995). In p p In this context, the government plays a role in encouraging individuals to willingly sacrifice their privacy in order to protect public health, thereby promoting a spirit of altruism (Kokkoris and Kamleitner, 2020). By willingly disclosing personal informa- tion to the government for crisis management purposes, HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2024) 11:451 | https://doi.org/10.1057/s41599-024-02969-8 2 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-024-02969-8 ARTICLE Shanghai lockdown due to the rampage of omicron variant of coronavirus from late March to early June 2020, the survey was designed to examine Shanghai citizens’ perception on the gov- ernance legitimacy of privacy tradeoff. From July 15th to August 14th 2022, the research team administered the questionnaire using online survey platform wenjuan.com which is one of the largest online research platforms in China. It has a sample library with over 8 million samples and conducts quality inspection and control on the entire sampling process through algorithms and manual procedures. In order to make the sample as representative as possible of the population distribution in Shanghai, we allo- cated quotas for each district based on the 7th population survey in Shanghai. The quotas for each district were as follows: 22.8% in Pudong District, 46.6% in Puxi (including Minhang, Jingan, Huangpu Putuo, Hongkou, Yangpu, Putuo and Baoshan district), and suburban areas 30.6% (including Jinshan, Chongming, Jiad- ing, Songjiang, and Qingpu districts) (See Fig. 1). These ques- tionnaires underwent a thorough screening process, including attention testing questions, to ensure the reliability of the col- lected data. We distributed 2583 questionnaires through the platform wenjuan.com, and received a total of 1074 responses, resulting in a questionnaire response rate of 41.58%. After excluding 25 incomplete responses, 36 with completion times less than 10 min, determined based on the platform’s assessment of the questionnaire’s average completion time and a pilot survey involving six participants, whose average completion time was also around ten minutes, and 5 with incomplete answers to open- ended questions, we obtained a final set of 1008 valid questionnaires. individuals contribute to the greater good and prioritize public interests over their own privacy concerns. Literature review h l Individuals with higher education levels are more likely to possess a deeper understanding of the broader societal implications and the necessity of privacy concessions during crisis events such as the COVID-19 pandemic. This knowledge can influence their decisions-making process and service as a mediator in the relationship between legitimacy perceptions and privacy disclosure. Educated individuals may engage in a more nuanced evaluation of the legitimacy of government actions, considering the potential trade-offs between privacy and public health benefits. To measure privacy disclosure, we utilized a measurement scale adapted from a previous study (Wang et al., 2021). The scale comprised four items aimed at assessing participants’ attitudes towards sharing their health information with pandemic preven- tion departments. These items focused on the willingness of individuals to disclose information when requested by author- ities.The four items are as belows, “I am willing to provide my health and travel history information when asked by pandemic prevention departments.” “I am willing to provide my personal pandemic prevention information to relevant authorities.” “I am willing to provide more personal pandemic prevention informa- tion for the convenience of travel.” “I am willing to provide any personal pandemic prevention information if it can help in pandemic control.” Participants indicated their level of agreement with these statements using a 5-point Likert-type scale, ranging from 1 (strongly disagree) to 5 (strongly agree). To create a composite variable, the scores of the four items were averaged. The reliability of the scale was deemed satisfactory (α = 0.83), and the majority of respondents expressed agreement with disclosing their information to pandemic prevention departments (M = 4.01, SD = 0.75). In summary, education level can act as a moderating variable between legitimacy and altruism by exerting its influence through multiple mechanisms. It can shape individuals’ perceptions of legitimacy, cultivate their moral values and altruistic tendencies, and equip them with the knowledge to make informed decisions regarding privacy disclosure in the context of pandemic governance. Considering the pivotal role of education, this paper proposes the following research hypotheses: H3a: Education level moderates the impact of cognitive legitimacy on altruism. H3b: Education level moderates the impact of moral legitimacy on altruism. Methods ll Data collection. This study employs a survey methodology to examine the research hypotheses. Against the backdrop of HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2024) 11:451 | https://doi.org/10.1057/s41599-024-02969-8 3 3 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-024-02969-8 Fig. 1 Sampling distribution in Shanghai (N = 1008). Displaying quota sampling distribution in the different areas of Shanghai; the orange represents Pudong district, suburban areas, and Puxi district respectively from light to dark. Fig. 1 Sampling distribution in Shanghai (N = 1008). Displaying quota sampling distribution in the different areas of Shanghai; the orange represents Pudong district, suburban areas, and Puxi district respectively from light to dark. To assess the legitimacy of public information collection by authorities during the lockdown, this study utilized a scale derived from prior research on legitimacy (Alexiou and Wiggins, 2019). Two dimensions of legitimacy were assessed: cognitive legitimacy and moral legitimacy. Cognitive legitimacy reflects individuals’ passive acceptance and perception that an organiza- tion is comprehensible, necessary, or taken for granted. Moral legitimacy pertains to individuals’ active evaluation of the extent to which an organization adheres to social norms and shared values for the betterment of society(Alexiou and Wiggins, 2019). Cognitive legitimacy was measured using a set of six items, “I would like to see institutions collect personal pandemic preven- tion information” .“I believe it is necessary for institutions to collect personal pandemic prevention information.” “ Overall, I believe that institutions collecting personal pandemic prevention information serves the purpose of controlling the pandemic.” “I believe there are valid reasons for institutions to collect personal pandemic prevention information.” “I think institutions collect- ing personal pandemic prevention information is an important component of overall pandemic control measures.” “In general, I believe that the collection of personal information by the pandemic prevention departments serves a purpose in effectively managing the pandemic.” Participants rated their agreement with each statement on a 5-point Likert-type scale, ranging from 1 (totally disagree) to 5 (strongly agree). The average score of the six items was calculated to create a composite variable, indicating satisfactory reliability (α = 0.90). The findings showed that a To assess the legitimacy of public information collection by authorities during the lockdown, this study utilized a scale derived from prior research on legitimacy (Alexiou and Wiggins, 2019). Two dimensions of legitimacy were assessed: cognitive legitimacy and moral legitimacy. Methods ll Cognitive legitimacy reflects individuals’ passive acceptance and perception that an organiza- tion is comprehensible, necessary, or taken for granted. Moral legitimacy pertains to individuals’ active evaluation of the extent to which an organization adheres to social norms and shared values for the betterment of society(Alexiou and Wiggins, 2019). majority of respondents agreed with the notion that the pandemic prevention departments possess cognitive legitimacy in collecting public information for pandemic governance (M = 3.89, SD = 0.79). Moral legitimacy was measured using a set of nine items, “Most of the public would agree with the way authorities collect personal pandemic prevention information.” “The collection of personal information by pandemic prevention departments adheres to acceptable ethical standards in their field.” “The way authorities collect personal pandemic prevention information promotes the public interest.” “The public would endorse the policies and procedures for authorities collecting personal pandemic preven- tion information.” “The policies for authorities to collect personal pandemic prevention information are reasonable.” “Most people would consider the practice of authorities collecting pandemic prevention information as ethical.” “I believe authorities consider public sentiments when collecting personal pandemic prevention information.” “Authorities follow regulations when collecting personal pandemic prevention information.” “If more organiza- tions adopt such policies and procedures, the world would become a better place.” Participants indicated their level of agreement with each statement on a 5-point Likert-type scale, ranging from 1 (totally disagree) to 5 (strongly agree). The scores of the nine items were averaged to create a composite variable, which demonstrated satisfactory reliability (α = 0.92). Methods ll A composite variable was created by averaging the responses to the nine items, and the scale exhibited satisfactory reliability (α = 0.80). Street official WeChat groups/blogs), international social media (e.g., Facebook, Twitter, TikTok), international news media (e.g., New York Times, Reuters), and We media (individuals and vloggers focusing on the pandemic in Shanghai, WeChat groups in public communities, phone calls with friends and family). Participants provided their responses on a 5-point Likert-type scale, ranging from 1 (never used) to 5 (several times a day). A composite variable was created by averaging the responses to the nine items, and the scale exhibited satisfactory reliability (α = 0.80). Altruism was measured in this study using a scale adapted from previous research (Feng et al., 2020; Rushton et al., 1981). Three items were employed to assess participants’ willingness to assist others during the Covid-19 pandemic. Participants were asked to indicate the degree to which they would be inclined to helpt others in specific situations, including “People can seek my assistance when they encounter difficulties in their lives during the pandemic.” “I have a responsibility for the health of others during the pandemic.” “During the pandemic, others can seek help from me if they have health problems.” The responses were recorded on a 5-point Likert-type scale, ranging from 1 (totally disagree) to 5 (strongly agree). A composite variable was created by averaging the scores of the three items. The reliability of the scale was satisfactory (α = 0.71). The results indicated that a majority of respondents expressed a willingness to help others during the pandemic (M = 3.88, SD = 0.71). Statistical analysis. The conceptual model was tested using structural equation modeling in R. The model consists of three stages: (1) the effect of cognitive legitimacy and moral legitimacy on privacy disclosure, (2) the mediating effect of altruism on the relationships of legitimacy and privacy disclosure, and (3) the moderating effect of education level on the relationships of legitimacy and altruism. In this study, education level was examined as a moderating variable. The variable encompassed seven categories, namely primary school, middle school, high school, junior college, undergraduate, master’s degree, and doctoral degree. To facilitate the analysis, the education levels were further grouped into two categories: low education level (primary school, middle school, and high school) and high education level (junior college, undergraduate, master’s and doctoral degrees). Methods ll To simplify the analysis, dummy variables were created to represent each education level category. The demographic characteristics of the sample are presented in Table 1, providing detailed information about the participants in the study. Table 1 illustrates a well-balanced distribution of gender, with women accounting for 50.90% of the sample. The majority of respondents belong to younger age groups, with the largest proportion in the 30–39 age range (39.00%), followed by 25–29 (17.20%), 18–24 (13.2%), 40–49 (12.5%), and 50 and above (18.2%). In terms of education level, 18.5% have completed high school or below, 70.8% have a junior college or bachelor’s degree, and 10.8% hold a master’s or doctoral degree. Regarding annual household income, 50.7% fall below RMB 200,000, 34.6% fall between RMB 200,000 and 500,000, and 14.7% have an income above RMB 500,000. In terms of housing type, the majority of residents own their house (36%), followed by those who rent a room (32.7%), live in a company or school dormitory (18.9%), share a room with others (11.0%), and reside in other types of accommodations (1.3%). Overall, the sample exhibits a relatively even distribution across various demographic categories. g y In addition to the primary variables under investigation (Liu, 2020; Koivula et al., 2023), this study accounted for four control variables in the analysis: gender, age, annual household income level, and media consumption. The respondents’ age was divided into three groups: 18−29 years old, 30–39 years old, and 40 years old and above. Similarly, the annual household income levels were categorized into three groups: below 200,000 RMB, 200,000–500,000 RMB, and above 500,000 RMB. These control variables were included to account for their potential influence on the relationship between the main variables of interest. Control variables were added into each cross-stage path. SEM results for the full model (with the interaction term) showed good fit (χ2 = 0.135, df = 2, χ2/df = 67.49, P > 0.05, CFI = 1.000, TLI = 1.014, RMSEA = 0.00, SRMR = 0.00). The SEM results are presented in Fig. 2. To assess participants’ media consumption during the pandemic lockdown, they were asked to indicate the frequency of their usage of various types of media. Methods ll The results revealed that a majority of respondents agreed that the pandemic prevention departments possess moral legitimacy in collecting y gg Cognitive legitimacy was measured using a set of six items, “I would like to see institutions collect personal pandemic preven- tion information” .“I believe it is necessary for institutions to collect personal pandemic prevention information.” “ Overall, I believe that institutions collecting personal pandemic prevention information serves the purpose of controlling the pandemic.” “I believe there are valid reasons for institutions to collect personal pandemic prevention information.” “I think institutions collect- ing personal pandemic prevention information is an important component of overall pandemic control measures.” “In general, I believe that the collection of personal information by the pandemic prevention departments serves a purpose in effectively managing the pandemic.” Participants rated their agreement with each statement on a 5-point Likert-type scale, ranging from 1 (totally disagree) to 5 (strongly agree). The average score of the six items was calculated to create a composite variable, indicating satisfactory reliability (α = 0.90). The findings showed that a HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2024) 11:451 | https://doi.org/10.1057/s41599-024-02969-8 4 ARTICLE ARTICLE Table 1 Descriptive statistics of variables (N = 1008). Table 1 Descriptive statistics of variables (N = 1008). Variable Names Variable types Frequency(%) Variable Names Variable types Frequency(%) Gender Male 495 (49.0) Districts Puxi 502 (49.8) Female 513 (50.9) Pudong 233 (23.1) Age 18–24 133 (13.2) Annual household income suburban shanghai 273 (27.) 25–39 173 (17.2) below 10,000RMB 1 (0.1) 30–39 393 (39.0) 40–49 126 (12.5) 10,000–50,000RMB 14 (1.4) 50–59 91 (9.0) 60–69 60 (6.0) 50,000–200,000RMB 496 (49.2) above 70 32 (3.2) Education Primary School 10 (1.0) 200,000–500,000RMB 349 (34.6) Middle School 43 (4.3) High School 133 (13.2) 500,000–10,000,000RMB 134 (13.3) Junior college 174 (17.3) Undergraduate 539 (53.5) Over10,000,000RMB 14 (1.4) Graduates 99 (10.8) public information for pandemic governance (M = 3.82, SD = 0.74). public information for pandemic governance (M = 3.82, SD = 0.74). Street official WeChat groups/blogs), international social media (e.g., Facebook, Twitter, TikTok), international news media (e.g., New York Times, Reuters), and We media (individuals and vloggers focusing on the pandemic in Shanghai, WeChat groups in public communities, phone calls with friends and family). Participants provided their responses on a 5-point Likert-type scale, ranging from 1 (never used) to 5 (several times a day). Discussion On May 5, 2023, WHO Director-General Tedros Adhanom Ghebreyesus announced that COVID-19 no longer constituted a Public Health Emergency of International Concern (PHEIC) (Burki, 2023). In the wake of this announcement, a global nar- rative unfolds—the return of individuals to their ordinary lives is both a relief and a prelude to a haunting uncertainty. It is against this backdrop of mounting global ambiguities that our journey into understanding the intricacies of personal information dis- closure unfolds. This study delves into the intricate mechanisms shaping individuals’ willingness to revealing personal informa- tion, weaving this puzzle into the core narrative of governance legitimacy. This legitimacy, intricately tied to perceptions of authority, stands as a linchpin in our ability to navigate and respond effectively to potential future crises. While our investi- gation is anchored in the nuanced data collected during the unique circumstances of the Shanghai lockdown, it unveils a tapestry of insights that extends beyond the immediate context: Based on the Fig. 3, the horizontal axis represents the strength of cognitive legitimacy, and the vertical axis represents altruism. We observe a trend that at higher education levels, the relationship between high cognitive legitimacy and altruism appears to be stronger compared to the link between low cognitive legitimacy and altruism. This observation suggests a potential association between cognitive legitimacy and altruistic behaviors among individuals with higher education. However, further research is needed to confirm and better understand this relationship. Fig. 2 SEM result with interaction effects of education and legitimacy. Note: *p < 0.05, **p < 0.01, ***p < 0.001. Firstly, governance legitimacy emerges as a key determinant influencing individuals’ inclination to share personal information with the government for the effective management of a pandemic. Notably, compared to the cognitive legitimacy, moral legitimacy takes precedence to influence the willingness to relinquish priv- acy, as evidenced by its larger path coefficient, particularly in nations like China where traditional moral and collective values hold significant weight. In such contexts, external perceptions and societal pressures shaped by moral norms and social order overshadow the influence of cognitive legitimacy. This Fig. 2 SEM result with interaction effects of education and legitimacy. Note: *p < 0.05, **p < 0.01, ***p < 0.001. Table 2 Results of SEM. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-024-02969-8 support to the first group of hypotheses we proposed. In the mediating effect model, both cognitive legitimacy (β = 0.16, SE = 0.07, P < 0.001) and moral legitimacy (β = 0.18, SE = 0.06, P < 0.001) had a positive influence on altruism; altruism had a positive influence on privacy disclosure (β = 0.08, SE = 0.02, P < 0.01); indirect total is significant (β = 0.03, SE = 0.01, P < 0.001). Thus, the second group of hypotheses were supported. Specifically, altruism serves as a mediator in the relationship between cognitive legitimacy and privacy disclosure, as well as in the relationship between moral legitimacy and privacy disclosure. In the moderating effect model, high education level negatively moderates the relationship between cognitive legitimacy and altruism (β = 0.44, SE = 0.15, P < 0.01); however, high education level positively moderates the relationship between moral legitimacy and altruism (β = −0.43, SE = 0.15, P < 0.01). Hence, the third group of hypotheses were supported. Based on the Fig. 4, the horizontal axis represents the strength of moral legitimacy, and the vertical axis represents altruism. In Fig. 3, there seems to be a tendency that among individuals with lower education levels, the impact of low moral legitimacy on altruism is more pronounced than the influence of high moral legitimacy on altruism. This observation hints at a possible connection between moral legitimacy and altruistic tendencies among those with lower educational attainment. Nevertheless, it’s essential to note that these are preliminary findings, and additional research is essential to substantiate these observations. Methods ll The media categories included central media (e.g., People’s Daily, CCTV, Xinhua News Agency), national commercial media (e.g., The Caixin, The Paper), Shanghai official media (e.g., Shanghai Post, Dragon TV), Shanghai official media at the district level (such as Shanghai Table 2 shows the results of the three stages. In the main effect model, results showed that both cognitive legitimacy (β = 0.33, SE = 0.03, P < 0.001) and moral legitimacy (β = 0.48, SE = 0.04, P < 0.001) had a positive influence on privacy disclosure, lending HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2024) 11:451 | https://doi.org/10.1057/s41599-024-02969-8 5 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-024-02969-8 Fig. 3 Interaction plot. Interaction between cognitive legitimacy and education on altruism. Fig. 4 Interaction plot. Interaction between moral legitimacy and education on altruism. Fig. 3 Interaction plot. Interaction between cognitive legitimacy and education on altruism. Fig. 3 Interaction plot. Interaction between cognitive legitimacy and education on altruism. Fig. 4 Interaction plot. Interaction between moral legitimacy and education on altruism. Fig. 4 Interaction plot. Interaction between moral legitimacy and education on altruism. Fig. 4 Interaction plot. Interaction between moral legitimacy and education on altruism. of governance alone. This line of inquiry holds promise for unraveling the intricate dynamics of the cultural roots of gov- ernance modes and legitimacy building in crisis management. observation provides indirect insights into the immediate impact of moral imperatives, such as the collective responsibility to wear masks and the importance of mutual assistance in crisis man- agement. Furthermore, this discovery stimulates further exploration, encouraging researchers to extend these conclusions to societies where collectivist moral perspectives remain influ- ential. It prompts an investigation into the intricate causal rela- tionship between moral legitimacy of governance and the willingness to disclose private information, shedding light on the universal applicability of these findings in societies characterized by obvious moral values. g y g g Thirdly, among individuals with higher levels of education, cognitive legitimacy emerges as a more powerful factor in fos- tering altruistic behavior. This phenomenon can be attributed to the enriching effect of higher education on cognitive abilities, critical thinking, and a deeper understanding of modern legal systems and regulations. Those with advanced education are more likely to possess the knowledge and skills necessary to comprehend the significance and benefits of adhering to legal rather than moral norms, thereby influencing their altruistic actions. Hence, cognitive legitimacy has a more significant impact in promoting altruism among individuals with higher education, who show increased receptivity to logical and cognitive justifi- cations for governance legitimacy. Conversely, moral legitimacy has a stronger influence on promoting altruism among indivi- duals with lower levels of education. This can be explained by the powerful influence of traditional and moral values among people who are less educated and more heavily on moral principles and societal expectations when making decisions. Secondly, altruism serves as a mediator in the intricate inter- play between legitimacy and individuals’ willingness to disclose personal information. Legitimacy acts as a catalyst that promotes altruistic behavior, thereby fostering the inclination to disclose personal information. Discussion N = 1008 Model1(Main effect) Model2 (Mediating effect) Model3(Moderating effect) Privacy disclosure Altruism Privacy disclosure Altruism Privacy disclosure Estimate (SE) Estimate (SE) Estimate (SE) Altruism 0.08 (0.02)*** 0.08 (0.02)*** Cognitive legitimacy 0.33 (0.03)*** 0.16 (0.07)** 0.32 (0.05)*** 0.12 (0.07)* 0.32 (0.05)*** Moral legitimacy 0.48 (0.04)*** 0.18 (0.06)** 0.47 (0.05)*** 0.22 (0.07)** 0.47 (0.05)*** Income1 −0.00 (0.04) −0.19 (0.06)*** 0.01 (0.04) −0.20 (0.07)** 0.01 (0.04) Income2 0.02 (0.04) −0.03 (0.06) 0.03 (0.04) −0.03 (0.07) 0.03 (0.04) Age1 0.33 (0.03) 0.04 (0.06) 0.03 (0.04) 0.05 (0.06) 0.03 (0.04) Age2 −0.00 (0.03) −0.02 (0.05) −0.00 (0.03) −0.01 (0.05) −0.01 (0.03) Gender (Female) −0.00 (0.03) −0.02 (0.04) −0.04 (0.03) −0.02 (0.04) −0.04 (0.03) Media consumption 0.00 (0.02) 0.16 (0.03)*** −0.01 (0.02) 0.16 (0.03)*** −0.01 (0.02) Education*Cognitive legitimacy 0.44 (0.15)*** Education*Moral legitimacy −0.43 (0.15)*** Adjusted R-squared 0.67 0.23 0.68 0.23 0.68 Indirect difference −0.00 (0.01) Indirect total 0.03 (0.01)*** Moderated mediation index (cognitive legitimacy) −0.03 (0.02)* Moderated mediation index (moral legitimacy) 0.03 (0.02)* Note: *p < 0 05 **p < 0 01 ***p < 0 001 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2024) 11:451 | https://doi.org/10.1057/s41599-024-02969-8 6 ARTICLE Conclusion I Dowthwaite L, Wagner HG, Babbage CM et al. (2022) The relationship between trust and attitudes towards the COVID-19 digital contact-tracing app in the UK. PloS One 17(10):e0276661. https://doi.org/10.1371/journal.pone. 0276661 In summary, this paper examined the intricate interplay of gov- ernance legitimacy, cultural dynamics, and educational influences on individuals’ responses to privacy trade-offs in times of crises. The discerned prominence of moral legitimacy in culturally grounded societies offers crucial insights into the governing fac- tors steering altruistic behavior. Furthermore, the identified lin- kages between governance legitimacy perceptions, privacy disclosure, and educational levels underscore the impact of modernization on crisis management efficacy. As we contemplate the trajectory from micro-level privacy protection to governance- oriented practices, this research contributes substantively to the- oretical discussions, introducing the concept of “governance- oriented privacy” and enriching our understanding of its role in the realm of global crises. Fahey RA, Hino A (2020) COVID-19, digital privacy, and the social limits on data- focused public health responses. Int J Inf Manag 55:102181. https://doi.org/ 10.1016/j.ijinfomgt.2020.102181 Feigin S, Owens G, Goodyear-Smith F (2014) Theories of human altruism: a systematic review. J. Psychiatry Brain Funct 1(1):1–9. https://doi.org/10.7243/ 2055-3447-1-5 Feng Y, Dong D, Zong M et al. (2020) When altruists cannot help: the influence of altruism on mental health during COVID-19 pandemic. Global Health 16(61):1–8. https://doi.org/10.1186/s12992-020-00587-y Gerber MM, Cuadrado C, Figueiredo A et al. (2021) Taking care of each other: how can we increase compliance with personal protective measures during the COVID‐19 pandemic in Chile? Political Psychol. 42(5):863–880. https://doi. org/10.1111/pops.12770 Hassandoust F, Akhlaghpour S, Johnston AC (2021) Individuals’ privacy concerns and adoption of contact tracing mobile applications in a pandemic: a situa- tional privacy calculus perspective. J Am Med Inform Assoc 28(3):463–471. https://doi.org/10.1093/jamia/ocaa240 g The present study has several limitations that should be con- sidered. First, the generalizability of the findings may be limited as the study focused on a specific case of a severe lockdown in Shanghai, China in 2022. The results may not fully apply to other regions or contexts that experienced different pandemic control measures or have different cultural backgrounds. Replicating the study in diverse settings would enhance the generalizability of the findings. Second, the reliance on self-reported data introduces potential biases. Participants’ willingness to disclose information and their beliefs about legitimacy and altruism were measured through self-report measures, which can be influenced by social desirability bias or memory recall errors. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-024-02969-8 Notably, the influence of moral legitimacy on cultivating altruistic behavior surpasses that of cognitive legitimacy. Consequently, individuals are more motivated to divulge personal information for the collective good of effective pandemic governance. Furthermore, this research finding sug- gests that altruistic strategies in governance legitimacy building might wield significant influence over individuals’ willingness and behavioral choices in disclosing privacy, superseding the efficacy The cumulative research findings bring to the forefront several theoretical considerations and avenues for future research. Firstly, HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2024) 11:451 | https://doi.org/10.1057/s41599-024-02969-8 7 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-024-02969-8 ARTICLE the study limits the ability to establish causal relationships or examine changes over time. The data were collected at a specific point in time, providing only a snapshot of participants’ attitudes and behaviors during the pandemic. Conducting longitudinal studies that capture data at multiple time points would provide a more comprehensive understanding of the dynamics between legitimacy, altruism, and other variables. Addressing these lim- itations in future research would further advance our under- standing of the underlying mechanisms in this area. in societies where traditional values heavily influence behavior, the research highlights that the moral legitimacy of governance may be more significant than cognitive legitimacy in shaping decisions related to pandemic governance and privacy trade-offs (Walker, 2022). This insight calls for further exploration into how public perceptions of governance legitimacy and privacy trade- offs differ across diverse cultural contexts. Future research can provide a deeper understanding of how cultural influences interact with governance structures to shape individual decision- making. Secondly, the robust connection found between per- ceptions of governance legitimacy, privacy disclosure, and edu- cational levels emphasizes that the level of modernization within a region significantly molds crisis management efficacy (Neblo and Wallace, 2021). Subsequent research could integrate macro- level modernization indicators into the examination of micro- level governance legitimacy and privacy data governance. This holistic approach offers the potential for a multi-dimensional understanding of the mechanisms underlying global crisis man- agement, as exemplified by responses to the COVID-19 pan- demic. The third point extends existing privacy management theories. Traditional theories, such as communication privacy management theory, privacy protection motivation theory, and privacy paradox theory, have predominantly focused on micro- level discussions of privacy disclosure and management (Petronio, 2002; Boerman et al., 2021; Kokolakis, 2017). 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Data availability The data analyzed during the current study could be available from the corresponding author on reasonable request. Received: 7 August 2023; Accepted: 19 March 2024; Received: 7 August 2023; Accepted: 19 March 2024; Acknowledgements This study was supported by the 2023 Shanghai Philosophy and Social Science Grant for Young Scholars (No. 2023EXW004), Young Scholar Grant in Humanities and Social Science from the Ministry of Education of People’s Republic of China (No. This study was supported by the 2023 Shanghai Philosophy and Social Science Grant for Young Scholars (No. 2023EXW004), Young Scholar Grant in Humanities and Social Science from the Ministry of Education of People’s Republic of China (No. g Lian Y, Zhou Y, Lian X et al. (2022) Cyber violence caused by the disclosure of route information during the COVID-19 pandemic. 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(2020) Ethics of instantaneous contact tracing using mobile phone apps in the control of the COVID-19 pandemic. J Med Ethics 46(7):427–431. https://doi.org/10.1136/medethics-2020-106314 Author Contributions XM designed the research, collected and analyze data, wrote and revised the manuscript, and provided funding. YL designed the research, collected data, wrote and revised the manuscript, and provided funding. QH designed the research, collected data, and revised the manuscript. All authors substantially contributed to the completion of the research. Liu PL (2020) COVID-19 information seeking on digital media and preventive beha- viors: the mediation role of worry. Cyberpsychol Behav Soc Netw 23(10):677–682 Martela F, Hankonen N, Ryan RM et al. (2021) Motivating voluntary compliance to behavioural restrictions: self-determination theory–based checklist of principles for COVID-19 and other emergency communications. 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Int J Hum Rights 25(1):52–78. https://doi.org/10. 1080/13642987.2020.1789109 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/. Walker KK (2022) Shining the light on altruism and its potential impact for behavior change strategies related to the COVID-19 pandemic and beyond. Health Commun 37(7):919–921. https://doi.org/10.1080/10410236.2021. 1872157 Wang Y, Zhou Y, Liao Z (2021) Health privacy information self-disclosure in online health community. Front Public Health 8(Feb):1–15. https://doi.org/ 10.3389/fpubh.2020.602792 White J (2016) Moral education and education in altruism: two replies to Michael Hand. J Philos Educ 50(3):448–460 von Wyl V, Höglinger M, Sieber C et al. (2021) Drivers of acceptance of COVID-19 proximity tracing apps in switzerland: panel survey analysis. JMIR Public Health Surveill 7(1):e25701. Additional information https://doi.org/10.2196/25701 © The Author(s) 2024 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2024) 11:451 | https://doi.org/10.1057/s41599-024-02969-8 9 9
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In vivo efficacy of artesunate–amodiaquine and artemether–lumefantrine for the treatment of uncomplicated falciparum malaria: an open-randomized, non-inferiority clinical trial in South Kivu, Democratic Republic of Congo
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de Wit et al. Malar J (2016) 15:455 DOI 10.1186/s12936-016-1444-x de Wit et al. Malar J (2016) 15:455 DOI 10.1186/s12936-016-1444-x Malaria Journal Open Access RESEARCH © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. In vivo efficacy of artesunate– amodiaquine and artemether–lumefantrine for the treatment of uncomplicated falciparum malaria: an open‑randomized, non‑inferiority clinical trial in South Kivu, Democratic Republic of Congo Marit de Wit1*  , Anna L. Funk1, Krystel Moussally1, David Aksanti Nkuba1, Ruby Siddiqui2, Karla Bil1, Erwan Piriou1, Aldert Bart3, Patrick Bahizi Bizoza4 and Teun Bousema5,6 Abstract Background:  Between 2009 and 2012, malaria cases diagnosed in a Médecins sans Frontières programme have increased fivefold in Baraka, South Kivu, Democratic Republic of the Congo (DRC). The cause of this increase is not known. An in vivo drug efficacy trial was conducted to determine whether increased treatment failure rates may have contributed to the apparent increase in malaria diagnoses. Methods:  In an open-randomized non-inferiority trial, the efficacy of artesunate–amodiaquine (ASAQ) was com- pared to artemether–lumefantrine (AL) for the treatment of uncomplicated falciparum malaria in 288 children aged 6–59 months. Included children had directly supervised treatment and were then followed for 42 days with weekly clinical and parasitological evaluations. The blood samples of children found to have recurring parasitaemia within 42 days were checked by PCR to confirm whether or not this was due to reinfection or recrudescence (i.e. treatment failure). Results:  Out of 873 children screened, 585 (67 %) were excluded and 288 children were randomized to either ASAQ or AL. At day 42 of follow up, the treatment efficacy of ASAQ was 78 % before and 95 % after PCR correction for re- infections. In the AL-arm, treatment efficacy was 84 % before and 99.0 % after PCR correction. Treatment efficacy after PCR correction was within the margin of non-inferiority as set for this study. Fewer children in the AL arm reported adverse reactions. Conclusions:  ASAQ is still effective as a treatment for uncomplicated malaria in Baraka, South Kivu, DRC. In this region, AL may have higher efficacy but additional trials are required to draw this conclusion with confidence. The high re-infection rate in South-Kivu indicates intense malaria transmission. Trial registration NCT02741024 Keywords:  Uncomplicated malaria, Artesunate–amodiaquine, Artemether–lumefantrine, Child, Treatment efficacy, DR Congo, Plasmodium falciparum, DRC *Correspondence: marit.de.wit@amsterdam.msf.org 1 Médecins sans Frontières (MSF), Plantage Middenlaan 14, 1018 DD Amsterdam, The Netherlands Full list of author information is available at the end of the article de Wit et al. Malar J (2016) 15:455 Page 2 of 10 Backgroundh Malaria is holo-endemic in most of DRC, with seasonal fluctuations in transmission intensity in the east and south of the country. Plasmodium falciparum is the predominant species causing malaria in DRC [5]. Since 2003, MSF has introduced artemisinin-based combination therapy (ACT) in all programmes worldwide. Artesunate–amodiaquine (ASAQ) has been the first-line treatment for uncomplicated malaria in DRC since 2005. Artemether–lumefantrine (AL) is the second line treatment for uncomplicated malaria [6, 7]. Targeted distribution of long-lasting insecticide-treated bed nets (LLIN) to pregnant women and children hospital- ized for severe malaria has been ongoing since 2003 in the MSF Baraka programme. The last mass LLIN distribution in this health zone was carried out in 2012. g The Democratic Republic of Congo (DRC) is one of the five countries with the highest malaria burden in the world [1]. The eastern part of DRC is suffering from armed conflict and internal political instability. The conflict has caused massive suffering for civilians, with estimates of millions of deaths, directly or indirectly, as a result of the fighting [2, 3]. In this humanitarian cri- sis there are acute health needs, with limited access to humanitarian assistance and violations of basic rights and freedoms. A survey conducted in 2014 in South Kivu indicated a crude mortality rate (CMR) of 2.80 per 10,000 per day [95 % CI (2.40–3.28)] and an under-five mortality rate (<5MR) of 5.58 per 10,000 per day [95 % CI (4.60– 6.76)]. Both of these mortality rates are more than two times above the emergency threshold [4]. Since 2009, MSF has seen an apparent rise in malaria incidence in most programmes in DRC. The National Malaria Control Programme (Programme National de Lutte contre le Paludisme, PNLP) has also observed an increase in malaria cases. In the Baraka programme, without any significant changes to the programme activi- ties, the number of parasitologically confirmed uncompli- cated malaria cases has risen from 7457 in 2009 to 44,317 cases in 2012 (Fig. 2). In order to investigate whether the Médecins sans Frontières–Operational Centre Amster- dam (MSF-OCA or MSF) has been working in the provinces of North Kivu and South Kivu since the early 1990s and in Katanga since 2003 (Fig. 1). In South Kivu, MSF supports primary and secondary health care in the Baraka and Kimbi hospitals and in six health centres, and provides response to outbreaks and emergencies. Fig. Backgroundh 1  Map of the Democratic Republic of Congo Fig. 1  Map of the Democratic Republic of Congo Fig. 1  Map of the Democratic Republic of Congo de Wit et al. Malar J (2016) 15:455 Page 3 of 10 0 5000 10000 15000 20000 25000 2009 2010 2011 2012 Baraka OPD malaria cases < 5 years >5 years Fig. 2  Confirmed malaria cases in Baraka project 2009–2012, DRC diagnostic test (RDT) were enrolled in the study if they had: (1) a body weight ≥5 kg, (2) a slide confirmation of mono-infection with P. falciparum with an asexual para- site density between 2000 and 200,000/µl of blood, (3) an ability to swallow (crushed or dissolved) oral medication, (4) a high probability of respecting follow up visits, and (5) a signed informed consent by their adult (≥18 years old) parent/caretaker. Children were excluded if they presented with general danger signs according to the WHO protocol “Methods for surveillance of antima- larial drug efficacy” [8]. These included signs of severe/ complicated malaria, including severe anaemia (Hb <5 g/ dL), history of convulsions, and jaundice. Children were also excluded if they had severe acute malnutrition (indi- cated by a weight-for-height Z score (WHZ) of <−3 Z, a middle upper arm circumference (MUAC) of  <115  cm, or bilateral oedema), a concomitant febrile or chronic ill- ness, a known allergy to one of the study medications, or had received a full course of one of the artemisinin-based combinations under study in the previous 28 days. observed increase in malaria over the years was associ- ated with increased treatment failure rates, the efficacy of the first-line treatment, ASAQ was evaluated, and com- pared to that of AL in children aged 6–59 months with confirmed uncomplicated falciparum malaria. Study design and site Sample size calculations were based on the data that was available before October 2012. At that moment, studies determined the 42-day risk of recurrent parasitaemia due to recrudescence (treatment failure) in children to range from 0.9 to 6 % with AL and ASAQ [9–12]. Based on a conservative estimated risk of recurrent parasitae- mia (due to recrudescence, PCR corrected) of 5 %, 120 patients per treatment arm would be needed to detect a difference in the risk of recrudescence between treatment arms of no greater than 7 % (one-sided type I error of 5, 80 % power) [13]. In order to account for undetermined PCR results, loss to follow up, withdrawal, and protocol violations, the total estimated sample size was increased by 20 % to 288 patients (144 per arm). An open-randomized non-inferiority trial comparing the efficacy of ASAQ to that of AL for the treatment of uncomplicated malaria, in children aged between six and 59  months, was conducted following the World Health Organization (WHO) protocols for surveillance of anti- malarial drug efficacy [8]. The study was carried out between October 2013 and December 2014 in the outpa- tient clinic of Baraka General Hospital and in the Health Centre of Baraka in South Kivu, DRC. Study procedure All patients who were eligible for the study had a medi- cal examination, and a MUAC and WHZ screening. A thick and a thin blood smear were performed and capil- lary blood was collected on a fast technology for analysis (FTA) card (Whatmann, UK). If the patient was eligible, informed consent was requested from the parent/care- taker after the explanation of the study by one of the study team members. Once the informed consent was obtained, patients were randomized to one of two treat- ment regimens according to which treatment had been randomly pre-allocated to that unique patient number. Treatment regimens consisted of: (1) ASAQ fixed dose (Winthrop Sanofi Aventis), given as 1 tab/day over three days (≥5–  <  9  kg, 1 tab of 25  mg artesunate/67.5  mg Ethical considerationsh The study was approved by the Ethical Review Board (ERB) of MSF on June 17th 2013 and by the Zone Chief Medical Officer (Médecin Chef de Zone) and the Pro- vincial Medical Inspector (Médecin Inspecteur Proven- çal MIP) of South Kivu (N251/969/B.MIP/SK/2013). It was registered at ClinicalTrials.gov under num- ber NCT02741024. Eligible patients were included in the study after an adult parent/caretaker gave written informed consent. If the parent/caretaker was illiterate, a literate witness was asked to sign next to the parent/care- taker’s fingerprint. Free health care for malaria and other illnesses is provided by MSF to the general population and was, therefore, also provided throughout the study follow-up period to all patients included in the study. Laboratory techniques Initial testing for malaria infection was done on fin- ger-prick blood using an RDT, the SD Bioline 05FK50 Malaria Ag P.f (Standard Diagnostics, Kyonggi, Republic of Korea). This RDT relies on detection of the Histidine Rich Protein 2 (HRP2), and is amongst the most sensitive according to the WHO product testing programme [14].h Thick and thin blood smears were stained with 10  % Giemsa for 15 min. Smears were read to 100 fields before they could be declared negative. Species were confirmed on the thin smear. Quantification of P. falciparum asexual parasitaemia on the thick smear was performed accord- ing to the WHO protocol “Methods for surveillance of antimalarial drug efficacy” [8]. Presence or absence of P. falciparum gametocytes was recorded. Each slide was read, independently, by two qualified microscopists in the Baraka hospital laboratory, Para- site densities were calculated by taking a mean of the two counts. Blood smears with discordant results (differ- ences in species diagnosis, in parasite density of >50 % or in the presence of parasites) were re-examined by a third microscopist who was blinded to the results of the first two, and parasite density was calculated by taking a mean of the two closest counts. In addition, external quality control of the slides was performed by blinded re-check- ing of 50 randomly selected malaria slides by an expert microscopist at the Epicentre Mbarare Research Base in Uganda. Haemoglobin levels on D0 or during follow-up were determined using the HemoCue® Hb 301 System (Äng- elholm, Sweden), according to manufacturer’s instruc- tions. PCR genotyping analysis was performed in order to distinguish true recrudescence (same parasite strain) from a newly acquired infection (different parasite strain) on capillary blood samples stored on FTA cards. The genotyping was performed at the Department of Medical Microbiology, at the Academic Medical Centre (Amster- dam, The Netherlands. PCR amplification of template DNA and analysis of glurp, msp2 and msp1 alleles in pre- (enrolment) and post-treatment (failure) samples was performed according to the WHO protocol [15]. Pre- and post-treatment pairs with similar genotype were classi- fied as recrudescence (true failure), and pairs with differ- ent genotypes were classified as re-infection according to WHO protocol “Methods and techniques for clinical tri- als on antimalarial drug efficacy: Genotyping to identify parasite populations” [15]. Species-specific PCRs were performed on samples with possible mixed re-infections according to Shokoples et al. [16]. Study population amodiaquine base; ≥9–<18 kg, 50 mg artesunate/135 mg amodiaquine base), or (2) AL fixed combination (Coar- tem®, 20 mg artemether/120 mg lumefantrine, Novartis) given with milk twice daily over three days (≥5– <15 kg, 1 tab of 20 mg artemether/120 mg lumefantrine BD, ≥15– <25  kg, 2 tabs of 20  mg artemether/120  mg lumefan- trine BD with fatty food) over 3 days. Drugs were given according to the manufacturer’s instructions and dosage. Study medication was imported and stored under strict regulations, ensuring control within the provided stor- age temperature and humidity ranges. The first dose of the study drug at day 0 (D0) was dispensed at the health clinic, and was taken by the child while under supervision of a study team member. For children who could not swal- low tablets, AL tablets were dissolved, and ASAQ tablets were crushed in minimal amounts of water and dispensed with a spoon. After intake, the child was observed for 30 min. If the child vomited or spat out the medication within the monitoring period, a resting period of 15 min was observed before re-administering a repeat dose. If the repeat dose was also vomited within 30 min, the child was administered a rescue treatment (which was defined as the alternative treatment that the child was not rand- omized to) and was excluded from the study. Patients returned to the clinic on days 1 and 2 for observed admin- istration of the study drug and a clinical assessment. In order to observe the intake of the evening doses of AL, patients randomized to receive AL were visited at home at around 8 h, 32 h and 56 h after the first dose. A fol- low up was scheduled for all patients on day 3, 7, 14, 21, 28, 35 and 42 for clinical assessment and blood smear evaluation. Study population Children aged between 6 and 59 months, presenting with fever (axillary temperature  ≥37.5  °C) or reported his- tory of fever in the last 24 h, and with a positive rapid de Wit et al. Malar J (2016) 15:455 Page 4 of 10 amodiaquine base; ≥9–<18 kg, 50 mg artesunate/135 mg amodiaquine base), or (2) AL fixed combination (Coar- tem®, 20 mg artemether/120 mg lumefantrine, Novartis) given with milk twice daily over three days (≥5– <15 kg, 1 tab of 20 mg artemether/120 mg lumefantrine BD, ≥15– <25  kg, 2 tabs of 20  mg artemether/120  mg lumefan- trine BD with fatty food) over 3 days. Drugs were given according to the manufacturer’s instructions and dosage. Study medication was imported and stored under strict regulations, ensuring control within the provided stor- age temperature and humidity ranges. The first dose of the study drug at day 0 (D0) was dispensed at the health clinic, and was taken by the child while under supervision of a study team member. For children who could not swal- low tablets, AL tablets were dissolved, and ASAQ tablets were crushed in minimal amounts of water and dispensed with a spoon. After intake, the child was observed for 30 min. If the child vomited or spat out the medication within the monitoring period, a resting period of 15 min was observed before re-administering a repeat dose. If the repeat dose was also vomited within 30 min, the child was administered a rescue treatment (which was defined as the alternative treatment that the child was not rand- omized to) and was excluded from the study. Patients returned to the clinic on days 1 and 2 for observed admin- istration of the study drug and a clinical assessment. In order to observe the intake of the evening doses of AL, patients randomized to receive AL were visited at home at around 8 h, 32 h and 56 h after the first dose. A fol- low up was scheduled for all patients on day 3, 7, 14, 21, 28, 35 and 42 for clinical assessment and blood smear evaluation. re-infection with a species other than P. falciparum) were not assigned an efficacy treatment outcome. Laboratory techniques If a clinical or parasitological treatment failure was observed, rescue treatment was initiated and a second blood sample was collected for PCR analysis. Caretak- ers were advised to present the child to the clinic at any time in case of illness. Patients who failed to return for scheduled follow up visits were traced immediately by home-visitors to minimize loss to follow up. Adverse events were evaluated by a physician and recorded at each visit. Study trial profile and baseline characteristics Study trial profile and baseline characteristics y pi A description of the study outline is shown in Fig. 3. Out of 873 children screened for uncomplicated malaria dur- ing the study period, 585 (67.0  %) were excluded. The main reasons for exclusion were ACT intake in the last 28  days (118, 20.2  %), parasitaemia outside the study range (116, 19.8 %) or co-morbidities such as pneumonia or urinary tract infections (86, 14.7 %). 288 children were included in the study and were randomized to receive ASAQ or AL. 18 children (6.3 %) in the ASAQ group and 13 children (4.5 %) in the AL group did not complete the three days of allocated treatment, due to the following: developing (apparent) signs of severe malaria (n = 5 ETF, n = 3 wrongfully admitted to ward), a late realisation of wrongful enrolment (n = 10), having an observed treat- ment dose missed (n = 7), vomiting of at least one of the treatment doses twice (n = 5), a dosage error (n = 1). Of the children who received their full course of supervised treatment (257), 19 children (7.0 %) did not reach an ana- lysable end result (censored), either due to being lost to follow up (n = 8), having had an anti-malarial adminis- tered by a 3rd party (n = 9), or having been re-infected with a malaria species other than P. falciparum (n = 2). Treatment outcome resultsi Five children were classified as early treatment failures as per study protocol. All of these children had a parasito- logical improvement, but had either anaemia or jaundice that warranted admission to the hospital. They received intravenous artesunate according to the study protocol and the WHO guidelines for the treatment of malaria [20] and were withdrawn from the study.fi Chi square tests or Fisher’s exact tests were used to compare categorical data and Student’s t test was used for continuous data, as appropriate. The log rank test was used to compare Kaplan–Meier survival curves. p < 0.05 were considered statistically significant. Treatment efficacy estimates are presented in Table 2 for days 28 and day 42 after initiation of treatment. Before PCR genotyping, 23 (9.7 %) children (13/119 on ASAQ and 10/119 on AL) were classified as late clinical failure and 23 (9.7 %) children (14/119 on ASAQ and 9/119 on AL) as late parasitological failure on day 42. Furthermore, during the study period, two children who were on AL were re-infected with other species (P. ovale), as con- firmed in weekly parasitological examinations; this was an involuntary protocol violation and these two children were censored in the final analysis. In total, 192 (80 %) children had an adequate clinical and parasitological response (92/119 for ASAQ and 100/119 for AL) at day 42. The proportion of recurring infection (including the two reinfections with other species) during the 42 days follow-up was higher in the ASAQ group (27/119, 22.7 %) compared to the AL group (21/119, 17.6 %), but this dif- ference was not statistically significant. Similarly, cumu- lative failure on day 28 was not statistically significantly different between arms. Treatment outcomes Study endpoints were classified according to the WHO guidelines [8] as adequate clinical and parasitologi- cal response (ACPR), early treatment failure (ETF), late clinical failure (LCF) and late parasitological failure (LPF). Patients who were lost to follow-up, who with- drew their consent at any time before reaching a study endpoint, or who had a protocol violation (ex. wrongful inclusion by study staff, non-completion of full treatment course, administration of a study drug by a third-party, de Wit et al. Malar J (2016) 15:455 Page 5 of 10 Data analysis The baseline characteristics of the patients enrolled in the study are presented in Table 1. Boys and girls were equally represented in the study population, with an average age of 31.5 months [(32.2 months for ASAQ (IQR 20.0–47.0) and 30.9 for AL (IQR 21.0–41.5)]. The average WHZ score was below international standards and simi- lar in both treatment groups (–0.59 for ASAQ and −0.68 for AL) [19]. The average axillary temperature at D0 was higher in the AL group (39.02 °C, IQR 38.0–39.7) than in the ASAQ group (38.77 °C, IQR 38.4–39.8). No other sta- tistically significant differences were observed between baseline characteristics for children randomized in both treatment arms. y All data were double entered in the WHO global database for anti-malarial drug efficacy by two different epidemi- ologists, as well as in the worldwide anti-malarial resist- ance network (WWARN) database provided online [17]. Statistical analysis was performed using STATA version 13.1. Data was analysed per-protocol and by intention- to-treat. Kaplan–Meier survival curves were generated to determine the cumulative probability of recurrence-free survival over the 42  days follow-up. These probability estimates were compared using a log rank test. For the per-protocol analysis, patients who were lost to follow up or withdrawn were removed from the denominator. Patients were considered withdrawn from the PCR-cor- rected analysis if the PCR results were unclassifiable or if the results of PCR indicated that the failure was due to a mono re-infection with a Plasmodium species other than the P. falciparum (such as Plasmodium vivax, Plasmo- dium malariae or Plasmodium ovale). Availability of data and materials Data are available on request in accordance with MSF’s data sharing policy [18]. PCR genotyping to determine re‑infection and recrudescencei 46 paired FTA filter paper samples were PCR genotyped, including all late clinical and parasitological failure sam- ples. Following the PCR genotyping, 21 of the 27 recur- rent parasitaemic episodes in the ASAQ arm (77.8  %) were classified as re-infections and 6 (22.2 %) as recru- descence. In the AL arm, 15 out of 19 samples (78.9 %) were classified as re-infections and 1 as (5.3 %) as recru- descence. There were no indications that treatment de Wit et al. Malar J (2016) 15:455 Page 6 of 10 dosing was lower for the children experiencing treatment Kaplan–Meier survival curves Trial profile Assessed for eligibility (n=873) Excluded (n=585) ♦Not meeting inclusion criteria (n=503) ♦Declined to participate (n=82) ♦Analysed (n=119) ♦Censored from analysis (n=0) ♦Completed 42 days follow up (n=119) ♦Did not complete 42 days follow up (n=7) Lost to follow-up (n=3) Protocol violation before end of follow up (n=4) Allocated to ASAQ (n=144) ♦Completed allocated treatment (n=126) ♦Did not complete allocated treatment (n=18) 4 with signs of severe malaria (D1) 5 protocol violations 5 vomiting 4 lost to follow up ♦Completed 42 days follow up (n=121) ♦Did not complete 42 days follow up (n=10) Lost to follow-up (n=5) Protocol violation before end of follow up (n=5) Allocated to AL (n=144) ♦Complete allocated treatment (n=131) ♦Did not complete allocated treatment (n=13) 1 with signs of severe malaria (D1) 5 protocol violation 3 vomiting 4 lost to follow up ♦Analysed (n=119) ♦Censored from analysis (n=2) Re-infection other species (n=2) Allocation Analysis Follow-Up Randomized (n=288) Enrollment Fig. 3  Trial profile efficacy study ASAQ–AL, Baraka, DRC Trial profile Assessed for eligibility (n=873) Excluded (n=585) ♦Not meeting inclusion criteria (n=503) ♦Declined to participate (n=82) ♦Completed 42 days follow up (n=121) ♦Did not complete 42 days follow up (n=10) Lost to follow-up (n=5) Protocol violation before end of follow up (n=5) ♦Completed 42 days follow up (n=119) ♦Did not complete 42 days follow up (n=7) Lost to follow-up (n=3) Protocol violation before end of follow up (n=4) Analysis ♦Analysed (n=119) ♦Censored from analysis (n=2) Re-infection other species (n=2) Fig. 3  Trial profile efficacy study ASAQ–AL, Baraka, DRC Kaplan–Meier survival curvesh dosing was lower for the children experiencing treatment failure. The cumulative probability of recurrence-free survival over the 42  days follow-up was determined by Kaplan– Meier survival analysis. The PCR un-adjusted curves shown in Fig. PCR genotyping to determine re‑infection and recrudescencei 4 indicate a non-significantly higher prob- ability of remaining free of recurrent parasitaemia in the AL arm compared to the ASAQ arm (p  =  0.16). After adjustment by PCR to distinguish between reinfection For three samples with recurring parasitaemia in the AL arm outcome could not be classified by PCR; these samples were censored at the last date of follow-up for which the child was still malaria negative. The propor- tions of children reaching each a study endpoint, before and after PCR adjustment, are presented in Table 2. de Wit et al. Malar J (2016) 15:455 Page 7 of 10 Table 1  Baseline characteristics of included patients All data is presented as the mean with the interquartile range (IQR) in brackets, unless otherwise stated ASAQ (n = 144) AL (n = 144) p value Age, in months 32.2 (20.0–47.0) 30.9 (21.0–41.5) 0.412 # Male (%) 65 (79) 65 (79) 1.000 Weight for height Z-score −0.59 (−1.39 to 0.22) −0.68 (−1.40–0.08) 0.484 Temperature in  °C 38.8 (38.0–39.7) 39.02 (38.4–39.8) 0.049 Parasite density/µl at day 0 63,637 (15, 211–95, 962) 45,154 (14, 225–94, 746) 0.826 Log parasite density at day 0 10.5 (9.6–11.5) 10.4 (9.6–11.5) 0.935 Haemoglobin in g/dl at day 0 9.5 (8.5–10.8) 9.7 (8.7–10.8) 0.316 Table 1  Baseline characteristics of included patients Table 2  Per-protocol crude/unadjusted and  PCR-adjusted study endpoints at  days 28 and  42 of  patient follow-up. Patients with re-infections and individuals for whom the outcome could not be assessed by PCR were censored from the PCR adjusted analysis crude/unadjusted and  PCR-adjusted study endpoints at  days 28 and  42 of  patient follow-up. ions and individuals for whom the outcome could not be assessed by PCR were censored from the Table 2  Per-protocol crude/unadjusted and  PCR-adjusted study endpoints at  days 28 and  42 of  patient follow-up. PCR genotyping to determine re‑infection and recrudescencei Patients with re-infections and individuals for whom the outcome could not be assessed by PCR were censored from the PCR adjusted analysis The analysis of the treatment outcomes by intention-to-treat are shown in Additional file 1: Annexure 1 Crude/no PCR Day 28 Day 42 ASAQ (n = 119) AL (n = 122) p value ASAQ (n = 119) AL (n = 119) p value n % n % n % n % Late clinical failure 8 6.7 4 3.3 0.250 13 10.9 10 8.4 0.510 Late parasitological failure 10 8.4 6 4.9 0.277 14 11.8 9 7.6 0.273 Adequate clinical and parasitological response 101 84.9 112 91.8 0.093 92 77.3 100 84.0 0.189 Cumulative failure 18 15.1 10 8.2 0.093 27 22.7 19 16.0 0.189 PCR adjusted/corrected Day 28 Day 42 ASAQ (n = 105)  AL (n = 111) p value ASAQ (n = 98)  AL (n = 101) p value n % n % n % n % Late clinical failure (recrudescence) 1 1.0 0 0 0.486 1 1.0 1 1.0 1.000 Late parasitological failure (recrudenscence) 3 2.9 0 0 0.113 5 5.1 0 0 0.027 Adequate clinical and parasitological response 101 96.2 111 100 0.038 92 93.9 100 99.0 0.049 Cumulative failure 4 3.8 0 0 0.054 6 6.1 1 1.0 0.062 Table 2  Per-protocol crude/unadjusted and  PCR-adjusted study endpoints at  days 28 and  42 Patients with re-infections and individuals for whom the outcome could not be assessed by PCR w PCR adjusted analysis The analysis of the treatment outcomes by intention-to-treat are shown in Additional file 1: Annexure 1 Fig. 4  Kaplan–Meier survival estimates PCR-unadjusted. Treatment efficacy for ASAQ and AL was 85.3 and 92.1 % at day 28 (p = 0.0933), and 77.7 and 84.5 % at day 42 (p = 0.1605), respectively Fig. 4  Kaplan–Meier survival estimates PCR-unadjusted. Treatment efficacy for ASAQ and AL was 85.3 and 92.1 % at day 28 (p = 0.0933), and 77.7 and 84.5 % at day 42 (p = 0.1605), respectively and recrudescence, the proportion of patients achieving ACPR at day 42 (treatment efficacy) was higher for the AL arm (99.0 %, 95CI 93.2–99.9) compared to the ASAQ arm (94.7 %, 95 CI 88.4–97.6), p = 0.0496 (Fig. 5). Regardless of the statistically significant differences between study arms, the difference in ACPR by day 42 after initiation of treatment was within the margin of non-inferiority. Discussion The results of this study indicate that both study drugs retain adequate efficacy (PCR corrected cure rate at day 42 of 94.7 % for ASAQ and 99.0 % for AL) in the treatment of uncomplicated P. falciparum malaria in children aged six to 59  months in the study setting. In 2004, a similar study in DRC (Pool Region) showed comparable results (98.5 % efficacy of ASAQ and 100 % efficacy of AL at 28 days) [21]. This was also the result of the efficacy study by Singana et al. [22] in Owando in Congo-Brazzaville done in 2012–2103 (efficacy of ASAQ 98.0 % and of AL 100.0 % at 28 days, after PCR correction). The 4ABC study group, performed in a comparable population and using the same methodol- ogy, showed a similar efficacy rate between 2007 and 2009 over seven sub-Saharan African countries (96.8 % efficacy of ASAQ and 95.5 % efficacy of AL at 28 days) [23]. In the 4ABC study, ASAQ was also shown to be effective in Eastern Africa, whereas other studies had previously casted doubts over its efficacy in this region [24, 25].hfi Fig. 5  Kaplan–Meier survival estimates PCR-adjusted. Treatment efficacy for ASAQ and AL was 96.6 and 100 % at day 28 (p = 0.0406), and 94.7 and 99.0 % at day 42 (p = 0.0496), respectively Fig. 5  Kaplan–Meier survival estimates PCR-adjusted. Treatment efficacy for ASAQ and AL was 96.6 and 100 % at day 28 (p = 0.0406), and 94.7 and 99.0 % at day 42 (p = 0.0496), respectively Table 3  Parasite clearance on Days 2 and 3 of treatment ASAQ AL p value Patients with parasite positivity at D2 18 (13.7 %) 27 (20.0 %) 0.173 Patients with parasite positivity at D3 2 (1.5 %) 6 (4.4 %) 0.167 Table 3  Parasite clearance on Days 2 and 3 of treatment The high efficacy of both ASAQ and AL in this region is reassuring. WHO only recommends a switch in the first line treatment if the efficacy falls below 90 % [26]. Although this study confirms that both artemisinin- based combinations retain good efficacy in this part of DRC, it does, however, demonstrate high re-infection levels. Discussion After PCR correction, this study showed that, over 6  weeks, 21/119 children (17.6  %) of those who were on ASAQ and 15/119 (12.6 %) of the children in the AL treatment group (p = 0.29) were re-infected within 42 days. Parasite clearance A relatively large proportion (13.7 % on ASAQ, 20.0 % on AL) of children still had parasites on day 2 of follow-up. This proportion fell to below 5 % in both arms on day 3. There was no significant difference in the proportion of parasites at D2 and D3 between the two treatment arms (Table 3). Fig. 4  Kaplan–Meier survival estimates PCR-unadjusted. Treatment efficacy for ASAQ and AL was 85.3 and 92.1 % at day 28 (p = 0.0933), and 77.7 and 84.5 % at day 42 (p = 0.1605), respectively Page 8 of 10 de Wit et al. Malar J (2016) 15:455 Page 8 of 10 Fig. 5  Kaplan–Meier survival estimates PCR-adjusted. Treatment efficacy for ASAQ and AL was 96.6 and 100 % at day 28 (p = 0.0406), and 94.7 and 99.0 % at day 42 (p = 0.0496), respectively Fig. 5  Kaplan–Meier survival estimates PCR-adjusted. Treatment efficacy for ASAQ and AL was 96.6 and 100 % at day 28 (p = 0.0406), and 94.7 and 99.0 % at day 42 (p = 0.0496), respectively Treatment safetyh There were no serious adverse events during the period of the study. However, minor adverse events occurred in both treatment arms during the treatment period (days 0–2). The adverse event most reported was asthenia (weakness, lack of energy and strength) for both arms. Children receiving ASAQ reported significantly more asthenia and anorexia (loss of appetite) than children receiving AL (Table 4). Whilst re-infections do not indicate failing anti-malar- ial drugs, they are of public health relevance and pose a considerable burden to the population and health sys- tems. The results of this study seem to support recent findings that the period over which lumefantrine and amodiaquine provide post-treatment protection is 10–14  days [26–28]. However, considering the public health impact of frequent malaria infections in infants, it would be useful to do further analysis into the specific protective period for each of ASAQ and AL in this set- ting. It is also worth considering if the use of an ACT with a longer protective half-life, such as dihydroartemisinin– piperaquine, may be beneficial to prevent malaria epi- sodes in the study population, exposed to intense malaria transmission [27–29]. Table 4  Adverse events = classified as related to the drug, for all patients during the study period ASAQ (n = 144) AL (n = 144) Total p value Asthenia 61 (42.4 %) 13 (9.0 %) 74 (25.7 %) <0.001 Anorexia 27 (18.8 %) 12 (8.3 %) 39 (13.5 %) 0.010 Vomiting 11 (7.6 %) 10 (6.9 %) 21 (7.3 %) 0.821 Cough 5 (3.5 %) 7 (4.9 %) 12 (4.2 %) 0.555 Abdominal Pain 6 (4.2 %) 2 (1.4 %) 8 (2.8 %) 0.151 Diarrhoea 4 (2.8 %) 2 (1.4 %) 6 (2.1 %) 0.409 Itching 3 (2.1 %) 1 (0.7 %) 4 (1.4 %) 0.314 Nausea 1 (0.7 %) 1 (0.7 %) 2 (0.7 %) 1.000 Table 4  Adverse events = classified as related to the drug, for all patients during the study period In this study’s setting children will normally receive ASAQ each time they have a malaria infection. Reas- suringly, Yeka et al. [29] showed that repeated treatment using both ASAQ and AL is effective, safe and well-tol- erated in children under 5 years of age and in a similar context [29]. de Wit et al. Malar J (2016) 15:455 de Wit et al. Availability of data and material D l bl Availability of data and material Data are available on request in accordance with MSF’s data sharing policy at http://www.fieldresearch.msf.org/msf/handle/10144/306488. Author details 1 Médecins sans Frontières (MSF), Plantage Middenlaan 14, 1018 DD Amster- dam, The Netherlands. 2 Manson Unit, Médecins Sans Frontières (MSF), 10 Furnival Street, London EC4A 1AB, UK. 3 Academisch Medisch Centrum, Amsterdam, The Netherlands. 4 Programme National de lutte contre le Paludisme, Kinshasa, South Kivu, Democratic Republic of the Congo. 5 London School of Hygiene and Tropical Medicine, London, UK. 6 Radboud university medical center, Nijmegen, The Netherlands. Additional file Additional file 1: Annexure 1. Intent-to-treat crude/unadjusted and PCR-adjusted study endpoints at days 28 and 42 of patient follow-up. Authors’ contributions RS, KB and MdW jointly drafted the proposal and supported the study. KM set-up and implemented the study in the field as lead field epidemiologist. KM and AF coordinated the field component of the study as field epidemiolo- gists. EP and AB gave input to and supervised the laboratory component. DAN was one of the study clinic supervisors. BB reviewed the proposal and the article. AF and MdW performed statistical analysis. MdW was the principal investigator and wrote the article. TB contributed to the statistical analysis and supervised the analysis of the study and the article. All authors reviewed and approved the article. All authors read and approved the final manuscript. There was a relatively high proportion (67 %) of children excluded from the study at the time of screening. There were two major reasons for exclusion: the high number of children with co-morbidities, and the high number of chil- dren who had taken ACT in the previous 28 days (either from a clinic or from a private vendor). In the children with co-morbidities, it is not clear what relation malaria had to their clinical illness; the children could be present- ing with clinical illness due to malaria, or have symptoms primarily from their co-morbidity but happen to have malaria parasites. This is a dilemma that is faced often in the field, and may lead to an overestimation of the burden of malaria. Furthermore, an RDT for malaria is often per- formed as a first means of triage for fever, and when the test is positive, there is the risk that the child may not be adequately examined for other illnesses. In this study, 49 of the 873 (5.6 %) children with a positive RDT (based on HRP2 detection) had a negative thick smear. This could be the result of circulating HRP2 from a previous, well treated malaria infection. This is expected considering that Grandesso et  al. (internal communication, Clinical Trial. gov Identifier NCT01325974) have shown that over 50 % of HRP2 RDTs remain positive more than 6 weeks after adequate treatment of a malaria infection. Conclusion The study was approved by the Ethical Review Board (ERB) of MSF on June 17th 2013 and by the Zone Chief Medical Officer (Médecin Chef de Zone) and the Provincial Medical Inspector (Médecin Inspecteur Provençal MIP) of South Kivu (N251/969/B.MIP/SK/2013). It was registered at ClinicalTrials.gov under number NCT02741024. Eligible patients were included in the study after an adult par-ent/caretaker gave written informed consent. If the parent/caretaker was illiterate, a literate witness was asked to sign next to the parent/caretaker’s fingerprint. Free health care for malaria and other illnesses is provided by MSF to the general population and was therefore also provided throughout the study follow-up period to all patients included in the study. ASAQ is still effective as a treatment for uncomplicated malaria in Baraka, South Kivu, DRC, 9  years after its introduction as a first-line treatment. There is no evi- dence that ASAQ is inferior to AL in this setting with the non-inferiority margin of 7 % that was set for the current study. AL may have higher efficacy but additional trials are required to draw this conclusion with confidence. There is a high rate of re-infection in South-Kivu within six weeks of adequate treatment of either ASAQ or AL, indicating intense malaria transmission. AL was better tolerated by patients than ASAQ. Although earlier esti- mates of anti-malarial drug efficacy are unavailable for Treatment safetyh Malar J (2016) 15:455 Page 9 of 10 With regards to tolerability, there were no serious adverse events registered during the trial. Asthenia and anorexia were the most commonly recorded minor adverse events with a marked difference between the two drugs. It was shown by the results of this study that AL was better tolerated. During the study it was observed that the parents/caretakers would prefer that their child be randomized to AL rather than ASAQ as the former was perceived as more tolerable by the children. In fact, the chance to be randomized to receive AL seemed to be a major driving force to participate in the study. Although asthenia and anorexia may be classified as minor adverse effects, it can have a negative effect on adherence, and thus prevent a complete cure.h the current population, the high efficacy of ASAQ makes it unlikely that declining drug efficacy is a reason for the apparent increase in malaria cases observed in the region. Acknowledgements The study team would like to thank the South Kivu coordination team and the Baraka field team (Joe Wazome, David Aksanti Nkuba, Alain Asukulu Pelochose, Claude Bwende Habinamwisho, Patrick Bujingo Murhula, Christian Bwira Buciza, Edwige Hali Zinduna, Jean-Jacques Mapenzi Ndahe, Jonathan Nusi Makyata Ndale, Henri Mwessa Ngumba, Franck Okundji Lomami, Kester Elembe Baruani, Sébastien Akilimali Mukandama, Olivier Situmani Ngun- gumazi) and all the young study participants and their caretakers. Many thanks to Nienke Verhaar for her practical work in the AMC lab. Competing interests h h d l h Competing interests Thr authors declare that they have no competing interests. References 1. WHO. World malaria report 2013. Geneva: World Heath Organization; 2013. p. 256. 2. Coghlan B, Brennan RJ, Ngoy P, Dofara D, Otto B, Clements M, et al. Mor- tality in the Democratic Republic of Congo: a nationwide survey. Lancet. 2006;367:44–51. 17. Worldwide antimalarial resistance network toolkit. http://www.wwarn. org/tools-resources/toolkit/plan 3. Coghlan B, Ngoy P, Mulumba F, Hardy C, Bemo VN, Stewart T, et al. Update on mortality in the Democratic Republic of Congo: results from a third nationwide survey. Disaster Med Public Health Prep. 2009;3:88–96. 18. MSF field research data sharing. http://fieldresearch.msf.org/msf/ handle/10144/306488 19. WHO. Weight-for-age. Geneva: World Heath Organization. http://www. who.int/childgrowth/standards/weight_for_age/en/ 4. Lenglet A. Retrospective Mortality Survey in the MSF Catchment Area in Fizi Health Zone, South Kivu. Amsterdam: Democratic Republic of Congo; 2014. g g g 20. WHO. Guidelines for the treatment of malaria. 2nd ed. Geneva: World Heath Organization; 2010. p. 197. 21. Van den Broek I, Kitz C, Al Attas S, Libama F, Balasegaram M, Guthmann JP. Efficacy of three artemisinin combination therapies for the treatment of uncomplicated Plasmodium falciparum malaria in the Republic of Congo. Malar J. 2006;5:113. 5. WHO. Malaria Country Profile DRC. Geneva: World Heath Organization; 2014. 6. PNLP DRC. Rapport annuel des activites de lutte contre le paludisme 2013. 2014. 22. Singana BP, Bogreau H, Matondo BD, Dossou-Yovo LR, Casimiro PN, Mbouka R, et al. Malaria burden and anti-malarial drug efficacy in Owando, northern Congo. Malar J. 2016;15:16. 22. Singana BP, Bogreau H, Matondo BD, Dossou-Yovo LR, Casimiro PN, Mbouka R, et al. Malaria burden and anti-malarial drug efficacy in Owando, northern Congo. Malar J. 2016;15:16. 7. Ministère de la Santé DRC. Profil pharmaceutique de la République Démocratique du Congo. 2011. q g 8. WHO. Methods for surveillance of antimalarial drug efficacy. Geneva: World Heath Organization; 2009. g 23. Group 4ABC. A head-to-head comparison of four artemisinin based combinations for treating uncomplicated malaria in African children: a randomized trial. PLoS Med. 2011;8:e1001119. 23. Group 4ABC. A head-to-head comparison of four artemisinin based combinations for treating uncomplicated malaria in African children: a randomized trial. PLoS Med. 2011;8:e1001119. 9. Mårtensson A, Strömberg J, Sisowath C, Msellem MI, Gil JP, Montgom- ery SM, et al. Efficacy of artesunate plus amodiaquine versus that of artemether-lumefantrine for the treatment of uncomplicated childhood Plasmodium falciparum malaria in Zanzibar, Tanzania. Clin Infect Dis. 2005;41:1079–86. 24. Clark TD, Njama-Meya D, Nzarubara B, Maiteki-Sebuguzi C, Greenhouse B, Staedke SG, et al. Funding The study was funded by MSF-OCA. Received: 20 April 2016 Accepted: 18 July 2016 Received: 20 April 2016 Accepted: 18 July 2016 Received: 20 April 2016 Accepted: 18 July 2016 de Wit et al. Malar J (2016) 15:455 Page 10 of 10 16. Shokoples SE, Ndao M, Kowalewska-Grochowska K, Yanow SK. Multiplexed real-time PCR assay for discrimination of Plasmodium species with improved sensitivity for mixed infections. J Clin Microbiol. 2009;47:975–80. References • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: References Incidence of malaria and efficacy of combination anti- malarial therapies over 4 years in an urban cohort of Ugandan children. PLoS ONE. 2010;5:e11759. 10. Espié E, Lima A, Atua B, Dhorda M, Flévaud L, Sompwe EM, et al. Efficacy of fixed-dose combination artesunate-amodiaquine versus artemether- lumefantrine for uncomplicated childhood Plasmodium falciparum malaria in Democratic Republic of Congo: a randomized non-inferiority trial. Malar J. 2012;11:174. 25. Dorsey G, Staedke S, Clark TD, Njama-Meya D, Nzarubara B, Maiteki-Sebu- guzi C, et al. Combination therapy for uncomplicated falciparum malaria in Ugandan children: a randomized trial. JAMA. 2007;297:2210–9. g 26. WHO. Guidelines for the treatment of malaria. 3rd ed. Geneva: World Heath Organization; 2015. 11. Yeka A, Dorsey G, Kamya MR, Talisuna A, Lugemwa M, Rwakimari JB, et al. Artemether-lumefantrine versus dihydroartemisinin-piperaquine for treating uncomplicated malaria: a randomized trial to guide policy in Uganda. PLoS ONE. 2008;3:e2390. 27. Okell LC, Cairns M, Griffin JT, Ferguson NM, Tarning J, Jagoe G, et al. Con- trasting benefits of different artemisinin combination therapies as first- line malaria treatments using model-based cost-effectiveness analysis. Nat Commun. 2014;5:5606. 12. Zongo I, Dorsey G, Rouamba N, Dokomajilar C, Séré Y, Rosenthal PJ, et al. Randomized comparison of amodiaquine plus sulfadoxine-pyrimeth- amine, artemether-lumefantrine, and dihydroartemisinin-piperaquine for the treatment of uncomplicated Plasmodium falciparum malaria in Burkina Faso. Clin Infect Dis. 2007;45:1453–61. 28. Bretscher MT, Griffin JT, Hugo P, Baker M, Ghani A, Okell L. A comparison of the duration of post-treatment protection of artemether-lumefantrine, dihydroartemisinin-piperaquine and artesunate-amodiaquine for the treatment of uncomplicated malaria. Malar J. 2014;13(Suppl 1):P19. 29. Yeka A, Lameyre V, Afizi K, Fredrick M, Lukwago R, Kamya MR, et al. Efficacy and safety of fixed-dose artesunate-amodiaquine vs. artemether- lumefantrine for repeated treatment of uncomplicated malaria in Ugan- dan children. PLoS ONE. 2014;9:e113311. 13. Centre for Clinical Trials. Sample size calculator: two parallel-sample proportions. http://www.cct.cuhk.edu.hk/stat/proportion/tspp_sup.htm 14. WHO. Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 6 (2014–2015). Geneva: World Heath Organization. 15. WHO. MMV methods and techniques for clinical trials on antimalarial drug efficacy: genotyping to identify parasite populations. Geneva: World Heath Organization; 2007. 15. WHO. MMV methods and techniques for clinical trials on antimalarial drug efficacy: genotyping to identify parasite populations. Geneva: World Heath Organization; 2007. 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https://www.frontiersin.org/articles/10.3389/fnsys.2017.00015/pdf
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Cortical Variability and Challenges for Modeling Approaches
Frontiers in systems neuroscience
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Keywords: metastability, excitation-inhibition balance, cortical variability, neuronal variability, recurrent networks, trial to trial variability Keywords: metastability, excitation-inhibition balance, cortical variability, neuronal variability, recurrent networks, trial to trial variability The functional role of the observed neuronal variability (the disparity in neural responses across multiple instances of the same experiment) is again receiving close attention in Computational and Systems Neuroscience (e.g., Durstewitz et al., 2010; Moreno-Bote et al., 2011; Oram, 2011; Beck et al., 2012; Churchland and Abbott, 2012; Brunton et al., 2013; Masquelier, 2013; Mattia et al., 2013; Balaguer-Ballester et al., 2014; Renart and Machens, 2014; Bujan et al., 2015; Lin et al., 2015; Pachitariu et al., 2015; Arandia-Romero et al., 2016; Doiron et al., 2016; McDonnell et al., 2016). Special consideration is currently given to understanding how spiking (Bujan et al., 2015; Deneve and Machens, 2016; Doiron et al., 2016; Hartmann et al., 2016; Landau et al., 2016) and phenomenological (Goris et al., 2014; Lin et al., 2015; Mochol et al., 2015; Arandia-Romero et al., 2016; Doiron et al., 2016) models account for the wide range of classical and new phenomena associated with trial-to-trial uncorrelated activity. Edited by: Reviewed by: Maurizio Mattia, Istituto Superiore di Sanità, Italy Arvind Kumar, Royal Institute of Technology, Sweden Iñigo Arandia-Romero, Pompeu Fabra University, Spain OPINION published: 04 April 2017 doi: 10.3389/fnsys.2017.00015 OPINION PREDICTABLE COMPONENTS OF NEURONAL VARIABILITY The view that the intrinsic stochasticity of single cell activity is the major source of variability has been questioned multiple times over the last decades by modeling (van Vreeswijk and Sompolinsky, 1996; Amit and Brunel, 1997; Shadlen and Newsome, 1998; Deneve et al., 2001; Stein et al., 2005; Faisal et al., 2008; Renart et al., 2010; Rabinovich and Varona, 2011; Masquelier, 2013; Stiefel et al., 2013; Rabinovich et al., 2014; Deneve and Machens, 2016; Hartmann et al., 2016) and empirical studies (e.g., Bryant and Segundo, 1976; Mainen and Sejnowski, 1995; Britten et al., 1996; Stein et al., 2005). It is well known that essentially deterministic networks of balanced excitation and inhibition are able to generate a weakly correlated, often chaotic attractor state which presents Poissonian statistical properties like the observed activity (van Vreeswijk and Sompolinsky, 1996; Amit and Brunel, 1997; Shadlen and Newsome, 1998; Sussillo and Abbott, 2009; Litwin-Kumar and Doiron, 2012). However, such a chaotic state is a non-mandatory modeling choice: recently, a range of models has shown that part of the observed variability may also be explained by a different class *Correspondence: Emili Balaguer-Ballester eb-ballester@bournemouth.ac.uk Received: 18 October 2016 Accepted: 06 March 2017 Published: 04 April 2017 Citation: Balaguer-Ballester E (2017) Cortical Variability and Challenges for Modeling Approaches. Front. Syst. Neurosci. 11:15. doi: 10.3389/fnsys.2017.00015 Received: 18 October 2016 Accepted: 06 March 2017 Published: 04 April 2017 Cortical Variability and Challenges for Modeling Approaches 1 Department of Computing and Informatics, Faculty of Science and Technology, Bournemouth University, Bournemouth, UK, 2 Bernstein Center for Computational Neuroscience, Medical Faculty Mannheim and Heidelberg University, Mannheim, Germany Specifically, it has often been proposed that a network state characterized by largely asynchronous spike times whilst maintaining slow oscillations in the firing-rates, may represent the default spontaneous cortical mode (e.g., Sanchez-Vives and Mattia, 2014; Deneve and Machens, 2016; Sancristobal et al., 2016); and similar states could also underlie observed stimulus-driven variability in rate (Litwin-Kumar and Doiron, 2012; Deneve and Machens, 2016; Hartmann et al., 2016). However, the way in which such a computationally advantageous network state for neural coding is achieved can differ substantially between modeling approaches; this challenge will be the focus of this manuscript. Specifically, it has often been proposed that a network state characterized by largely asynchronous spike times whilst maintaining slow oscillations in the firing-rates, may represent the default spontaneous cortical mode (e.g., Sanchez-Vives and Mattia, 2014; Deneve and Machens, Edited by: Gilad Silberberg, Karolinska Institutet, Sweden DIVERSITY OF THEORETICAL APPROACHES The consensus on the network origin of a substantial part of cortical variability led to the development of a multitude of models for explaining the underlying neuronal mechanisms of the asynchronous state (e.g., Boerlin et al., 2013; Deco et al., 2014; Ostojic, 2014; Barral and Reyes, 2016; Hartmann et al., 2016; Rosenbaum et al., 2017). A linking theme in these approaches is the crucial contribution of fast inhibition in recurrent networks; which is negatively correlated with excitation and strong enough to counterbalance it to different degrees (Renart et al., 2010; Deneve and Machens, 2016). Nevertheless, despite such recent advances, the mapping between the cognitive state and variability is still challenging. For instance, on the one hand, correlated rate variability between pairs of neurons is often reduced by top-down attentional processes (e.g., Cohen and Maunsell, 2009; Mitchell et al., 2009; Cohen and Kohn, 2011; Doiron et al., 2016). On the other hand, the opposite can be observed when attention is highly variable across trials (Roelfsema et al., 2004; Renart and Machens, 2014; Ruffand Cohen, 2014); and such noise correlation analyses show a variety of mixed results (Cohen and Kohn, 2011; Eyherabide and Samengo, 2013; Moreno-Bote et al., 2014; Ruffand Cohen, 2014, 2016; Doiron et al., 2016). FIGURE 1 | Three dimensions in models of neuronal variability. The diamond shows an example of a spiking model with random connectivity and a tight Excitation/Inhibition (E/I) balance as in Renart et al., 2010; the circle represents a substantially different modeling choice such as in Hartmann et al., 2016. A range of modeling approaches typically fall between these two examples (triangles); such as semi-structured connectivity architectures which modulate the E/I balanced dynamics in realistic networks (Litwin-Kumar and Doiron, 2012; Landau et al., 2016). Importantly, compelling evidence suggests that a substantial portion of the spontaneous and evoked total and shared variability is attributable to global fluctuations (Ecker et al., 2014, 2016; Goris et al., 2014; Mochol et al., 2015; Pachitariu et al., 2015; Schölvinck et al., 2015; Arandia-Romero et al., 2016); and this has direct implications in neural coding in visual (Lin et al., 2015; Arandia-Romero et al., 2016; Ecker et al., 2016) and in auditory areas (Mochol et al., 2015; Pachitariu et al., 2015). For instance, high population activity in monkey V1 increases the information that a subset of neuronal ensembles carry about stimulus orientation, only the ones that show a strong multiplicative modulation. Citation: April 2017 | Volume 11 | Article 15 Frontiers in Systems Neuroscience | www.frontiersin.org Cortical Variability and Modeling Challenges Balaguer-Ballester of deterministic processes (Beck et al., 2012; Mattia et al., 2013; Renart and Machens, 2014; Bujan et al., 2015; Abbott et al., 2016; Deneve and Machens, 2016; Doiron et al., 2016; Gillary and Niebur, 2016; Hartmann et al., 2016) such as the lack of specificity in top-down processing of cognitively complex tasks (Beck et al., 2012). global network state (Oram, 2011; Bujan et al., 2015; Pachitariu et al., 2015; Doiron et al., 2016; Landau et al., 2016) and explain noise correlations dynamics (Bujan et al., 2015). This complex variety of results has been recently analyzed using a range of phenomenological and spiking models. These recent modeling efforts aim to pin down when precisely during the course of the trial (Moreno-Bote et al., 2014; Bujan et al., 2015; Doiron et al., 2016) and in which specific network state (Arandia-Romero et al., 2016) noise correlations are informative or deleterious for neural coding (Ecker et al., 2014, 2016; Moreno-Bote et al., 2014; Lin et al., 2015; Pachitariu et al., 2015; Schölvinck et al., 2015; Arandia-Romero et al., 2016; Doiron et al., 2016). At the same time, empirical studies found mounting evidence of deterministic patterns for some of the trial to trial variability. For example, a range of indexes (Shadlen and Newsome, 1998; Churchland and Abbott, 2012; Marcos et al., 2013) suggest that variance is systematically reduced at the stimulus onset (Churchland et al., 2010); and in general shows a predictable trend during different events of the task (Churchland et al., 2006, 2010; Churchland and Abbott, 2012; Ledberg et al., 2012; Renart and Machens, 2014). Thus, there seems to be an increasing consensus in that at least part of the trial to trial variability shows a deterministic pattern which may play a functional role; and hence cannot be simply neglected (Balaguer-Ballester et al., 2011, 2014; Masquelier, 2013; Ecker et al., 2014; Goris et al., 2014; Renart and Machens, 2014; Lin et al., 2015; Schölvinck et al., 2015; Arandia-Romero et al., 2016; Hartmann et al., 2016). Frontiers in Systems Neuroscience | www.frontiersin.org FUTURE CHALLENGES FOR MODELS This non-exhaustive summary of few recent examples suggests the availability of a plethora of recurrent and feedforward network models for understanding the source of variability during cognitive processing and in the resting state. These configurations often differ at least in the level of detail of the computational units, in the connectivity structure and in the degree of balance between excitation and inhibition (Figure 1). This challenging scenario perhaps compels to the development of novel approaches for probing the networks in order to identify the suitable architecture or architectures for each specific cognitive process and cortical area. However, the question remains how to effectively dissect a recurrent network, beyond the linearization of the network dynamics, in order to investigate the components originating the asynchronous state (Sussillo, 2014). Recently, Doiron et al. (2016) proposed a framework to identify the physiological processes underlying decorrelation in feedforward circuits by analysing state- dependent correlations in different time windows. However, applying this approach is more problematic in recurrent circuits when coupling is not weak and is highly nonlinear; as is often the case in models. j In contrast, other recent approaches rely on non-closely balanced excitatory-inhibitory dynamics in networks of simplified units. For instance, Hartmann et al. (2016) proposed a fully deterministic approach to describe spontaneous and stimulus evoked variability; consisting of an architecture of schematic noise-free units. In this approach, excitatory connectivity is specifically set by plasticity and homeostasis; and is not necessarily balanced. In Deco et al. (2014), the spontaneous state also stems from not necessarily tightly balanced architectures, where elements are field equations derived from spiking units with background input noise. In this and related models, connectivity is also set; but in the latter case local inhibition is regulated by a different homeostatic control. In addition, it has been recently shown that most of the evoked variability could be accounted for by essentially feedforward architectures (Bujan et al., 2015; Doiron et al., 2016). Hence, inferring which level of detail and architecture are mostly probably responsible for the neuronal variability phenomena is perhaps one of the major challenges for the next years, which possibly requires the development of novel theoretical tools for scrutinizing network behavior. DIVERSITY OF THEORETICAL APPROACHES In contrast, the stimulus-decoding information of such multiplicative ensembles plummets for low global activity states; whilst information increases in the group additively-modulated neurons in the population (Arandia- Romero et al., 2016). Global modulations could either stem from the default up/down state of ongoing activity (Mochol et al., 2015) or from fluctuations within a single state (Arandia-Romero et al., 2016). When controlled for this global co-modulations, noise correlations are often negligible (Renart et al., 2010), but not always (Pachitariu et al., 2015; Arandia-Romero et al., 2016). Moreover, stimulus-driven input statistics can also have a strong contribution to the observed evoked variability in parallel to the E 1 | Three dimensions in models of neuronal variability. The FIGURE 1 | Three dimensions in models of neuronal variability. The diamond shows an example of a spiking model with random connectivity and a tight Excitation/Inhibition (E/I) balance as in Renart et al., 2010; the circle represents a substantially different modeling choice such as in Hartmann et al., 2016. A range of modeling approaches typically fall between these two examples (triangles); such as semi-structured connectivity architectures which modulate the E/I balanced dynamics in realistic networks (Litwin-Kumar and Doiron, 2012; Landau et al., 2016). April 2017 | Volume 11 | Article 15 2 Cortical Variability and Modeling Challenges Balaguer-Ballester This scenario is currently the subject of a lively debate; and a variety of processing architectures of spiking units have been developed to explain the observed variability phenomena from different perspectives. It has recently been proposed that a much tighter synchronization between excitation and inhibition than considered so far, at the spike level, has an even stronger experimental support and would enable the network to operate optimally by reducing the minimum coding error (Renart et al., 2010; Boerlin et al., 2013; Abbott et al., 2016; Deneve and Machens, 2016). The precise way in which the asynchronous state is achieved however, is not unique. For example, connectivity weights are specifically learnt in the design termed spike-coding network (Boerlin et al., 2013; Schwemmer et al., 2015; Abbott et al., 2016; Deneve and Machens, 2016); whilst connectivity is clustered in Litwin-Kumar and Doiron (2012), shaped by plasticity in Vogels et al. (2011) and Landau et al. (2016) and much less structured in many other dense (Renart et al., 2010; Abbott et al., 2016) or sparse (Ostojic, 2014) networks. computations could also underlie motor plans (Mattia et al., 2013). Abbott, L. F., DePasquale, B., and Memmesheimer, R.-M. M. (2016). Building functional networks of spiking model neurons. Nat. Neurosci. 19, 350–355. doi: 10.1038/nn.4241 DIVERSITY OF THEORETICAL APPROACHES Other state-dependent computational ingredients such as neuromodulation could also play a major role in shaping the observed variability (Mattia et al., 2013; Lapish et al., 2015; Doiron et al., 2016). ACKNOWLEDGMENTS The author wants to thank very much the three reviewers for their valuable comments. FUNDING This paper has been funded by Bournemouth University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The author conceived and wrote the manuscript. The author conceived and wrote the manuscript. AUTHOR CONTRIBUTIONS Asymmetry and slightly unbalanced configurations are also considered to promote the so called metastable state (Mattia et al., 2013; Tognoli and Kelso, 2014a,b; Deco and Kringelbach, 2016), in which high dimensional ensembles flexibly re-organize, synchronize and disengage, possibly by changing their role in a high-dimensional setting (Lapish et al., 2015; Fusi et al., 2016). Such state also exhibits advantageous computational properties (Hellyer et al., 2015; Deco and Kringelbach, 2016). In an instantiation of such ideas, essentially deterministic structures of simplified units generate itinerancy through robust transient states. These states enable the model to process cognitive entities without the compelling need for attractors (Rabinovich et al., 2008, 2014; Varona and Rabinovich, 2016). Moreover, combinations of attractor-based and transient-based Arandia-Romero, I., Tanabe, S., Drugowitsch, J., Kohn, A., and Moreno-Bote, R. (2016). Multiplicative and additive modulation of neuronal tuning with Amit, D. J., and Brunel, N. (1997). Model of global spontaneous activity and local structured activity during delay periods in the cerebral cortex. Cereb. Cortex 7, 237–252. doi: 10.1093/cercor/7.3.237 Arandia-Romero, I., Tanabe, S., Drugowitsch, J., Kohn, A., and Moreno-Bote, R. (2016). Multiplicative and additive modulation of neuronal tuning with Amit, D. J., and Brunel, N. (1997). 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Cathodized Stainless Steel Mesh for Binder-Free NiFe&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;/NiFe Layer Double Hydroxides Oxygen Evolution Reaction Electrode
Engineering innovations
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Keywords: Electrocatalyst; Oxygen evolution reaction; Cathodization Abstract. Oxygen evolution reaction (OER) is an essential reaction commonly applied in various energy storage and conversion technologies. One of the common issues of OER lies in its low kinetic activity. Therefore, developing durable, low-cost, and high-performance OER catalysts is critical. Recently, many attempts have used stainless steel mesh (SSM) as the substrate for OER electrodes because SSM is abundant, cheap, and durable. Nickel/iron-based materials, i.e., NiFe2O4/NiFe layer double hydroxides (LDHs), are regarded as one of the most excellent OER catalysts in alkaline electrolytes, making them attractive low-cost materials for OER catalysts. However, synthesizing NiFe2O4/NiFe LDHs directly on the surface of SSM is challenging. Modifying the SSM surface through cathodization has proved to enhance the adhesion and OER activity. Moreover, the cathodization technique is facile and cost-effective. In this work, the surface of SSM is modified by cathodization treatment. Subsequently, NiFe2O4/NiFe LDHs are deposited onto the surface of treated SSM via a low-temperature one-step chemical bath deposition technique. This synthesis is a binder- free method; the resulted electrodes show excellent OER performance without the binder effects. The as-prepared electrodes have a small Tafel slope of 125.4 mV/dec (1 M KOH) and high durability (10 mA/cm2 for 50 hours). Engineering Innovations ISSN: 2813-1002, Vol. 9, pp 23-30 doi:10.4028/p-1BMxJg © 2024 The Author(s). Published by Engineering Innovations ISSN: 2813-1002, Vol. 9, pp 23-30 doi:10.4028/p-1BMxJg © 2024 The Author(s). Published by Submitted: 2023-06-26 Revised: 2023-10-04 Accepted: 2023-10-04 Online: 2024-02-12 Cathodized Stainless Steel Mesh for Binder-Free NiFe2O4/NiFe Layer Double Hydroxides Oxygen Evolution Reaction Electrode Natthapon Sripallawita and Soorathep Kheawhomb* Research Unit of Advanced Materials for Energy Storage, Chulalongkorn University, Bangkok 10330, Thailand a6370081821@student.chula.ac.th, bsoorathep.k@chula.ac.th This article is an open access article under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0) Introduction Due to the obvious rising demand for energy, the demand for technology related to energy conversion and storage is increasing [1, 2]. As a result, the development of efficient, cost-effective, and sustainable renewable energy technology is important. Lithium-ion batteries are now widely employed in a variety of applications, including mobile phones, laptops, and electric vehicles, and they have almost dominated the rechargeable battery market. However, lithium-ion batteries' development and applications have been hampered by their low energy density (<350 Wh/kg), limited lithium resources, and safety concerns [3, 4]. Metal-air battery is one of the more intriguing batteries. since the reactants (O2) are not kept in the cell but retrieved from the environment, the energy density of metal-air batteries is substantially higher than that of lithium-ion batteries [5]. Metal anodes for metal-air batteries come in a variety of materials, including lithium (Li), magnesium (Mg), aluminum (Al), zinc (Zn), and iron (Fe). Zn–air batteries (ZAB) have risen to the forefront of current rechargeable battery development due to their many advantages, such as: zinc is abundant in nature, has low toxicity, is extremely stable, can be handled safely in oxygen and humid environments, is economically priced, and is safe to use with aqueous electrolytes. Furthermore, gadgets made of zinc have a high specific and volumetric energy density [3, 6, 7]. Although Li-air batteries have the highest specific energy density, they are unstable when exposed to oxygen or water, necessitating an inert atmosphere and the use of organic electrolyte systems, which adds to the manufacturing complexity and safety issues. The safety concerns arise from the explosive reactivity of lithium with air or water, as well as the flammable organic electrolyte. Another disadvantage of Li-air batteries is that they are unsuitable for commercial application due to their high cost and limited lithium resources [3, 4]. Engineering Innovations Vol. 9 24 However, ZABs still require improvement in the oxygen evolution reaction (OER) and oxygen reduction reaction (ORR), which occur during the charge and discharge processes [8, 9]. The problem with OER is that it has inert kinetics as it relates to the occurrence of four sequential electron transfers and the formation of an oxygen-oxygen bond [10, 11]. As a result, the use of a catalyst is required. Introduction Many studies have shown that iridium dioxide (IrO2) and ruthenium dioxide (RuO2) are highly efficient OER electrocatalysts in alkaline electrolytes, but the problem with precious metals is their low natural reserves and high cost [12]. Most catalysts are prepared in powder form, which requires a polymer binder to help the catalyst bind to a conductive substrate. The binders may deteriorate from continuous cycling and from the formation of O2 gas bubbles during the OER reaction, resulting in catalyst detachment and dramatically decreased battery lifespan [3]. Based on the aforementioned problems, efforts were made to design a well-performed, inexpensive, and binder-free OER electrode. Nickel/iron-based materials are regarded as one of the most excellent OER catalysts in alkaline electrolytes, making them one of the attractive low-cost materials as catalysts [13, 14]. As a result, numerous investigations have concentrated on the synthesis of nickel/iron-based electrocatalysts. Layered double hydroxides (LDHs) are great suggestions among them as high-performance catalysts because they have many advantages, such as having a large number of active sites and a large specific surface area [12]. At present, there are many research that use stainless steel mesh (SSM) as the substrate of electrodes because SSM is an easy-to-find, cheap, and durable material. There are also reports that it can increase the OER activity of SSM by improving its surface. Examples of interesting surface improvements are thermal treatment and cathodization treatment. Improving its surface through cathodization can increase OER activity, and this method is also very easy to implement [15]. In this work, we created electrodes from SSM that were surface enhanced by cathodization treatment and then coated with electrocatalysts to serve as OER electrode for ZAB with high efficiency and durability. Experimental Section Materials. Chemicals and Materials used in this work include stainless steel (304) 20 mesh was purchased from Lee Ngiab Seng (Thailand), Nickel (II) sulfate hexahydrate (NiSO4∙6H2O) was purchased from Kemaus, Iron (II) Chloride tetrahydrate (FeCl2∙4H2O) was purchased from Merck, Ammonia solution 25% (NH4OH) was purchased from Loba Chemie Pvt. Ltd., Potassium hydroxide (KOH) was purchased from Kemaus. Preparation of cathodized SSM. The SSM substrate (with dimensions of 1 cm × 1.5 cm) was ultrasonically cleaned in ethanol for 30 minutes. After that, it was cleaned with deionized water and then dried. The cathodization of SSMs was done utilizing a three-electrode setup and repetitive potential cycling, with SSM being used as the working electrode, platinum as the counter electrode, and Hg/HgO as the reference electrode. The cathodization method was carried out in -1.8 to 0.6 V (V vs. Hg/HgO), 0.1 M KOH electrolyte for up to 5 cycles at a scan rate of 10mV/s [15]. Preparation of NiFe2O4/NiFe LDH on cathodized SSM. Cathodized SSM were utilized as a substrate. The precursor solutions of 25 mL (0.05 M) NiSO4∙6H2O and 25 mL (0.1 M) FeCl2∙4H2O were transferred to a beaker, and the pH was adjusted to 10 by adding NH4OH solution dropwise under vigorous stirring while the temperature was kept at 60 °C using a water bath. The cathodized SSM were submerged in the aforementioned solution for 2 hours, with the reaction bath temperature kept at 60 °C. Then the NiFe2O4/NiFe LDH on cathodized SSM were washed with deionized water multiple times and dried in a hot air oven at 75 °C for 1 hour [16]. Physical characterizations. The morphology and elements distribution of SSM surface was studied using field emission scanning electron microscopes (FE-SEM). The composition and Engineering Innovations Vol. 9 25 oxidation state of the synthesized NiFe2O4/NiFe LDH electrocatalyst was determined using x-ray photoelectron spectroscopy (XPS). oxidation state of the synthesized NiFe2O4/NiFe LDH electrocatalyst was determined using x-ray photoelectron spectroscopy (XPS). Electrochemical measurements. Squidstat plus was used to perform electrochemical measurements using a three-electrode system in the 1 M KOH electrolyte. Working electrodes were OER electrode samples, which had dimensions of 1×1 cm. Pt as the reference electrode, and KCl- saturated Ag/AgCl as the counter electrode. The OER activity was measured using linear sweep voltammetry (LSV) curves and Tafel slope is utilized to detect the rate-determining step in a four- electron-transfer process during oxygen evolution. Experimental Section The LSV measurements were performed with a potential range of -0.1 to 1.0 V vs Ref and a scan rate of 10 mV/s. Electrochemical impedance spectroscopy (EIS) was used on samples to acquire a better understanding of the OER kinetics, and it was performed from 1 Hz to 1000 kHz. In addition, the stability of the electrode was measured by a chronopotentiometry test and was conducted at 10 mA/cm2 current density for 50 hours. Results and Discussions A FE-SEM characterization of pristine and cathodized SSM was performed to demonstrate that a treatment affects the surface morphology, as shown in Fig 1A, 1B. From FE-SEM image with a lowest magnification, it was not possible to clearly show the difference in the surface of SSM before and after treatment. When observing the high magnification, it is found that the SSM surface after cathodization has more irregularly shaped particles attached to the surface than ever before. Fig. 1C, 1D displays FE-SEM images of the electrocatalyst on pristine SSM and cathodized SSM. When observing NiFe2O4/NiFe LDH on cathodized SSM at low magnification, the electrocatalyst was found to have a thicker surface covering and formed higher than pristine SSM in some areas. At high magnification, both pristine SSM and cathodized SSM have the same electrocatalyst growth characteristics. The electrocatalyst that is on the pristine SSM and cathodized SSM was synthesized perpendicular to the surface as randomly bonded plates, and on those plates, it contained a lot of small spherical particles perched on it. Figure 2 shows x-ray photoelectron spectra of Ni 2p, Fe 2p, and O 1s for electrocatalyst. According to the results of XPS, Peak of Ni 2p3/2 and Ni 2p1/2 found that it was at positions 855.252, 856.199 and 872.915, 873.945 eV, respectively. These deconvoluted peak pairs relate to Ni2+ and Ni3+, respectively. Next, Peak of Fe 2p3/2 and Fe 2p1/2 found that it was at positions 710.027, 711.709 and 723.776, 725.592 eV, respectively. These deconvoluted peak pairs relate to Fe2+ and Fe3+, respectively. Next, deconvoluted peaks of O 1s with centers at 529.489, 531.060 eV are involved for metal oxides and hydroxides, respectively. The binding energies of nickel-ions, iron- ions and oxygen in this XPS spectra were proven the synthesis of the NiFe2O4/NiFe LDH film on cathodized SSM, in which it has peaks of Ni 2p, Fe 2p, and O 1s that well match the literature reports [16-17]. Engineering Innovations Vol. 9 Engineering Innovations Vol. 9 26 Fig. 1. Low- and high-resolution FE-SEM image for (A) pristine SSM, (B) cathodized SSM, (C) NiFe2O4/NiFe LDH on SSM, (D) NiFe2O4/NiFe LDH on cathodized SSM. Fig. 1. Low- and high-resolution FE-SEM image for (A) pristine SSM, (B) cathodized SSM, (C) NiFe2O4/NiFe LDH on SSM, (D) NiFe2O4/NiFe LDH on cathodized SSM. Fig. 1. Results and Discussions Low- and high-resolution FE-SEM image for (A) pristine SSM, (B) cathodized SSM, (C) NiFe2O4/NiFe LDH on SSM, (D) NiFe2O4/NiFe LDH on cathodized SSM. Fig. 2. XPS spectra of NiFe2O4/NiFe LDH on cathodized-no.4 SSM: (A) Ni 2p, (B) Fe 2p, and (C) O 1s. Fig. 2. XPS spectra of NiFe2O4/NiFe LDH on cathodized-no.4 SSM: (A) Ni 2p, (B) Fe 2p, and (C) O 1s. A three-electrode setup in a 1 M KOH was used to assess the OER performance of the OER electrode. The LSV can show the difference in the OER activity of the samples. Fig. 3A shows the LSV curve comparison of bare SSM, NiFe2O4/NiFe LDH on SSM, and NiFe2O4/NiFe LDH on cathodized SSM. The best samples from all samples are NiFe2O4/NiFe LDH on cathodized SSM, it can be said that cathodization treatment results in a remarkable increase in OER activity. In addition, we have determined the Tafel slope of the bare SSM, NiFe2O4/NiFe LDH on SSM, and NiFe2O4/NiFe LDH on cathodized SSM shown in Fig. 3B. The result of calculating the Tafel slope of the SSM is Engineering Innovations Vol. 9 27 190.5 mV/dec, NiFe2O4/NiFe LDH on SSM is 171 mV/dec, and NiFe2O4/NiFe LDH on cathodized SSM is 125.4 mV/dec. As the results show, it can be interpreted that the NiFe2O4/NiFe LDH on cathodized SSM, showing a small Tafel slope, has the fastest electron transfer rate during oxygen evolution. 190.5 mV/dec, NiFe2O4/NiFe LDH on SSM is 171 mV/dec, and NiFe2O4/NiFe LDH on cathodized SSM is 125.4 mV/dec. As the results show, it can be interpreted that the NiFe2O4/NiFe LDH on cathodized SSM, showing a small Tafel slope, has the fastest electron transfer rate during oxygen evolution. Fig. 3. Graphs of (A) LSV, (B) Tafel slope, (C) EIS, and (D) Chronoamperometric measurements. aphs of (A) LSV, (B) Tafel slope, (C) EIS, and (D) Chronoamperometric measurements The SSM that improves the surface with cathodization was found to have an electrocatalyst that grows higher on the surface than pristine SSM, possibly resulting in an even greater OER activity. In addition, the improvement of SSM surfaces with cathodization also causes OER active site (Ni(OH)2 and NiOOH) to occur on the surface [15], which promotes increased OER activity. The SSM that improves the surface with cathodization was found to have an electrocatalyst that grows higher on the surface than pristine SSM, possibly resulting in an even greater OER activity. Results and Discussions In addition, the improvement of SSM surfaces with cathodization also causes OER active site (Ni(OH)2 and NiOOH) to occur on the surface [15], which promotes increased OER activity. Electrochemical impedance spectroscopy (EIS) was used to determine the behavior of electrode and electrolyte interfaces to obtain additional information about OER kinetics [15-16]. To assess stability, Chronoamperometric measurements were performed. In this work, the EIS and stability measurements were focused on the study of NiFe2O4/NiFe LDH on cathodized SSM, which has the best OER activity. Fig. 3C shows a Nyquist plots, that showed the OER kinetics improved when the SSM was improved with cathodization treatment. The charge transfer resistance (Rct) for NiFe2O4/NiFe LDH on cathodized SSM (5.165 Ω) is lower than Rct for bare SSM (7.193 Ω) and Rct for cathodized SSM (5.856 Ω). This suggests that improving the surface of SSM by cathodization treatment and synthesis of the NiFe2O4/NiFe LDH to grow on its surface allows for greater charge transferability during OER, and when both methods are used together, it allows for a greater charge transfer than ever before. Fig. 3D shows chronoamperometry (i−t) curve of NiFe2O4/NiFe LDH on cathodized SSM at 10 mA/cm2 for 50 hours and demonstrated the high stability of this sample. We also investigated further on the morphology of the electrocatalyst after stability test. Fig. 4 shows the comparison between NiFe2O4/NiFe LDH on cathodized SSM before and after 50 hours of Engineering Innovations Vol. 9 28 stability testing and using the low- and high-resolution FE-SEM to see the electrocatalyst characteristics on the surface. At low magnification, it was found that the highly grown electrocatalyst was detached after testing, but the low-rise electrocatalyst remained well attached to the surface of the SSM surface. At high magnification, it was found that the characteristics of the electrocatalyst remained the same, with its morphology as a plate that rises perpendicular to the surface and is randomly connected, with small spherical particles on those plates. Fig. 4. Low- and high-resolution FE-SEM image for (A) NiFe2O4/NiFe LDH on cathodized SSM and (B) NiFe2O4/NiFe LDH on cathodized SSM after 50 hours of stability testing. Fig. 4. Low- and high-resolution FE-SEM image for (A) NiFe2O4/NiFe LDH on cathodized SSM and (B) NiFe2O4/NiFe LDH on cathodized SSM after 50 hours of stability testing. Fig. Engineering Innovations Vol. 9 Engineering Innovations Vol. 9 29 Conclusion In this work, the effects of cathodization were studied for improving the surface of SSM to greater OER activity for air electrodes in zinc air batteries. The NiFe2O4/NiFe LDH was used as an electrocatalyst, in which it was coated on the surface of SSM samples by one-step chemical bath deposition technique. According to the results, NiFe2O4/NiFe LDH on cathodized SSM (Tafel slope = 125.4 mV/dec) has a greater OER activity than NiFe2O4/NiFe LDH on SSM (Tafel slope = 171 mV/dec). The results are due to the fact that electrocatalyst can deposit on the surface of the cathodized SSM, which is thicker than the surface of the pristine SSM, and the surface of cathodized SSM has a greater OER active site (Ni(OH)2 and NiOOH). In addition, NiFe2O4/NiFe LDH on cathodized SSM also has high durability (10 mA/cm2 for 50 hours). Therefore, SSM that improves the surface with the cathodized can be used as a better substrate for binder-free NiFe2O4/NiFe LDH oxygen evolution reaction electrode. Results and Discussions 5 shows a Nyquist plot of NiFe2O4/NiFe LDH on cathodized SSM after stability test at 10, 20, 30, 40, and 50 hours to confirm its stability, with the results from the figure showing that the NiFe2O4/NiFe LDH on cathodized SSM has constant OER kinetics for 50 hours. The charge transfer resistance (Rct) of NiFe2O4/NiFe LDH on cathodized SSM after 10, 20, 30, 40, and 50 hours is 9.912 Ω, 10.66 Ω, 10.34 Ω, 10.45 Ω, and 10.22 Ω, respectively. Such results show that it has constant charge transferability and support the results of stability tests well. Fig. 5. EIS of NiFe2O4/NiFe LDH on cathodized SSM after 10, 20, 30, 40, and 50 hours of stability testing. Fig. 5. EIS of NiFe2O4/NiFe LDH on cathodized SSM after 10, 20, 30, 40, and 50 hours of stability testing. References [1] J. Cho, S. Jeong, and Y. Kim, Commercial and research battery technologies for electrical energy storage applications, Progress in Energy and Combustion Science. 48 (2015) 84-101. [2] M.S. Ghazvini1, G. Pulletikurthi1, T. Cui1, C. Kuhl1, F. Endres, Electrodeposition of zinc from 1-ethyl-3-methylimidazolium acetate-water mixtures: investigations on the applicability of the electrolyte for Zn-air batteries, Journal of The Electrochemical Society. 165(9) (2018) 354. [3] X. Yan, Y. Ha, R. Wu, Binder‐Free Air Electrodes for Rechargeable Zinc‐Air Batteries: Recent Progress and Future Perspectives, Small Methods. 5(4) (2021) 2000827. [4] J. Pan, Y. Xu, H. Yang, Z. Dong, H. Liu, B. Xia, Advanced architectures and relatives of air electrodes in Zn–air batteries, Advanced Science. 5(4) (2018) 1700691. [5] X. Cai, L. Lai, J. Linb, Z. Shen, Recent advances in air electrodes for Zn–air batteries: electrocatalysis and structural design, Materials Horizons. 4(6) (2017) 945-796. [6] K. Harting, U. Kunz, T. Turek, Zinc-air batteries: prospects and challenges for future improvement, Zeitschrift für Physikalische Chemie. 226(2) (2012) 151-166. [7] B. Lee, H. Lee, H. Kim, K. Chung, B. Choa, S. Hyoung, Elucidating the intercalation mechanism of zinc ions into αMnO 2 for rechargeable zinc batteries, Chemical communications. 51(45) (2015) 9265-9268. [8] T. Zhang, Z. Tao, J. Chen, Magnesium–air batteries: from principle to application, Materials Horizons. 1(2) (2014) 196-206. [9] L. Ma, S. Chen, D. Wang, Q. Yang, F. Mo, G. Liang, N. Li, H. Zhang, J.A. Zapien, C. Zhi, Super‐stretchable zinc–air batteries based on an alkaline‐ tolerant dual‐network hydrogel electrolyte, Advanced Energy Materials. 9(12) (2019) 1803046. [10] B. Zhang, X. Zheng, O. Voznyy, R. Comin, M. Bajdich, M. García-Melchor, L. Han, J. Xu, M. Liu, L. Zheng, F.G. de Arquer, C.T. Dinh, F. Fan, M. Yuan, E. Emre Yassitepe, N. Chen, T. Regier, P. Liu, Y. Li, P. De Luna, A. Janmohamed, H. L Xin, H. Yang, A. Vojvodic, E. Edward H Sargent, Homogeneously dispersed multimetal oxygen-evolving catalysts, Science. 352(6283) (2016) 333-337. [11] L. Han, S. Dong, E. Wang, Transition‐metal (Co, Ni, and Fe)‐based electrocatalysts for the water oxidation reaction, Advanced materials. 28(42) (2016) 9266-9291. [12] F. Liao, X. Zhao, G. Yang, Q. Cheng, L. Mao, L. Chen, Recent advances on two-dimensional NiFe-LDHs and their composites for electrochemical energy conversion and storage, Journal of Alloys and Compounds. 872 (2021) 159649. Engineering Innovations Vol. 9 30 [13] S. Niu, Y. Sun, G. Sun, D. Rakov, Y Li, Y Ma, J. Chu, P. References Xu, Stepwise electrochemical construction of FeOOH/Ni (OH) 2 on Ni foam for enhanced electrocatalytic oxygen evolution, ACS Applied Energy Materials. 2(5) (2019) 3927-3935. [14] M.B. Stevens, C. D. M. Trang, L.J. Enman, J. Deng, S.W. Boettcher, Reactive Fe-sites in Ni/Fe (oxy) hydroxide are responsible for exceptional oxygen electrocatalysis activity, Journal of the American Chemical Society. 139(33) (2017) 11361-11364. [15] G. Zhang, L Shen, P. Schmatz, K. Krois, B. J.M. Etzold, Cathodic activated stainless steel mesh as a highly active electrocatalyst for the oxygen evolution reaction with self-healing possibility, Journal of Energy Chemistry. 49 (2020) 153-160. [16] A.A. Kashale, C. Yi, K. Cheng, J. Guo, Y. Pan, I.P. Chen, Binder-free heterostructured NiFe2O4/NiFe LDH nanosheet composite electrocatalysts for oxygen evolution reactions, ACS Applied Energy Materials. 3(11) (2020) 10831-10840. [17] Z. Cai, D. Zhou, M Wang, S. Bak, Y. Wu, Z. Wu, Y. Tian, X. Xiong, Y. Li, W. Liu, S. Siahrostami, Y. Kuang, X. Yang, H. Duan, Z. Feng, H. Wang, X. Sun, Introducing Fe2+ into nickel–iron layered double hydroxide: local structure modulated water oxidation activity, Angewandte Chemie. 130(30) (2018) 9536-9540.
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Superconductivity assisted change of the perpendicular magnetic anisotropy in V/MgO/Fe junctions
Scientific reports
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To cite this version: César González-Ruano, Diego Caso, Lina Johnsen, Coriolan Tiusan, Michel Hehn, et al.. Supercon- ductivity assisted change of the perpendicular magnetic anisotropy in V/MgO/Fe junctions. Scientific Reports, 2021, 11 (1), pp.19041. ￿10.1038/s41598-021-98079-5￿. ￿hal-04370026￿ Superconductivity assisted change of the perpendicular magnetic anisotropy in V/MgO/Fe junctions César González-Ruano, Diego Caso, Lina Johnsen, Coriolan Tiusan, Michel Hehn, Niladri Banerjee, Jacob Linder, Farkhad Aliev Distributed under a Creative Commons Attribution 4.0 International License Superconductivity assisted change of the perpendicular magnetic anisotropy in V/MgO/Fe junctions César González‑Ruano1, Diego Caso1, Lina G. Johnsen2, Coriolan Tiusan3,4, Michel Hehn4, Niladri Banerjee5, Jacob Linder2 & Farkhad G. Aliev1* César González‑Ruano1, Diego Caso1, Lina G. Johnsen2, Coriolan Tiusan3,4, Michel Hehn4, Niladri Banerjee5, Jacob Linder2 & Farkhad G. Aliev1* Controlling the perpendicular magnetic anisotropy (PMA) in thin films has received considerable attention in recent years due to its technological importance. PMA based devices usually involve heavy-metal (oxide)/ferromagnetic-metal bilayers, where, thanks to interfacial spin-orbit coupling (SOC), the in-plane (IP) stability of the magnetisation is broken. Here we show that in V/MgO/Fe(001) epitaxial junctions with competing in-plane and out-of-plane (OOP) magnetic anisotropies, the SOC mediated interaction between a ferromagnet (FM) and a superconductor (SC) enhances the effective PMA below the superconducting transition. This produces a partial magnetisation reorientation without any applied field for all but the largest junctions, where the IP anisotropy is more robust; for the smallest junctions there is a reduction of the field required to induce a complete OOP transition ( HOOP ) due to the stronger competition between the IP and OOP anisotropies. Our results suggest that the degree of effective PMA could be controlled by the junction lateral size in the presence of superconductivity and an applied electric field. We also discuss how the HOOP field could be affected by the interaction between magnetic stray fields and superconducting vortices. Our experimental findings, supported by numerical modelling of the ferromagnet-superconductor interaction, open pathways to active control of magnetic anisotropy in the emerging dissipation-free superconducting spin electronics. Control of out-of-plane (OOP) anisotropies in ultra thin ferromagnetic multilayer films have revolutionized magnetic storage and spintronics technologies by mitigating the impact of the demagnetizing energy as the bit and magnetic tunnel junction sizes ­diminished1,2. Tuning of perpendicular magnetic anisotropy (PMA) by careful selection of structure ­design3,4 and ­size5 has been among the main challenges of spintronics. Besides the variation of the ferromagnet thickness and interface with oxides, OOP magnetisation reorientation can be achieved by a temporary reduction of the IP-OOP barrier using, for example, heat and microwave ­pulses6,7 or a combination of magnetic field and low ­temperature8. gi p Recently, we demonstrated a fundamentally different route to magnetisation reorientation through the influ- ence of superconductivity on the IP magnetisation ­anisotropy9. The key idea behind this effect is that the mag- netisation aligns to minimize the weakening of the superconducting condensate associated with the creation of spin triplet (ST) Cooper ­pairs10. The spin triplet generation depends on the magnetisation direction relative to the interfacial Rashba spin-orbit field. HAL Id: hal-04370026 https://hal.science/hal-04370026v1 Submitted on 3 Feb 2024 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 Figure 1 shows the experimental configuration and the different types of OOP transition observed above the vanadium TC . Figure 1a shows the V(40 nm)/MgO(2 nm)/Fe(10 nm)/MgO(2 nm)/Fe(10 nm)/Co(20 nm) (N(SC)/FM1/FM2) junctions with a hard Fe/Co layer (FM2) sensing the magnetisation alignment of the 10 nm thick Fe(001) soft layer (FM1). Details about the sample growth, characterization and the experimental set-up are explained in the “Methods” section. All junctions were saturated with a 3 kOe IP magnetic field (see the alignment calibration procedure in Supplementary Material, Sect. 1) before each of the OOP magnetoresistance (TMR) measurements, in order to eliminate magnetic inhomogeneities from previous OOP measurements. All except one of the studied junctions showed OOP anisotropy below 3 kOe. On the right side of the vertical axes of Fig. 1b–d, we indicate the TMR values corresponding to the well established parallel (P), perpendicular out-of-plane (OOP) and antiparallel (AP) states for each sample, which are used to calibrate the angle between the two FM layers ( φ = φFM1 −φFM2 , where φFM1 and φFM2 are the angles of each FM layer with respect to the plane of the layers, as shown in Fig. 1a) with the same procedure as described in Refs.8,9. This indicates that the IP-OOP transition also triggers a total or partial reorientation of the sensing (hard) FM2 layer, providing a resistance close to that of an AP state. Previous OOP ­measurements8 above TC made on only two 20 × 20 µm2 junctions revealed asymmetric transitions into the perpendicular alignment of the soft FM1 layer, without any subsequent transition of the sensing layer with perpendicular fields up to 3 kOe. The present study is made with a total of 16 junctions of four different lateral sizes, where about half of them also demonstrate a transition to an AP configuration when the magnetic field is further increased after the transition to the OOP state has been completed. This AP configuration could potentially be either with the two layers oriented OOP or IP, although it seems rather unlikely that both layers reorient to an IP configuration while the applied OOP field increases. We believe that the high-field-induced transition from OOP to AP alignment or, in some cases, a nearly direct P to AP transition in N/FM1/FM2 junctions could be a consequence of the enhanced antiferromagnetic coupling reported for MgO magnetic tunnel junctions with perpendicular magnetic anisotropy ­(see26). www.nature.com/scientificreports/ non-epitaxial SC/FM ­structures20,21 and Rashba SOC in epitaxial V/MgO/Fe ­junctions9,22 where ST Cooper pairs are generated depending on the magnetisation orientation relative to the Rashba field.h i Theoretically, it has been ­shown10 that a superconductor coupled to a ferromagnet by SOC could stimulate the modification not only of the ­IP9, but also of the OOP magnetic anisotropy below the superconducting critical temperature ( TC ). Due to the stray fields, however, ferromagnetic films are expected to have a stronger interac- tion with the superconductor when an OOP magnetisation is present, compared to a simple IP ­variation23,24. Therefore, a careful consideration of the interaction of these stray field generated by the OOP magnetisation and superconducting vortices is essential to fully capture the factors influencing the effective OOP anisotropy. lf Here, we investigate the superconductivity-induced OOP magnetisation reorientation in epitaxial Fe(001) films with competing IP and OOP anisotropies, both at zero field and in the presence of out-of-plane applied magnetic fields. The V/MgO/Fe(001) junctions are ideal candidates to verify the predicted modification of the effective perpendicular anisotropy in the superconducting state for several ­reasons10. Firstly, the Fe(001) has the ­required10 cubic symmetry; secondly, previous studies show that the normal state IP-OOP reorientation transi- tion takes place at a well-defined critical ­field8; thirdly, the system has Rashba type SOC, which is responsible for the PMA in MgO/Fe25; fourthly, the relative contribution of the IP and OOP magnetisation anisotropies can be tuned by changing the junction lateral size, and SOC can be varied by applying an external electric field; finally, the change in magnetisation can be determined with high precision by studying the transport characteristics using a second magnetically hard Fe/Co layer which is magnetostatically decoupled from the soft Fe ­layer8. h ll h h d l k bl b g g y y g yt y For the smallest junctions, where the IP and OOP anisotropies strongly compete, we remarkably observe the full superconductivity-induced IP-OOP magnetisation reorientation predicted in Ref.10. This results in (i) a decreasing of the required field to induce the full IP-OOP transition below TC , which is not observed in bigger junctions; and (ii) a spontaneous increasing of the misalignment angle between the two FM layers below TC in the absence of applied field, which is consistently observed in all but the largest junctions. www.nature.com/scientificreports/ These differences in the observed behaviour depending on the junctions dimensions are most likely due to the IP anisotropy becoming more dominant with increasing lateral size. We discard the magnetostatic interaction between supeconducting vortices and the FM layers as the main cause of the observed effects. Superconductivity assisted change of the perpendicular magnetic anisotropy in V/MgO/Fe junctions César González‑Ruano1, Diego Caso1, Lina G. Johnsen2, Coriolan Tiusan3,4, Michel Hehn4, Niladri Banerjee5, Jacob Linder2 & Farkhad G. Aliev1* Understanding the factors influencing this superconductivity-induced change of magnetic anisotropy is crucial for designing the next generation of cryogenic memories in the emerg- ing field of superconducting spintronics, where control over non-volatile magnetisation states still remains a major ­challenge11–14. j g The main underlying physical mechanisms for the transformation of ST Cooper pairs from singlet to mixed- spin and equal-spin triplet pairs are magnetic ­inhomogeneities15,16, two misaligned FM ­layers17,18 or ­SOC19. Previous experiments focusing on SOC-driven generation of triplets have focused on heavy metal (Pt) layers in 1Departamento Física de la Materia Condensada C‑III, Instituto Nicolás Cabrera (INC) and Condensed Matter Physics Institute (IFIMAC), Universidad Autónoma de Madrid, Madrid  28049, Spain. 2Department of Physics, Center for Quantum Spintronics, Norwegian University of Science and Technology, 7491 Trondheim, Norway. 3Department of Physics and Chemistry, Center of Superconductivity Spintronics and Surface Science C4S, Technical University of Cluj-Napoca, Cluj‑Napoca 400114, Romania. 4Institut Jean Lamour, Nancy Universitè, 54506  Vandoeuvre‑les‑Nancy Cedex, France. 5Department of Physics, Loughborough University, Epinal Way, Loughborough LE11 3TU, UK. *email: farkhad.aliev@uam.es | https://doi.org/10.1038/s41598-021-98079-5 Scientific Reports | (2021) 11:19041 www.nature.com/scientificreports/ Scientific Reports | (2021) 11:19041 | Results We cannot exclude the possibility that the AP alignment could be triggered by a partial reorientation of the hard Fe/Co layer (with only the Fe part or the atomic layers closer to the Fe/MgO interface in the hard layer orienting OOP, as shown in the sketches in Fig. 1b,c). However, since we measure the total resistance of the junctions, it is impossible to distinguish between these two cases from transport measurements alone. Therefore, we mainly focus on the influence of superconductivity on the transition between IP and OOP states and the partial OOP reorientation at zero magnetic field. Consequently, for the OOP field range reported here, we will assume that the φFM2 angle of the FM2 layer with respect to the in-plane configuration is fixed and close to 0. y p pi gi Figure 1c shows typical OOP TMR cycles measured in two 20 × 20 µm2 junctions, one of them switching to an AP alignment following an OOP orientation (blue) and the other one only switches to the OOP state (red). Figure 1d shows an OOP TMR for a 30 × 30 µm2 junction where the OOP alignment of the FM1 and FM2 elec- trodes remains stable up to 3 kOe. Note that all junctions showed remanent OOP alignment of the soft Fe(001) electrode once the perpendicular magnetic field is removed (Fig. 1). This indicates the relatively small number of interfacial defects present in our junctions, as supported by numerical simulations analyzing the OOP configura- tion robustness as a function of the density of interfacial defects by studying the inverse OOP to IP transition, which are discussed in the Supplementary Material, S2.2.h pp y The symmetry broken spin reorientation observed in the OOP TMR experiments shown in Fig. 1 b–d, has been previously explained in Ref.8 by the difference in the dislocation density present at the top and bottom Scientific Reports | (2021) 11:19041 | https://doi.org/10.1038/s41598-021-98079-5 www.nature.com/scientificreports/ Figure 1. (a) Sketch of the junctions under study where Fe(10 nm) (FM1) is the soft ferromagnet undergoing spin reorientation transitions, while Fe(10 nm)Co(20 nm) (FM2) is the hard (sensing) layer. φFM1 and φFM2 are the OOP angles of each FM layer (i.e. the angle with respect to the plane of the layers). Since the FM2 layer is normally fixed to act as a sensor, φFM2 is assumed to be very close to 0 unless otherwise stated. Results (b–d) show TMR experiments where the field is applied in the OOP direction in 10 × 10 , 20 × 20 and 30 × 30 µm2 junctions respectively, showing the field-induced transition into the nonvolatile OOP state. The right vertical axes indicate the parallel (P), antiparallel (AP) and OOP states for each junction, marked with dotted lines. The inset sketches depict the proposed configuration of the two FM layers in the P (only shown in panel (d)), OOP and AP configurations of the spin valve stack. Figure 1. (a) Sketch of the junctions under study where Fe(10 nm) (FM1) is the soft ferromagnet undergoing spin reorientation transitions, while Fe(10 nm)Co(20 nm) (FM2) is the hard (sensing) layer. φFM1 and φFM2 are the OOP angles of each FM layer (i.e. the angle with respect to the plane of the layers). Since the FM2 layer is normally fixed to act as a sensor, φFM2 is assumed to be very close to 0 unless otherwise stated. (b–d) show TMR experiments where the field is applied in the OOP direction in 10 × 10 , 20 × 20 and 30 × 30 µm2 junctions respectively, showing the field-induced transition into the nonvolatile OOP state. The right vertical axes indicate the parallel (P), antiparallel (AP) and OOP states for each junction, marked with dotted lines. The inset sketches depict the proposed configuration of the two FM layers in the P (only shown in panel (d)), OOP and AP configurations of the spin valve stack. surfaces of the soft Fe(001) layer due to the growth process. This differently affects the top and bottom surface anisotropies, which leads to different intensities at each interface resulting in the magnetisation being more easily reoriented into the OOP configuration for one field direction than the other. This asymmetric field behaviour might seem at odds with the Stoner–Rashba model developed in Ref.27. This model suggests that a net Rashba field related to the asymmetric top and bottom interfaces of a ferromagnetic film leads to a pseudo-dipolar contribution to the anisotropy, which would mainly favor an in-plane magnetisation and an uniaxial-like ani- sotropy favouring the/a perpendicular magnetisation configuration. Correspondingly, the hysteresis curve of a single magnetic (here Fe) layer is expected to be an even function with respect to the external magnetic field. Scientific Reports | (2021) 11:19041 | Results However, we note that the model does not fully account for the complexities discussed below that could lead to the asymmetric hysteresis we observe in our multilayer structures.hi y y y The fact that the hysteresis curve is not an even function of the external magnetic field is simply related to the fact that the model is developed for a single ferromagnetic layer while in our complex heterostructure, we do not reverse the Fe/Co interface magnetisation. This is not unreasonable considering a large interface anisotropy. A full magnetisation reversal including interfacial magnetisation would only result in an asymmetric hysteretic response. Secondly, in our structures stray fields play a relevant/crucial/central role and importantly the stray fields seen by both interfaces are not similar. The bottom interface experiences the stray field of the Fe/Co top bilayer, while the top interface sees the contribution from the bottom Fe layer. In a macrospin model, increasing the stray fields would decrease the perpendicular anisotropy. To fully understand the complexities of the asym- metric magnetisation response, future studies such as direct OOP magnetisation measurements on the MgO/ Fe/MgO structures in the absence of the sensing Fe/Co and V/MgO layers, could be performed. g g g y p It is worth mentioning a distinct feature of our junctions, having a strongly preferred IP magnetisation at room ­temperature8, with the OOP configuration of the soft 10 nm Fe layer only becoming non-volatile below 80 K. In the temperature range in which this study takes place (0.3 to 7 K), the magnetic field required to induce an OOP transition in the soft layer does not typically exceed 2 kOe. These relatively low values (with respect to continu- ous 10 nm thick Fe films) could be explained by the combined influence of a few factors. Firstly, the variation of the relation between the IP and OOP anisotropy energies could vary with temperature, possibly favouring Scientific Reports | (2021) 11:19041 | https://doi.org/10.1038/s41598-021-98079-5 www.nature.com/scientificreports/ Figure 2. (a) Field induced OOP magnetisation transition in a 10 × 10 µm2 SC/FM1/FM2 junction at different temperatures from above to below TC . A strong reduction of HOOP takes place below TC . (b) Shows a similar experiment in a 30 × 30 µm2 junction. In this case, some increasesting in the low field TMR is observed, but not enough to be attributed to a complete OOP reorientation. Results (c) Temperature dependence of the normalized HOOP anisotropy field for junctions with four different lateral sizes. (d) Represents the temperature dependence of the misalignment angle between the two FM layers ( φ = φFM1 −φFM2 , calibrated following the procedure outlined in Ref.9) at zero field for the four different sized samples, using the same color legend as in (c). The inset shows a comparison of the zero field φ angle at T = 5 K (above TC ) and at T = 0.3 K (well below TC ) as a function of the samples’ lateral size. The gradual decrease of the zero-field angle above TC with increasing lateral size points towards a small equilibrium initial angle already existing in the normal state, which we attribute to competing OOP and IP anisotropies. When superconductivity develops below TC , an additional magnetisation reorientation is observed in all except the bigger samples. The colored dashed lines are guides for the eyes, while the vertical, black, dashed lines indicate the critical temperature. Figure 2. (a) Field induced OOP magnetisation transition in a 10 × 10 µm2 SC/FM1/FM2 junction at different temperatures from above to below TC . A strong reduction of HOOP takes place below TC . (b) Shows a similar experiment in a 30 × 30 µm2 junction. In this case, some increasesting in the low field TMR is observed, but not enough to be attributed to a complete OOP reorientation. (c) Temperature dependence of the normalized HOOP anisotropy field for junctions with four different lateral sizes. (d) Represents the temperature dependence of the misalignment angle between the two FM layers ( φ = φFM1 −φFM2 , calibrated following the procedure outlined in Ref.9) at zero field for the four different sized samples, using the same color legend as in (c). The inset shows a comparison of the zero field φ angle at T = 5 K (above TC ) and at T = 0.3 K (well below TC ) as a function of the samples’ lateral size. The gradual decrease of the zero-field angle above TC with increasing lateral size points towards a small equilibrium initial angle already existing in the normal state, which we attribute to competing OOP and IP anisotropies. When superconductivity develops below TC , an additional magnetisation reorientation is observed in all except the bigger samples. Results The colored dashed lines are guides for the eyes, while the vertical, black, dashed lines indicate the critical temperature. the OOP configuration at low ­temperatures28. Secondly, interfacial strain has also demonstrated the potential to induce changes in the perpendicular anisotropy in thin ferromagnetic ­films29. Thirdly, the IP saturation in this study was carried out with a field of 3 kOe. This value was considered sufficiently high since the resistance values were stable above 1 kOe, but it could be insufficient to induce a perfect IP alignment at low temperatures. This factor could be more relevant for the smallest junctions where edge magnetic charges would have a relatively higher influence on the measured OOP switching field, qualitatively explaining the dependence of this field with the junctions lateral size, as supported by numerical simulations (see Supplementary Material Fig. S5). Finally, as mentioned before, as long as we measure the total resistance of the junction, we can’t exclude that the OOP reorientation might take place preferently in the atomic layers closer to the Fe/MgO interface (where it would be easier to reorient the magnetic moments due to the surface anisotropy). Thus, the surface OOP state (with a thickness of a few nm, close to that of the Fe magnetic exchange ­length30) might be realized with the aid of interface anisotropy at the Fe/MgO interface and an external OOP magnetic field. This is shown in the sketches of the spin valve configuration in Fig. 1b–d. the OOP configuration at low ­temperatures28. Secondly, interfacial strain has also demonstrated the potential to induce changes in the perpendicular anisotropy in thin ferromagnetic ­films29. Thirdly, the IP saturation in this study was carried out with a field of 3 kOe. This value was considered sufficiently high since the resistance values were stable above 1 kOe, but it could be insufficient to induce a perfect IP alignment at low temperatures. This factor could be more relevant for the smallest junctions where edge magnetic charges would have a relatively higher influence on the measured OOP switching field, qualitatively explaining the dependence of this field with the junctions lateral size, as supported by numerical simulations (see Supplementary Material Fig. S5). Results Influence of the electric field on the magnetisation reorientation transition HOOP , above ( T = 5 K) d b l T (T 0 3 K) Th t iti i h f li d bi f 600 V ( l t i fi ld f Figure 3. Influence of the electric field on the magnetisation reorientation transition HOOP , above ( T = 5 K) and below TC ( T = 0.3 K). The transition is shown for an applied bias of 600 mV (electric field of about 2.5 × 108 V/m), with both positive and negative polarities, for samples with varying lateral sizes: (a) 10 × 10 µm2 , (b) 20 × 20 µm2 and (c) 30 × 30 µm2 . (d) shows the difference of TMR with temperature (calculated as TMR0.3 K −TMR5 K ) for both polarities and in the absence of applied electric field ( V = 5 mV) as a function of the lateral size, for an applied field of H = −50 Oe. The superconducting transition seems to have bigger effects on the magnetic OOP reorientation for smaller samples. the HOOP anisotropy field turned out to be nearly independent of temperature (Fig. 2c). Interestingly, our junc- tions also revealed spontaneous zero field TMR emerging below TC (corresponding to a partial magnetic reori- entation of the soft FM1 layer), which is more pronounced for the smaller samples and diminishes with lateral size, abruptly disappearing for the largest junctions. This is shown in Fig. 2d, where instead of the TMR, the calculated angle between the two FM layer is plotted. It is worth noting that this relative angle calculation is similar to our previous ­work8,9, and assumes a uniform magnetisation in the whole FM layer. However, the real scenario could be more complex (see Supplementary Material, S2.1). Influence of electric field on the out‑of‑plane reorientation. The presence of the MgO barriers allows us to explore the possible influence of high electric fields on the magnetic-field-induced IP-OOP transi- tions above and below TC . High electric field influences the PMA anisotropy by modifying the SOC Rashba field in magnetic tunnel ­junctions27. Our previous ­study8 revealed that roughly two thirds of the voltage drop in our junctions occurs at the V/MgO/Fe barrier, resulting in a high electric field across this interface. Results Finally, as mentioned before, as long as we measure the total resistance of the junction, we can’t exclude that the OOP reorientation might take place preferently in the atomic layers closer to the Fe/MgO interface (where it would be easier to reorient the magnetic moments due to the surface anisotropy). Thus, the surface OOP state (with a thickness of a few nm, close to that of the Fe magnetic exchange ­length30) might be realized with the aid of interface anisotropy at the Fe/MgO interface and an external OOP magnetic field. This is shown in the sketches of the spin valve configuration in Fig. 1b–d. Superconductivity induced change of the out‑of‑plane anisotropy field. Figure 2a shows the temperature dependence of OOP TMRs in a 10 × 10 µm2 junction, in a field range where the field-induced magnetisation reorientation of the FM1 layer (measured at 5 mV) takes place. A decrease of the characteristic HOOP field (defined as the applied magnetic field providing a complete OOP reorientation of the FM1 layer) just below TC can be observed upon lowering the temperature, as represented in Fig. 2c. We note that the SC-induced full IP-OOP transitions have been clearly observed in the smallest 10 × 10 µm2 lateral size junctions. The larger junctions showed a small low field TMR increase below TC , which could be interpreted either as a partial FM1 layer reorientation or an inhomogeneous OOP alignment (Fig. 2b). For the 20 × 20 µm2 and larger junctions, https://doi.org/10.1038/s41598-021-98079-5 Scientific Reports | (2021) 11:19041 | www.nature.com/scientificreports/ Figure 3. Influence of the electric field on the magnetisation reorientation transition HOOP , above ( T = 5 K) and below TC ( T = 0.3 K). The transition is shown for an applied bias of 600 mV (electric field of about 2.5 × 108 V/m), with both positive and negative polarities, for samples with varying lateral sizes: (a) 10 × 10 µm2 , (b) 20 × 20 µm2 and (c) 30 × 30 µm2 . (d) shows the difference of TMR with temperature (calculated as TMR0.3 K −TMR5 K ) for both polarities and in the absence of applied electric field ( V = 5 mV) as a function of the lateral size, for an applied field of H = −50 Oe. The superconducting transition seems to have bigger effects on the magnetic OOP reorientation for smaller samples. Figure 3. Discussion E l ti Evaluation of magnetostatic coupling between superconducting vortices and ferromag‑ net. Let us start our discussion by considering different scenarios involving the possible magnetostatic cou- pling between the superconducting vortices and the ­ferromagnet31. It is tempting to consider the device edges as mainly responsible for the superconductivity-induced spin reorientation, as the edge has a more important contribution for the smallest samples, in which the minimum applied field is enough to fully reorient the mag- netisation. However, a few experimental facts contradict this scenario. Firstly, the superconductivity-induced additional zero field OOP angle variation is similar for 10 × 10 to 30 × 30 µm2 junctions (see inset in Fig. 2 d), which would not be the case if the change comes from the device’s edges. The superconductivity induced spin reorientation effect abruptly diminishes for the 40 × 40 µm2 junction only (Fig. 2d). Secondly, numerical simu- lations show that the OOP reorientation due to magnetostatic coupling, if relevant, could potentially be triggered by the nucleation of OOP domains in the interior of the samples rather than at the edges; even if we assume the edges as the initial OOP nucleation places, the resulting vortex distribution would affect the whole FM layer (see Supplementary Material S2.1). Finally, electric field stimulates the OOP transition for relatively small junctions with competing anisotropies (see Fig. 3) which points towards the possible role of the Rashba field. p g p g p pi We have seen from micromagnetic simulations and as an experimental trend that, on average, the normal state HOOP increases with the junctions area (Supplementary Material S2.3). This is in agreement with the gradual decrease of the partial OOP magnetisation reorientation with increasing lateral size seen in the normal state, just above the critical temperature (see Fig. 2d). Within the above picture, a lower HOOP field is required to reorient the magnetisation perpendicularly in the smallest junctions, and therefore one would expect a weaker magnetostatic coupling to SC vortices. g p g Numerical simulations of the magnetostatic interaction of the V/MgO/Fe system during an OOP TMR experi- ment such as the ones shown in Fig. 1, where a varying OOP magnetic field is applied, is a complex problem which requires self-consistent treatment of the interaction between magnetic charges and stray fields of super- conducting ­vortices32. The Supplementary Material (Sect. Discussion E l ti 2.1) introduces a simplified simulation scheme which evaluates this interaction in the presence of the Meissner effect. These results show that the vortex-mediated magnetostatic interaction might only explain a weak enhancement of HOOP in the superconducting state in the largest junctions (Fig. 2c). However, we note that varying the superconducting hysteresis strength or width in the magnetostatic simulations could not explain the strong decrease of HOOP below TC which was experimentally observed in the smaller junctions. Moreover, a dominant magnetostatic coupling would contradict the observed influence of electric field on TMR below TC for the smallest junctions (Figs. 2, 3). Microscopic model. To explain the strong decrease in the OOP anisotropy field below TC for the smallest junctions and the superconductivity-induced zero field magnetic reorientation in all except the largest ones, as well as the influence of the SOC strength through the application of an electric field, we present a microscopic model describing the observed superconductivity-assisted OOP magnetic reorientation. In heterostructures consisting of superconducting and magnetic layers, the superconducting condensate is weakened as Cooper pairs leak into the magnetic ­regions33. This leakage is more efficient when the spin-singlets are transformed into equal-spin triplet pairs polarized along the same axis as the magnetisation. In our system, the MgO layer boosts the Rashba SOC at the SC/FM interface allowing for a generation of equal-spin triplets that depends on the orientation of the magnetisation with respect to the ­interface10,20.fifi g To show how the efficiency of the triplet leakage affects the critical field for reorienting the magnetisation OOP, we calculate the free energy of the system from a tight-binding Bogoliubov–de Gennes (BdG) Hamiltonian (see Sect. 3 in the Supplementary Material for a complete description of our method). The V/MgO/Fe structure is modelled as a cubic lattice with electron hopping between neighboring sites. We include conventional s-wave on-site superconducting pairing potential in the V layer, Rashba SOC in the MgO layer, and an exchange split- ting between spins in the Fe layer. Although this model is valid in the ballistic limit, we expect similar results for diffusive materials since spin singlets are partially converted into odd-frequency s-wave triplets that are robust to impurity scattering. www.nature.com/scientificreports/ we observed a larger influence of the electric field on the IP-OOP transition for the smaller junctions. This field is about an order of magnitude below the first critical field of our Vanadium films, which was estimated to be close to 400 ­Oe22, therefore minimizing the presence of vortices in the superconducting layer. We believe that the electric field effect asymmetry could be due to the combined influence of the relatively more dominant proximity effects between the SC and FM states at the V/MgO/Fe interface in smaller junctions, and the electric-field- induced variation of the Rashba field influencing the OOP anisotropy for the non-equivalent interfaces MgO/ Fe and Fe/MgO in the ­junctions27. Results The remaining voltage drops at the Fe/MgO/Fe interface, which is responsible for the change in the resistance providing the measured TMR depending on the relative magnetic configuration of the two FM layers.l p g gi g y We have therefore investigated the influence of high bias and its polarity on the IP-OOP transition in junctions with different lateral sizes. Figure 3a–d show that an applied bias of 600 mV (generating an electric field at the V/MgO/Fe interface exceeding 2 × 108 V/m) hardly affects HOOP above TC , independently of the junctions size. Moreover, the application of a large electric field has also a negligible effect on the superconductivity-induced IP- OOP transition in the larger than 30 × 30 µm2 junctions, with a dominant IP magnetisation alignment (Fig. 3c). However this changes for the smaller junctions, where IP and OOP anisotropy values are comparable leading to an entirely different behaviour. Strikingly, we observe that for 10 × 10 µm2 and 20 × 20 µm2 junctions, the electric field stimulates an IP-OOP transition below TC at very small values of the applied magnetic field (below 100 Oe). Fi d h i fl f l i bi l 6 V i h diff l i i h i C y pp gi ( Figure 3d compares the influence of an electric bias close to 600 mV with different polarities on the m netisation alignment below TC (0.3 K) with an applied magnetic field of −50 Oe, within the field range in wh Scientific Reports | (2021) 11:19041 | https://doi.org/10.1038/s41598-021-98079-5 www.nature.com/scientificreports/ Discussion E l ti Figure 4. When the magnetisation of the soft ferromagnet is rotated from a parallel to an OOP alignment with respect to the hard ferromagnet, as sketched above (a) (In the upper right part, only the part of the soft FM layer closer to the V is depicted, not to scale, in order to show the possible magnetisation configuration. In the theoretical modelling the magnetisation is considered to be uniform for simplicity, altough, as mentioned before, experimentally the magnetisation reorientation is more likely to happen only close to the interface. The FM2 layer is considered to be fixed with an IP orientation), the SOC assisted conversion (white arrows) of singlet Cooper pairs (orange) into equal-spin triplets (blue) is at its minimum for the OOP orientation. The superconducting condensate is therefore stronger when the magnetisation is OOP, causing a decrease in the OOP free energy as the temperature is decreased below TC (panel a)). The deepening of the OOP minimum causes a decrease in HOOP (b)). In (a), KIP = 1.5 and KOOP = 1.6 , while in (b) Kanis = 0.8 favoring the IP orientation. The free energy is scaled by the hopping parameter t. For further details about the parameters used in the BdG calculations, see Sect. 3 in the Supplementary Material. In Fig. 4a), we demonstrate how the local free energy minimum for an OOP magnetisation deepens as the temperature is decreased below TC . As a simple qualitative model, we calculate the external magnetic field that can be used to force the magnetisation into the OOP orientation as HOOP = (Kanis + FOOP −FIP)/µ0µtot , where µtot is the total magnetic moment, Kanis is a constant anisotropy favoring the IP orientation that includes the above mentioned parameters for the normal-state IP and OOP anisotropies, KIP and KOOP , as well as an energy barrier associated with the reorientation; and FOOP and FIP are the calculated free energies in the OOP and IP states of the soft layer respectively. In Fig. 4b, we show how HOOP decreases below TC as observed for the 10 × 10  µm2 junction in Fig. 2c. We have thus demonstrated that the proximity effect enables a strong decrease in HOOP that cannot be explained by the coupling of the ferromagnet to superconducting vortices discussed in the previous section. Discussion E l ti Moreover, since this variation in HOOP requires that SOC is present, it also explains the dependence on the electric field observed for the smaller junctions (Fig. 3). The fact that HOOP decreases over a longer temperature interval than in the experiments, rather than flattening out for low T, is caused by the downscaling of the lattice that is necessary in our theoretical model. In order to scale down the superconduct- ing coherence length so that it remains comparable to the thickness of the superconducting layer, the on-site interaction must be increased, leading to a higher TC . Since the temperature interval is larger, a smaller fraction of the temperatures exist in the low-temperature limit where the free energy is temperature independent. Keep- ing in mind that our measurements of HOOP show a dependence on the magnitude of the Rashba SOC, we can conclude that the SOC induced change in magnetic anisotropy below TC shown here must strongly contribute to the suppression in HOOP for the 10 × 10  µm2 junctions. Discussion E l ti Moreover, the variation in the singlet population under IP to OOP reorientation of the magnetisation have previously been demonstrated both experimentally and by dirty limit ­calculations20.h The free energy determined from this model captures the contribution from the superconducting proximity effect, and also includes a normal-state contribution favoring an IP magnetisation. In addition, we include a nor- mal-state anisotropy KIP[1 −cos4(FM1)] + KOOP[1 −sin2(FM1)] , where FM1 ranges from 0◦ (corresponding to an IP magnetisation of the soft ferromagnet) to 90◦ (OOP magnetisation). In total this gives a normal-state anisotropy favoring an IP magnetisation, with an additional local minimum for the OOP magnetisation direc- tion. Here we only focus on the superconductivity-assisted deepening of these OOP quasi-minima associated with the spin singlet to spin triplet conversion. The increase in HOOP below TC discussed in the previous section is not covered by this theoretical framework, as it does not take into account formation of vortices or the size of the junction. The discussion here is therefore relevant to the smaller junctions where the superconductivity- assisted decrease in HOOP dominates. https://doi.org/10.1038/s41598-021-98079-5 Scientific Reports | (2021) 11:19041 | www.nature.com/scientificreports/ Figure 4. When the magnetisation of the soft ferromagnet is rotated from a parallel to an OOP alignment with respect to the hard ferromagnet, as sketched above (a) (In the upper right part, only the part of the soft FM layer closer to the V is depicted, not to scale, in order to show the possible magnetisation configuration. In the theoretical modelling the magnetisation is considered to be uniform for simplicity, altough, as mentioned before, experimentally the magnetisation reorientation is more likely to happen only close to the interface. The FM2 layer is considered to be fixed with an IP orientation), the SOC assisted conversion (white arrows) of singlet Cooper pairs (orange) into equal-spin triplets (blue) is at its minimum for the OOP orientation. The superconducting condensate is therefore stronger when the magnetisation is OOP, causing a decrease in the OOP free energy as the temperature is decreased below TC (panel a)). The deepening of the OOP minimum causes a decrease in HOOP (b)). In (a), KIP = 1.5 and KOOP = 1.6 , while in (b) Kanis = 0.8 favoring the IP orientation. The free energy is scaled by the hopping parameter t. For further details about the parameters used in the BdG calculations, see Sect. 3 in the Supplementary Material. Conclusions Our experiments point towards the superconductivity induced modification of the perpendicular magnetic ani- sotropy in the epitaxial Fe(001) films in the V(40 nm)/MgO(2 nm)/Fe(10 nm) system. The behaviour depends on the lateral dimensions of the junctions in the following way: First, for the smallest junctions, the magnetic field necessary for a full OOP magnetisation reorientation drops by an order of magnitude in the supercon- ducting state, while for the rest of the junctions it varies only slightly. Second, in all but the largest junctions, an increase in the OOP misalignment angle between the soft Fe(10 nm) layer and the hard one is observed when the temperature is decreased below TC without any applied field. This spontaneous reorientation is similar for 10 × 10 to 30 × 30  µm2 junctions and disappears in the largest ones, suggesting that superconductivity could be affecting the competition between the IP and OOP anisotropies (which is more pronounced for the smaller junctions) rather than being the result of the reorientation taking place at the edges of the samples. The decreasing https://doi.org/10.1038/s41598-021-98079-5 Scientific Reports | (2021) 11:19041 | www.nature.com/scientificreports/ of HOOP transition field in the superconducting state, which could also be stimulated by the application of electric field changing the Rashba SOC, is consistent with the theoretical ­prediction10 of the absolute minimum of free energy corresponding to the OOP spin direction in SC/SOC/FM hybrids with competing (IP vs OOP) anisotropies just below TC . The magnetostatic interaction between vortices and magnetic inhomogeneities could explain a weak hardening of the OOP transition in the largest junctions. A detailed theoretical analysis of the mutual interplay between the inhomogeneous magnetisation of the soft ferromagnet and the superconductor is, however, beyond the scope of this work. Our results open a route to active manipulation of perpendicular magnetic anisotropy in the expanding field of dissipation-free superconducting electronics involving ­spin34–36 or spin polarized ­supercurrents37. References References 1. Piramanayagam, S. N. Perpendicular recording media for hard disk drives. J. Appl. Phys. 102, 011301 (2007). 1. Piramanayagam, S. N. Perpendicular recording media for hard disk drives. J. Appl. Phys. 102, 011301 (2007). 1. Piramanayagam, S. N. Perpendicular recording media for hard disk drives. J. Appl. Phys. 102, 011301 (2007). y g g pp y 2. Dieny, B. & Chshiev, M. 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The V(40 nm)/MgO(2 nm)/Fe(10 nm)/MgO(2 nm)/Fe(10 nm)/ Co(20 nm) MTJ multilayer stacks have been grown by molecular beam epitaxy (MBE) in a chamber with a base pressure of 5 × 10−11 mbar following the procedure described in Ref.38. The samples were grown on (001) MgO substrates. A 10 nm thick seed of anti-diffusion MgO underlayer is grown on the substrate to trap the C from it before the deposition of the Fe (or V). The MgO insulating layer is then epitaxially grown by e-beam evaporation up to a thickness of approximately ∼2 nm and the same process is then executed for the rest of the layers. Each layer is annealed at 450 °C for 20 min for flattening. After the MBE growth, all the MTJ multilayer stacks are patterned in micrometre-sized square junctions by UV lithography and Ar ion etching, controlled step-by-step in situ by Auger spectroscopy. Experimental measurement methods. The measurements are performed inside a JANIS® He3 cryostat (the minimum attainable temperature is 0.3 K). The magnetic field is varied using a 3D vector magnet consist- ing of one solenoid (Z axis) with Hmax = 3.5 T and two Helmholtz coils (X and Y axis) with Hmax = 1 T. In our system the different magnetic states can be distinguished by looking at the resistance, so the relative orientation between two electrodes can be measured. The magnetoresistance measurements are performed by first setting the magnetic field to the desired value, then applying positive and negative current up to the desired voltage (5 mV unless otherwise stated), and averaging the absolute values of the measured voltage for the positive and negative current, obtaining a mean voltage which is used to calculate the resistance at that point. The tempera- ture is measured and controlled with a LakeShore 340 thermometer. Received: 17 June 2021; Accepted: 30 August 2021 www.nature.com/scientificreports/ Tailored flux pinning in superconductor-ferromagnet multilayers with engineered magnetic domain morphology from stripes to skyrmions. Phys. Rev. Appl. 13, 014043 (2020). 34. Palermo, X. et al. Tailored flux pinning in superconductor-ferromagnet multilayers with engineered magnetic domain morphology from stripes to skyrmions. Phys. Rev. Appl. 13, 014043 (2020). p y y pp 35. Shafraniuk, S. E., Nevirkovets, I. P. & Mukhanov, O. A. Modeling computer memory based on ferromagnetic/superconductor multilayers. Phys. Rev. Appl. 11, 064018 (2019). p y y pp 35. Shafraniuk, S. E., Nevirkovets, I. P. & Mukhanov, O. A. Modeling computer memory based on ferromagnetic/superconductor multilayers. Phys. Rev. Appl. 11, 064018 (2019). y y pp ( ) 6. Golod, T., Kapran, O. M. & Krasnov, V. M. Planar superconductor-ferromagnet-superconductor josephson junctions as scanning- probe sensors. Phys. Rev. Appl. 11, 014062 (2019).fl p y pp 37. Jeon, K.-R. et al. Effect of Meissner screening and trapped magnetic flux on magnetization dynamics in thick Nb /Ni80 Fe20/Nb trilayers. Phys. Rev. Appl. 11, 014061 (2019). y pp 37. Jeon, K.-R. et al. Effect of Meissner screening and trapped magnetic flux on magnetization dynamics in thick Nb /Ni80 Fe20/Nb trilayers. Phys. Rev. Appl. 11, 014061 (2019). y y pp 38. Tiusan, C. et al. Spin tunneling phenomena in single crystal magnetic tunnel junction systems. J. Phys. Condens. Matter 19, 165201 (2007). 38. Tiusan, C. et al. Spin tunneling phenomena in single crystal magnetic tunnel junction systems. J. Phys. Condens. Matter 19, 165201 (2007). Acknowledgements g We acknowledge Antonio Lara and Miguel Granda for help with simulations and Yuan Lu for help in sample preparations. The work in Madrid was supported by Spanish Ministerio de Ciencia (RTI2018-095303-B-C55) and Consejería de Educación e Investigación de la Comunidad de Madrid (NANOMAGCOST-CM Ref. P2018/ NMT-4321) Grants. FGA acknowledges financial support from the Spanish Ministry of Science and Innovation, through the “María de Maeztu” Program for Units of Excellence in R&D (CEX2018-000805-M) and “Acción financiada por la Comunidad de Madrid en el marco del convenio plurianual con la Universidad Autónoma de Madrid en Línea 3: Excelencia para el Profesorado Universitario”. D.C. has been supported by Comunidad de Madrid by contract through Consejería de Ciencia, Universidades e Investigación y Fondo Social Europeo (PEJ- 2018-AI/IND-10364). NB was supported by EPSRC through the New Investigator Grant EP/S016430/1. The work in Norway was supported by the Research Council of Norway through its Centres of Excellence funding scheme Grant 262633 QuSpin. C.T. acknowledges “EMERSPIN” grant ID PN-IIIP4-ID-PCE-2016-0143, No. UEFISCDI: 22/12.07.2017 and “MODESKY” Grant ID PN-III-P4-ID-PCE-2020-0230 No. UEFISCDI: 4/04.01.2021. The work in Nancy was supported by CPER MatDS and the French PIA project “Lorraine Université d’Excellence”, reference ANR-15-IDEX-04-LUE. References Phys. Rev. B 97, 214509 (2018). https://doi.org/10.1038/s41598-021-98079-5 Scientific Reports | (2021) 11:19041 | Competing interests h p g The authors declare no competing interests. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 23. Dubonos, S. V., Geim, A. K., Novoselov, K. S. & Grigorieva, I. V. Spontaneous magnetization changes and nonlocal effects in mesoscopic ferromagnet-superconductor structures. Phys. Rev. B 65, 220513(R) (2002). p g p y 24. Milošević, M. V. & Peeters, F. M. Interaction between a superconducting vortex and an out-of-plane magnetized ferromag disk: Influence of the magnet geometry. Phys. Rev. B 68, 094510 (2003). disk: Influence of the magnet geometry. Phys. Rev. B 68, 094510 l g g y y 25. Yang, H. X., Chshiev, M. & Dieny, B. First-principles investigation of the very large perpendicular magnetic anisotropy at Fe–MgO and Co–MgO interfaces. Phys. Rev. B 84, 054401 (2011). 6. Nistor, L. E. et al. Oscillatory interlayer exchange coupling in MgO tunnel junctions with perpendicular magnetic anisotropy. Phys Rev. B 81, 220407(R) (2010).i Rev. B 81, 220407(R) (2010). 27. Barnes, S. E., Ieda, J. & Maekawa, S. Rashba spin-orbit anisotropy and the electric field control of magnetism. Sci. Rep. 4, 4105 (2014). 27. Barnes, S. E., Ieda, J. & Maekawa, S. Rashba spin-orbit anisotropy and the electric field control of magnetism. Sci. Rep. 4, (2014). ( ) 8. Wang, W. et al. Out-of-plane magnetic anisotropy enhancement in La1−x Srx CoO3−δ/La2/3 Sr1/3 MnO3/La1−x Srx CoO3−δ thin films. Phys. Rev. B 101, 024406 (2020).i i y 29. Wolloch, M. & Suess, D. Strain-induced control of magnetocrystalline anisotropy energy in FeCo thin films. J. Magn. Magn. M 522, 167542 (2021).i ( ) 30. Abo, G. S. et al. Definition of magnetic exchange length. IEEE Trans. Magn. 49, 8 (2013). 30. Abo, G. S. et al. Definition of magnetic exchange length. IEEE Trans. Magn. 49, 8 (2013). i g g g g 31. Fritzsche, J., Kramer, R. B. G. & Moshchalkov, V. V. Visualization of the vortex-mediated pinning of ferromagnetic domai superconductor-ferromagnet hybrids. Phys. Rev. B 79, 132501 (2009). 31. Fritzsche, J., Kramer, R. B. G. & Moshchalkov, V. V. Visualization of the vortex-mediate superconductor-ferromagnet hybrids. Phys. Rev. B 79, 132501 (2009). p g y y 32. Niedzielski, B. & Berakdar, J. Controlled vortex formation at nanostructured superconductor/ferromagnetic junctions. Phys. S Solidi B 257, 7 (2019). 32. Niedzielski, B. & Berakdar, J. Controlled vortex formation at nanostructured superconductor/ferromagnetic junctions. Phys. Status Solidi B 257, 7 (2019). ( ) 33. Eschrig, M. Spin-polarized supercurrents for spintronics: A review of current progress. Rep. Prog. Phys. 78, 104501 (2015).l 34. Palermo, X. et al. Author contributions F.G.A. and C.T. designed the junctions, and C.T. and M.H. fabricated the junctions. The measurements were performed by C.G.-R. with the participation of D.C. The modelling was developed by L.G.J. and J.L. Micro- magnetic and TDGL calculations have been carried out by D.C. and C.G-R. F.G.A., C.G.-R., D.C., L.G.J., N.B. and J.L. analyzed the data and wrote the manuscript. All authors contributed to the study, discussed the results, commented and revised the manuscript. F.G.A. supervised the work. 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Additional informationh Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​021-​98079-5. Correspondence and requests for materials should be addressed to F.G.A. Correspondence and requests for materials should be addressed to F.G.A. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps institutional affiliations. https://doi.org/10.1038/s41598-021-98079-5 Scientific Reports | (2021) 11:19041 | 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 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. © The Author(s) 2021 https://doi.org/10.1038/s41598-021-98079-5 Scientific Reports | (2021) 11:19041 |
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Screening and Identification of an H-2Kb-Restricted CTL Epitope within the Glycoprotein of Hantaan Virus
Frontiers in cellular and infection microbiology
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Screening and Identification of an H-2Kb-Restricted CTL Epitope within the Glycoprotein of Hantaan Virus The cytotoxic T lymphocyte (CTL) response plays a key role in controlling viral infection, but only a few epitopes within the HTNV glycoprotein (GP) that are recognized by CTLs have been reported. In this study, we identified one murine HTNV GP-derived H2-Kb-restricted CTL epitope in C57BL/6 mice, which could be used to design preclinical studies of vaccines for HTNV infection. First, 15 8-mer peptides were selected from the HTNV GP amino acid sequence based on a percentile rank of <=1% by IEDB which is the most comprehensive collection of epitope prediction and analysis tool. A lower percentile rank indicates higher affinity and higher immune response. In the case of the consensus method, we also evaluated the binding score of peptide-binding affinity by the BIMAS software to confirm that all peptides were able to bind H2-Kb. Second, one novel GP-derived CTL epitope, GP6 aa456-aa463 (ITSLFSLL), was identified in the splenocytes of HTNV-infected mice using the IFN-γ ELISPOT assay. Third, a single peptide vaccine was administered to C57BL/6 mice to evaluate the immunogenic potential of the identified peptides. ELISPOT and cell-mediated cytotoxicity assays showed that this peptide vaccine induced a strong IFN-γ response and potent cytotoxicity in immunized mice. Last, we demonstrated that the peptide-vaccinated mice had partial protection from challenge with HTNV. In conclusion, we identified an H2-Kb-restricted CTL epitope with involvement in the host immune response to HTNV infection. ORIGINAL RESEARCH published: 25 November 2016 doi: 10.3389/fcimb.2016.00151 Received: 10 September 2016 Accepted: 31 October 2016 Published: 25 November 2016 Received: 10 September 2016 Accepted: 31 October 2016 Published: 25 November 2016 Keywords: HTNV, epitope, glycoprotein, IFN-γ, infection INTRODUCTION Hantaan virus (HTNV), the prototype of the Hantavirus genus, is a rodent-borne pathogen that is the major causative agent of hemorrhagic fever with renal syndrome (HFRS; Abbott et al., 1999). The genome of HTNV consists of L, M, and S segments, which encode the viral RNA-dependent RNA polymerase, glycoprotein, and nucleoprotein, respectively (Cheng et al., 2014). HFRS is a severe, life-threatening illness characterized by fever, hemorrhage, and renal failure and is a serious public health threat in China (Hansen et al., 2015). A total of 136,012 cases were reported during 2005–2015 in mainland China, with a case-fatality rate as high as 10–15% (Zhang et al., 2010; Kang et al., 2012). To date, several types of inactivated vaccines targeting the pathogens that cause HFRS have been licensed (Liu et al., 2016). However, these inactivated vaccines have many shortcomings, Edited by: Gong Cheng, Tsinghua University, China Reviewed by: Wei Hou, Wuhan University, China Penghua Wang, New York Medical College, USA *Correspondence: Fang Wang wangf07@fmmu.edu.cn Xing-an Wu wuxingan@fmmu.edu.cn †These authors have contributed equally to this work and should be considered co-first authors. Edited by: Gong Cheng, Tsinghua University, China Reviewed by: Wei Hou, Wuhan University, China Penghua Wang, New York Medical College, USA *Correspondence: Fang Wang wangf07@fmmu.edu.cn Xing-an Wu wuxingan@fmmu.edu.cn †These authors have contributed equally to this work and should be considered co-first authors. Keywords: HTNV, epitope, glycoprotein, IFN-γ, infection Received: 10 September 2016 Accepted: 31 October 2016 Synthesis of Peptides The 15 8-mer peptides used in the ELISPOT screens correspond to the glycoprotein of HTNV strain 76–118 (Table 1). All of these peptides were synthesized with greater than 98% purity as determined by high-performance liquid chromatography (HPLC) and resuspended in sterile phosphate-buffered saline (PBS) solution containing DMSO (1 mg/mL). Each peptide is H-2Kb-restricted. HTNV nucleoprotein (NP) aa221–aa228 (SVIGFLAL) derived from the HTNV nucleoprotein was used as a positive control epitope (Park et al., 2000). We used computer algorithms in Immune Epitope Database and Analysis Resources (IEDB-http://tools.iedb.org/main/) to identify optimal peptide epitopes within the reactive 8-mer peptides. Citation: Ma R-x, Cheng L-f, Ying Q-k, Liu R-r, Ma T-j, Zhang X-x, Liu Z-y, Zhang L, Ye W, Zhang F-l, Xu Z-k, Wang F and Wu X-a (2016) Screening and Identification of an H-2Kb-Restricted CTL Epitope within the Glycoprotein of Hantaan Virus. Front. Cell. Infect. Microbiol. 6:151. doi: 10.3389/fcimb.2016.00151 November 2016 | Volume 6 | Article 151 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Identification of the HTNV-GP CTL-Epitope Ma et al. Viruses and Cells HTNV strain 76–118 was obtained from our repository. EL- 4 and P815 cells, which express H-2b and H-2d as MHC class I molecules, were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). EL-4 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% horse serum (HyClone, Logan, UT, USA). All other cells were maintained in DMEM that was supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA). The antigenicity of Hantaviruses largely depends on two structural proteins, the nucleocapsid protein (NP) and envelope glycoprotein (GP) (Zeier et al., 2005). NP is highly immunogenic, inducing vigorous cellular and humoral immune responses in humans, and conserved among viral species. GP is not only responsible for receptor binding and membrane fusion, but is also considered the main target of neutralizing antibodies (Wang et al., 2011). T cells are essential components of the immune response to most viral infections. Therefore, epitopes of HTNV NP that elicit CD4+ and CD8+ cytotoxic T lymphocyte responses have been extensively studied (Ennis et al., 1997; Van Epps et al., 1999; Lee et al., 2002). Viral GP-specific T cell responses have also been discovered (Ma et al., 2015). Protective immunity elicited through infection with recombinant HTNV- glycoprotein in murine models has also been demonstrated (Yu et al., 2013). Recent interest has emerged in the concept of HTNV glycoprotein serving as a potent immunogen to induce T cell responses (Manigold et al., 2010). However, T cell epitopes on the HTNV glycoprotein have not been fully identified, and specific responses to these immunogens remain largely unknown. Animals Pathogen-free C57BL/6 female mice between 8 and 10 weeks old were purchased from the animal research center of the Fourth Military Medical University (Xi’an, China). The mice were cared for in accordance with the Guide for the Care and Use of Laboratory Animals. The animals were acclimated to the laboratory environment for 5–7 d before use. While in their home cage environment, the animals were allowed free access to a standard animal diet and tap water. The room was maintained at 20–23◦C with a 12 h/12 h light/dark cycle. The animals were deeply anesthetized using inhaled isoflurane (1–3% or as needed) before all operations. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo experiments whenever appropriate or feasible. The animal protocol was approved by the Fourth Military Medical University Medical Ethics Committee (Xi’an, Shaanxi, China; approval no.XJYYLL-2015510). Experiments were performed in age-matched groups. MATERIALS AND METHODS including poor immunogenicity, which hinders the production of neutralizing antibodies or cell-mediated immunity (Song et al., 1992), and the concern of safety when using inactivated vaccines, which may contain some infectious particles. Furthermore, there is no study reporting that it could establish long-term memory immunity (Jiang et al., 2015). Thus, it is necessary to develop an alternative immunotherapy strategy that can induce robust humoral immune response to produce therapeutically effective antiviral antibodies and T cell-mediated autoimmune responses. However, epitope based vaccines represent a powerful approach to induce a specific immune response against the selected epitopes, avoiding the side effects of other unfavorable epitopes in the complete antigen. What’s more, epitope based vaccine has other considerable advantages, including increased safety, the opportunity to engineer the epitopes rationally for increased potency and breadth, and the ability to focus the immune response on conserved epitopes CTL epitope-specific T cell immunity demonstrate to be a long-term approach immune strategy. Moreover, the epitope based vaccine approach has been shown to be successful in various infectious diseases, such as Neisseria meningitides infection, HIV (Park et al., 2016), RSV, and tuberculosis (Paul et al., 2013). Therefore, a potent T cell-activating peptide vaccine based on the HTNV structural protein may be a promising method of disease control (Ma et al., 2013). Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Ex vivo IFN-γ Enzyme-Linked Immunospot (ELISPOT) Assay During viral infection, CTLs are important for viral clearance and preventing immunopathology in humans (Kiepiela et al., 2007). To create a mouse model for studying the role of HTNV-specific T cells in vivo, we employed a newly identified mouse CTL epitope on the HTNV glycoprotein. In our study, we describe a CTL epitope on HTNV GP and analyze the cellular immune response against HTNV. Our findings could support the production of an effective peptide vaccine for HTNV infection in humans. To our knowledge, this is the first report about mouse CTL epitopes on the glycoprotein of HTNV. Identification of the HTNV GP-specific CTL epitopes was performed using the IFN-γ ELISPOT assay (Mabtech, Büro Deutschland, Germany) according to the manufacturer’s instructions. After 7 days of infection with HTNV, mice were sacrificed and their spleens harvested. Briefly, splenocytes were prepared and assayed for their ability to secrete IFN-γ during in vitro restimulation with antigenic peptides. The positive peptides in the first round screen were subsequently characterized in CD4- or CD8-depleted splenocytes using anti-CD4-coated November 2016 | Volume 6 | Article 151 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 2 Identification of the HTNV-GP CTL-Epitope Ma et al. TABLE 1 | Synthetic peptides on the glycoprotein of HTNV predicted to bind with H2-Kb. Serial number 8-mer Octamer Percentile_ Binding of peptide peptide sequencea rankb scorec GP 1 39∼46: SVIGYVEL 0.75 7.2 GP 2 44∼51 VELPPVPL 0.75 4.5 GP 3 64∼71 SMDNHQSL 0.75 7.4 GP 4 208∼215 IVCFFVAV 0.55 3.7 GP 5 420∼427 VNFVCQRV 0.5 5.7 GP 6 456∼463 ITSLFSLL 0.25 10.3 GP 7 489∼496 VTFCFGWV 0.2 6.8 GP 8 499∼506 PAITFIIL 0.5 8.1 GP 9 797∼804 ITIRYSRR 0.4 4.9 GP 10 845∼852 TLLFFGPL 0.4 8.4 GP 11 925∼932 QSFNTSTM 0.4 3.4 GP 12 987∼994 VGFTLTCL 0.5 4.3 GP 13 1102∼1109 FSGNWIVL 0.7 3.6 GP 14 1113∼1120 CVFLLFSL 0.85 4.9 GP 15 1114∼1121 VFLLFSLV 0.25 3.1 NP 1 221–228 SVIGFLAL 0.2 11.0 aCorresponding to HTNV (76–118) GP. bA percentile rank in H-2Kb restriction, was calculated by IEDB. cThe binding score in H-2Kb restriction, expressed as arbitrary units, was calculated by the BIMAS software. TABLE 1 | Synthetic peptides on the glycoprotein of HTNV predicted to bind with H2-Kb. splenocytes (effector cells) were added to a final volume of 50 µL at effector/target (E/T) ratios of 100:1, 50:1, 20:1, and 10:1. Cell-Mediated Cytotoxicity Assay Cell-Mediated Cytotoxicity Assay The lactate dehydrogenase (LDH)-releasing CytoTox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI, USA) was performed to detect the level of specific cytotoxicity, as described in the manufacturer’s protocol. CD8+ T cells isolated with anti-CD8-coated Dynalbeads from the splenocytes of HTNV-immunized mice were used as effector cells. Target cells used in this study were peptide-pulsed EL-4 cells, HTNV- inoculated macrophages, and control cells (P815). Target cells were plated at 1 × 104 cells per well in a volume of 50 µL in wells of a 96-well U-bottomed microtiter plate. The Ex vivo IFN-γ Enzyme-Linked Immunospot (ELISPOT) Assay The assay plate included the following controls: spontaneous lactate dehydrogenase (LDH) release from effector cells alone (50 µL of effector cells and 50 µL of 10% FBS RPMI-1640 medium), spontaneous LDH release from target cells alone (50 µL of target cells and 50 µL of 10% FBS RPMI-1640 medium), maximum LDH release from target cells alone (50 µL of target cells, 50 µL of 10% FBS RPMI-1640 medium and 10 µL of the lysis solution), a volume correction control (100 µL of 10% FBS RPMI-1640 medium and 10 µL of the lysis solution), and a culture medium background control (100 µL of 10% FBS RPMI-1640 medium). The percentage of cells lysis was calculated according to the following formula: % cytotoxicity = [(E −St −Se)/(M −St)] × 100% (E, LDH release from effector-target co-culture cells; St, spontaneous LDH release from target cells; Se, spontaneous LDH release from effector cells; M, maximum LDH release from target cells). All of the CTL assays were performed in triplicate and repeated at least twice. Intracellular Cytokine Staining y g Two million freshly isolated splenocytes from HTNV-infected mice were stimulated for 4 h with HTNV GP-specific peptides (10 µg/mL for each peptide). Cells stimulated with PHA (0.1 µg/ml, Sigma-Aldrich, St. Louis, MO) or medium alone were used as positive and negative controls, respectively. After washing, splenocytes were stained with antibodies against surface markers, CD3-FITC and CD8-PerCP/Cy5.5 (BD Pharmingen), in different combinations for 40 min at 4◦C, followed by washing, fixation and permeabilization with a buffer (BD Biosciences, San Jose, CA). The cells were subsequently stained with antibodies against intracellular markers IFN-γ-PE (BD Pharmingen), for 40 min at 4◦C, followed by washing, and acquisition using a FACS Calibur flow cytometer (Becton Dickinson). A total of 100,000 events per sample from the lymphocyte gate were collected for each analysis. Dynalbeads (Invitrogen Dynal AS, Oslo, Norway) and anti-CD8- coated Dynalbeads (Invitrogen Dynal AS, Oslo, Norway). Total splenocytes or the isolated T cells were seeded in ELISPOT plates at 5 × 105 cells/well and stimulated with 15 8-mer peptides at a final concentration of 10 µg/mL. Cells with nonspecific phytohemagglutinin stimulation (PHA, 10 µg/mL, Sigma- Aldrich, St. Louis, MO) or without peptide stimulation served as positive and negative controls, respectively. Additionally, spleen cells pulsed with the immunodominant NP221–228 peptide (Park et al., 2000) was used as an epitope positive control. For quantification of ex vivo responses, the assay was performed in duplicate. An automated ELISPOT reader (Cellular Technology Limited, USA) was used to count the spots. Spot-forming cells (SFC) were adjusted by subtracting average negative values and expressed as SFC/106 splenocytes. A positive response was defined as having at least 100 SFC/106 input cells. The SFC/106 splenocytes in unstimulated control wells never exceeded 5 spots per well. Ex vivo Proliferation Assay y The CFSE-labeled proliferation assay was performed as previously described. Briefly, 1 × 107/ml PBMCs were labeled with 10 µM 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, OR) at 37◦C for 15 min. Labeling was terminated upon the addition of fetal bovine serum and the cells were stimulated with HTNV GP-specific peptides (10 µg/mL). Anti-mouse CD3 (Biolegend) stimulation of PBMCs and no peptide stimulation served as positive and negative controls, respectively. After 2 days, 10% exogenous IL-2 was added. After 5 days, the cells were harvested to detect their expansion capacity. Approximately 100,000 cells were acquired using a FACS Calibur (BD Immunocytometry Systems, California). Identification of Novel H-2Kb-Restricted CTL Epitopes within the Glycoprotein of HTNV by ELISPOT Assay Each tissue sample was weighed, diluted in PBS, ground, and then freeze-thawed (−80/37◦C) three times to prepare a 10% (g/mL) tissue suspension. The suspension was centrifuged at 12,000 rpm for 10 min at 4◦C to collect the supernatant. HTNV antigens in the supernatant were detected by sandwich ELISA. The mAb 1A8 was used as a coating antibody, and HRP-conjugated 1A8 was used as the detecting antibody. Normal tissue supernatant was used as a negative control. The absorbance was measured at a wavelength of 450 nm using a Synergy HT ELISA plate reader (Biotec, Dresden, Germany). Absorbance of >0.1 was required as positive and positive/negative (P/N) >2.1 was considered significant. y y To examine the T cell responses specific for HTNV-glycoprotein in C57BL/6 mice, we first screened T cell epitopes on HTNV- glycoprotein. Splenocytes isolated from HTNV-infected mice were tested against each peptide by enumeration of peptide- specific IFN-γ SFC using an ex vivo ELISPOT assay. Peptides GP1, GP2, GP3, GP6, GP8, and GP10 were found to induce IFN-γ production with a spot magnitude of 225, 130, 257, 461, 196, and 162 SFC/106 input cells, respectively, whereas the remaining peptides only elicited a weak response (Figure 1A). Notably, the GP6 peptide elicited the strongest IFN-γ response among the various peptides tested, with a frequency of 461 SFC/106 input cells, which was similar to the 516 SFC/106 input cells of NP1. This finding indicates that six of 15 HTNV GP-specific peptides could induce a strong IFN-γ response. HTNV Nucleic Acid Detection in Tissues by qRT-PCR Total cellular RNA was extracted from each tissue sample using an RNAprep Pure Tissue Kit (Tiangen, Beijing, China), then 1 µg of total cellular RNA from each sample was used in a qRT-PCR using a SYBR Premix Ex Taq II Kit and the following HTNV RNA-specific primers: HTNV (forward) 5′-GATCAGTCACAGTCTAGTCA-3′ and HTNV (reverse) 5′- TGATTCTTCCACCATTTTGT-3′. GAPDH was included in the qRT-PCR as an internal control for cellular RNA using the following mouse GAPDH-specific primers: GAPDH (forward) 5′-AGGCCGGTGCTGAGTATG TC-3′ and GAPDH (reverse) 5′-TGCCTGCTTCACCACCTTCT-3′. p Then, to determine a more precise understanding of the epitope-specific T cell responses, CD4+ or CD8+ T cell- depleted splenocytes from HTNV-infected mice were employed as effector cells. The results showed that one peptide (GP6 aa456∼463: ITSLFSLL) induced significant numbers of IFN-γ- producing cells in CD4+ T cell-depleted cultures and limited the response magnitude in CD8+ T cell-depleted cultures. Identification of Novel H-2Kb-Restricted CTL Epitopes within the Glycoprotein of HTNV by ELISPOT Assay GP6 peptide induced a significantly higher numbers of IFN-γ spots in total splenocytes cultures compared to the spots in CD8+ T cell-depleted cultures (p < 0.0001), but significant difference has not been observed when compared to the spots in CD4+ T cell-depleted cultures. This finding confirms that GP6 is a CTL epitope on HTNV GP. However, CD4+ T cell depletion completely abrogated the IFN-γ responses induced by another 5 Flow Cytometric Analysis Flow Cytometric Analysis Flow cytometric analysis was performed immediately with FlowJo version 9.2 (TreeStar). FITC-, PE-, and PerCP-Cy5.5- conjugated mouse IgG1, κ were used as isotype controls of the 3-color staining method. Lymphocytes were defined by FSC/SSC, and CD8+ T cells were defined as CD3+CD8+ events, which were then displayed on flow cytometric plot. The cytokine response was considered positive when the percentage of cells producing the cytokine was greater than 0.1% after background subtraction. vaccine per mouse consisted of 100 µL (100 µg) of GP6 packaged with either 100 µL complete Freund’s adjuvant (CFA) at primary immunization or incomplete Freund’s adjuvant (IFA) at boost immunization. Each vaccine solution was emulsified. Four control groups were used: the first two groups were immunized with 100 µL (100 µg) of NP 1 packaged with 100 µL Freud’s adjuvant or 200 µL (100 µg) of commercially purchased inactivated Hantavirus vaccine (Tianyuan, Hangzhou, Zhejiang, China) per mouse; and the other two groups were given either 200 µL of PBS or 200 µL of Freud’s adjuvant per mouse. At 10 d after the last immunization, 5 mice of each group were sacrificed and their splenocytes were isolated for subsequent assays. Detection of HTNV Antigens in Mouse Tissues by Detection of HTNV Antigens in Mouse Tissues by ELISA Immunizations As shown in Figure 5A, 8-week-old C57BL/6 (H-2b) mice (10 mice/group) were subcutaneously immunized four times with mixed peptide vaccines at 10 day intervals. The mixed peptide November 2016 | Volume 6 | Article 151 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 3 Identification of the HTNV-GP CTL-Epitope Ma et al. Statistical Analysis Data were analyzed for statistical significance using GraphPad Prism 5 software. All data are expressed as the mean ± the standard error of mean (SEM) and are representative of at least three independent experiments. Comparisons of positive rate were analyzed by one way ANOVA. Results of ELISPOT assay were compared by one way ANOVA, and CTL assay was compared using two way ANOVA. Protective efficacy was compared using one way ANOVA. A p-value of 0.05 or less was considered significant. Animal Challenge/Protection Assay At 10 d after the final boost immunization, the remaining mice of each group received an infectious dose (1 × 105 pfu/mouse) of HTNV strain 76–118 intramuscularly. After 3 days of infection, the mice were killed, and the major tissues, including the heart, liver, spleen, lung, kidney, and cerebrum, were individually collected for subsequent assays. Histopathological Analysis Each tissue sample was embedded in a paraffin block and cut with a microtome into 5-µm sections, which were subsequently mounted onto glass slides and stained with hematoxylin and eosin (H&E). November 2016 | Volume 6 | Article 151 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 4 Ma et al. Ma et al. Identification of the HTNV-GP CTL-Epitope FIGURE 1 | Identification of an HTNV GP-specific CD8+ T cell epitope by IFN-γ ELISPOT assay. (A) IFN-γ ELISPOT analysis of splenocytes obtained 7 d after HTNV infection (n = 6). Cells were stimulated with 15 8-mer peptides corresponding to HTNV glycoprotein. (B) IFN-γ ELISPOT analysis of splenocytes obtained 7 d after infection (n = 6) with the depletion of CD4+ T cells (CD4 depleted SP) and CD8+ T cells (CD8 depleted SP) or without depletion (Total SP). ***P < 0.0001. FIGURE 1 | Identification of an HTNV GP-specific CD8+ T cell epitope by IFN-γ ELISPOT assay. (A) IFN-γ ELISPOT analysis of splenocytes obtained 7 d after HTNV infection (n = 6). Cells were stimulated with 15 8-mer peptides corresponding to HTNV glycoprotein. (B) IFN-γ ELISPOT analysis of splenocytes obtained 7 d after infection (n = 6) with the depletion of CD4+ T cells (CD4 depleted SP) and CD8+ T cells (CD8 depleted SP) or without depletion (Total SP). ***P < 0.0001. peptides (GP1, GP2, GP3, GP8, and GP10; Figure 1B), indicating that these peptides are CD4+ T cell epitopes that could be immunodominant. macrophages were also lysed by the primary antiviral CTLs at higher rates of 41.4, 32.7, 20.8, and 15.8%, at each E/T ratio mentioned above, respectively. These results indicate that GP6 can elicit CTL responses and plays a role in the primary antiviral response of HTNV-infected mice. The Cytotoxic Capacity of HTNV Glycoprotein-Specific CD8+ T Cells A second cell-mediated cytotoxicity assay was performed to evaluate whether GP6 could enhance peptide-specific cytotoxicity of CTLs. Splenocytes from HTNV-infected mice were restimulated in vitro with GP6 and NP1. Figure 2C shows that restimulation with GP6 resulted in strong CTL responses with percentages of cell death of 63.2, 58.6, 50.3, and 31.2% at E/T ratios of 100:1, 50:1, 20:1, and 10:1, respectively. NP1 peptide-specific cytotoxicity was greater with higher percentages of 71.1, 59.6, 53.9, and 45.9%, respectively (Figure 2D). Although, GP6 induced a relatively weaker CTL response compared to NP1, this peptide caused strong cytotoxic activity, which was especially significant at the E/T ratios of To further characterize the GP6 peptide as a CTL epitope, cytotoxic assays using GP6 and NP1 were performed. As described in methods, HTNV-infected macrophages, peptide- pulsed EL-4 cells and P815 cells (control cells) were used as target cells. As shown in Figure 2A, EL-4 cells pulsed with GP6 were killed by primary antiviral CTLs, with cell death percentages of 25.5, 19.3, 13.2, and 10.5% at E/T ratios of 100:1, 50:1, 20:1, and 10:1, respectively. The NP1 peptide sensitized target cells for lysis by anti-HTNV CTLs, with percentages of 33.5, 21.7, 15.5, and 10.2% at E/T ratios of 100:1, 50:1, 20:1, and 10:1, respectively (Figure 2B). As endogenous target cells, the HTNV-infected Frontiers in Cellular and Infection Microbiology | www.frontiersin.org November 2016 | Volume 6 | Article 151 5 Identification of the HTNV-GP CTL-Epitope Ma et al. FIGURE 2 | Cytotoxic activity of splenocytes derived from HTNV-infected mice in vitro. The primary antiviral CTL responses were first detected. Splenocytes from C57BL/6 mice infected with HTNV (1 × 105 pfu/head) were used as effector cells. As targets, EL-4 cells were pulsed with GP6 (A) and NP1 (B) at a concentration of 10 µg/mL. Splenocytes that were restimulated with GP6 (C) and NP1 (D) were used as effector cells in the second round of the CTL assay. Macrophages infected with HTNV (■) and peptide-pulsed EL-4 cells (•) were used as target cells. P815 cells (▲) were used as negative controls. (E) Evaluation of the mean lysis percentage of naïve CD8+T cells to kill no peptide-pulsed target cells. Macrophages (■), EL-4 cells (•), and P815 cells (▲) were served as target cells, respectively. Data are expressed as the mean ± SEM (n = 6). **P < 0.001, ***P < 0.0001. The Cytotoxic Capacity of HTNV Glycoprotein-Specific CD8+ T Cells FIGURE 2 | Cytotoxic activity of splenocytes derived from HTNV-infected mice in vitro. The primary antiviral CTL responses were first detected. Splenocytes from C57BL/6 mice infected with HTNV (1 × 105 pfu/head) were used as effector cells. As targets, EL-4 cells were pulsed with GP6 (A) and NP1 (B) at a concentration of 10 µg/mL. Splenocytes that were restimulated with GP6 (C) and NP1 (D) were used as effector cells in the second round of the CTL assay. Macrophages infected with HTNV (■) and peptide-pulsed EL-4 cells (•) were used as target cells. P815 cells (▲) were used as negative controls. (E) Evaluation of the mean lysis percentage of naïve CD8+T cells to kill no peptide-pulsed target cells. Macrophages (■), EL-4 cells (•), and P815 cells (▲) were served as target cells, respectively. Data are expressed as the mean ± SEM (n = 6). **P < 0.001, ***P < 0.0001. 50:1 and 20:1. Significant CTL activity could not be found in the non-specific group (Figure 2E). Other peptides were also tested, but CTL activity was undetectable. Collectively, these results suggest that the GP6 peptide is able to provide restimulation for primary antiviral CTLs to maintain specific cytotoxicity ability. of total CD8+T cells). The frequency of IFN-γ-producing cells upon recognition of NP1 peptide was also observed (15.98% of total CD8+T cells). CD8+T cells restimulated with PHA also showed a high level of IFN-γ secretion with 15.25 percent of the total. However, a few cells producing IFN-γ were detected in both negative control groups (4.64% of total CD8+T cells in mock-infected mice with no peptide restimulation and 4.73% of total CD8+T cells in HTNV-infected mice with no peptide restimulation; Figure 3A). This assay was performed and repeated six times. We have given the statistical results in Figure 3B. HTNV-infected mice with GP6 restimulation group induced a significantly IFN-γ response compared to both negative control groups (the mock-infected mice with no peptide restimulation, p < 0.0001; HTNV-infected mice with no peptide Effector Functions of HTNV GP Epitope-Specific CD8+T Cells Given the importance of IFN-γ production in the defense against HTNV infection, we further investigated the production of IFN-γ by CD8+ T cells specific for HTNV GP by intracellular cytokine staining. This assay showed that the GP6 peptide induced a high percentage of IFN-γ production among CD8+ T cells (11.97% November 2016 | Volume 6 | Article 151 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 6 Identification of the HTNV-GP CTL-Epitope Ma et al. FIGURE 3 | The rapid secretion of IFN-γ after recognition of the HTNV-specific peptide. (A) Representative flow cytometric plots of IFN-γ production by peptide-specific CD8+ T cells. The upper lane (A) shows the results 4 h after peptide restimulation and both negative and positive controls, and the lower lane (B) shows the frequency of IFN-γ production in CD8+ T cell populations. Data are expressed as the mean ± SEM (n = 6). ***P < 0.0001. FIGURE 3 | The rapid secretion of IFN-γ after recognition of the HTNV-specific peptide. (A) Representative flow cytometric plots of IFN-γ production by peptide-specific CD8+ T cells. The upper lane (A) shows the results 4 h after peptide restimulation and both negative and positive controls, and the lower lane (B) shows the frequency of IFN-γ production in CD8+ T cell populations. Data are expressed as the mean ± SEM (n = 6). ***P < 0.0001. FIGURE 3 | The rapid secretion of IFN-γ after recognition of the HTNV-specific peptide. (A) Representative flow cytometric plots of IFN-γ production by peptide-specific CD8+ T cells. The upper lane (A) shows the results 4 h after peptide restimulation and both negative and positive controls, and the lower lane (B) shows the frequency of IFN-γ production in CD8+ T cell populations. Data are expressed as the mean ± SEM (n = 6). ***P < 0.0001. demonstrate the proliferative potential and capacity of specific CD8+ T cells in protecting from HTNV infection. restimulation, p < 0.0001). Our result is consistent with the data obtained in the ELISPOT assay (Figure 1). Ex vivo CD8+ T Cell Expansion against HTNV Glycoprotein First, we measured cytokine production by ELISPOT assay as an indicator of cellular immune responses to reflect T cell responses to HTNV GP peptides. As shown in Figure 5B, splenocytes from GP6 peptide-immunized mice, restimulated with purified HTNV NP, presented a significantly higher number of IFN-γ spots compared to both the HTNV NP1-immunized group and inactivated Hantavirus vaccine group. HTNV-immunized mice showed the highest number. In contrast, we also observed that the numbers of IFN-γ spots from the two negative control groups (resolver control and adjuvant control) were extremely low. We next assessed whether the antiviral effect of CTLs was dependent on the capacity of expansion. The CFSE staining assay showed that expansion of HTNV peptide-specific CTLs was readily detectable. As shown in Figure 4, PBMCs of HTNV- infected mice after stimulation with GP6 had a considerable expansion potential with 49.23% cells belonging to Generation 3 (Figure 4A). A similar level of expansion potential was observed in the NP1 stimulation group (Figure 4B). However, when stimulated with anti-mouse CD3 as a control, PBMCs had proliferative activity with 59.1% of cells belonging to Generation 3 (Figure 4C), which was higher than cells stimulated with HTNV-specific epitopes. The negative control of no peptide stimulation showed a low expansion coefficient with 39.99% (Figure 4D). The statistical results were provided in Figure 4E. HTNV-immunized mice with GP6 stimulation group showed a significant proliferation ability compared to the group with no peptide stimulation (P < 0.05). Previous studies have demonstrated a strong linkage between HTNV-specific CD8+T cell expansion and the control of HTNV infection. These data Next, the GP peptide-specific CTL activity was detected by an LDH release assay. The cytotoxicity of splenocytes from mice immunized with GP6 was enhanced in accordance with the E/T ratio, which was the most significant at the ratio of 100:1 (Figure 5C). Splenocytes from the NP1-immunized mice showed the highest specific cytotoxic activity at E/T ratios of 100:1, 50:1, and 20:1 compared to those of the other groups. Additionally, the specific cytotoxic activities were not significant in the vaccine group. Perhaps, poor immunogenicity of these vaccines to elicit November 2016 | Volume 6 | Article 151 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 7 Identification of the HTNV-GP CTL-Epitope Ma et al. FIGURE 4 | The proliferative capacity of HTNV peptide-specific CD8+ T cells. Ex vivo CD8+ T Cell Expansion against HTNV Glycoprotein Representative flow cytometric histogram of the expansion percentage of CD8+ T cells stimulated by the GP6 (A) and NP1 (B). Anti-mouse CD3 (Biolegend) (D) stimulation or no peptide (C) stimulation served as positive or negative controls, respectively. (E) The statistical data of the proliferation assay. Data are expressed as the mean percentage ± SEM (n = 6). *P < 0.05. FIGURE 4 | The proliferative capacity of HTNV peptide-specific CD8+ T cells. Representative flow cytometric histogram of the expansion percentage of CD8+ T cells stimulated by the GP6 (A) and NP1 (B). Anti-mouse CD3 (Biolegend) (D) stimulation or no peptide (C) stimulation served as positive or negative controls, respectively. (E) The statistical data of the proliferation assay. Data are expressed as the mean percentage ± SEM (n = 6). *P < 0.05. FIGURE 4 | The proliferative capacity of HTNV peptide-specific CD8+ T cells. Representative flow cytometric histogram of the expansion percentage of CD8+ T cells stimulated by the GP6 (A) and NP1 (B). Anti-mouse CD3 (Biolegend) (D) stimulation or no peptide (C) stimulation served as positive or negative controls, respectively. (E) The statistical data of the proliferation assay. Data are expressed as the mean percentage ± SEM (n = 6). *P < 0.05. Immunization with Recombinant CTL Epitope Peptides Induced Protective Immunity in C57BL/6 Mice against HTNV Challenge cell-mediated immunity is the main cause. However, non-specific cytotoxicity in the two negative control groups was extremely weak at E/T ratios of 100:1, 50:1, and 20:1. Based on these results, immunization with GP6 peptide induced a high level of IFN-γ response and strongly specific cytotoxic activity, suggesting that immunization with HTNV-GP specific peptides enhances cellular immune responses. cell-mediated immunity is the main cause. However, non-specific cytotoxicity in the two negative control groups was extremely weak at E/T ratios of 100:1, 50:1, and 20:1. Based on these results, immunization with GP6 peptide induced a high level of IFN-γ response and strongly specific cytotoxic activity, suggesting that immunization with HTNV-GP specific peptides enhances cellular immune responses. Protective immunity was evaluated in peptide-immunized mice. Three days after infection with HTNV, the major tissues of the mice were isolated to test against HTNV antigens by ELISA. We November 2016 | Volume 6 | Article 151 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Identification of the HTNV-GP CTL-Epitope Ma et al. Ma et al. FIGURE 5 | The enhanced cellular immune responses in peptide-immune C57BL/6 and the timeline of immunization. (A) Scheme of peptide immunization. Mice were primed and boosted with GP6 and NP1 packaged with Freud’s adjuvants (Pep Immun group) or PBS (Mock group). Ten days after the final boost, splenocytes from mice were harvested for subsequent assays. (B) IFN-γ ELISPOT analysis of splenocytes obtained from HTNV peptide-immunized mice. Cells were stimulated with purified HTNV NP. (C) Splenocytes from immunized mice, restimulated with relative peptide were used as effector cells. As targets, EL-4 cells were pulsed with GP6 or NP1 at a concentration of 10 µg/ml. (■) represents NP1 immunization; (•) represents GP6 immunization; (▲) represents a positive control from the HTNV-challenged mice; (□) represents vaccine control; (◦) represents PBS control; (▼) represents adjuvant control. Data are expressed as the mean ± SEM (n = 6). ***P < 0.0001. FIGURE 5 | The enhanced cellular immune responses in peptide-immune C57BL/6 and the timeline of immunization. (A) Scheme of peptide immunization. Mice were primed and boosted with GP6 and NP1 packaged with Freud’s adjuvants (Pep Immun group) or PBS (Mock group). Ten days after the final boost, splenocytes from mice were harvested for subsequent assays. (B) IFN-γ ELISPOT analysis of splenocytes obtained from HTNV peptide-immunized mice. Cells were stimulated with purified HTNV NP. Immunization with Recombinant CTL Epitope Peptides Induced Protective Immunity in C57BL/6 Mice against HTNV Challenge Interestingly, this method detected lesser amounts of HTNV antigens in the liver, spleen and kidney of GP6-immunized mice compared with negative control groups that had absolutely no protection (P/N = 5.01, 4.90, and 2.57, respectively). Additionally, the HTNV antigens in NP1 peptide- immunized mice were found at low values of 3.67, 4.15, and 2.74, respectively. HTNV-specific antigens could not be detected in the heart, lung, or cerebrum of C57BL/6 mice from all of the groups (Figure 6). These results indicate that the immunization of C57BL/6 mice with the recombinant positive peptides provides protective immunity against HTNV challenge. Taken together, these results show that immunization with the recombinant positive peptides induces protective immunity in C57BL/6 mice against HTNV challenge. Immunization with Recombinant CTL Epitope Peptides Induced Protective Immunity in C57BL/6 Mice against HTNV Challenge (C) Splenocytes from immunized mice, restimulated with relative peptide were used as effector cells. As targets, EL-4 cells were pulsed with GP6 or NP1 at a concentration of 10 µg/ml. (■) represents NP1 immunization; (•) represents GP6 immunization; (▲) represents a positive control from the HTNV-challenged mice; (□) represents vaccine control; (◦) represents PBS control; (▼) represents adjuvant control. Data are expressed as the mean ± SEM (n = 6). ***P < 0.0001. found that HTNV antigens were detected in the liver, spleen, and kidney of C57BL/6 mice in both negative control groups (adjuvant control and PBS control; P/N = 10.49, 11.57, and 5.13 in adjuvant control group or 9.89, 11.3, and 4.89 in PBS control group, respectively). However, antigens could not be detected in the liver, spleen or kidney of mice in the inactivated Hantavirus vaccine control group. Interestingly, this method detected lesser amounts of HTNV antigens in the liver, spleen and kidney of GP6-immunized mice compared with negative control groups that had absolutely no protection (P/N = 5.01, 4.90, and 2.57, respectively). Additionally, the HTNV antigens in NP1 peptide- immunized mice were found at low values of 3.67, 4.15, and 2.74, respectively. HTNV-specific antigens could not be detected in the heart, lung, or cerebrum of C57BL/6 mice from all of the groups (Figure 6). These results indicate that the immunization of C57BL/6 mice with the recombinant positive peptides provides protective immunity against HTNV challenge. Histopathological analysis by H&E staining showed that no significant alterations were found in the tissues from all of the groups except for the spleens of the two negative control groups (PBS control and adjuvant control). The results indicated some pathological changes including diffused lymphocyte infiltration, diffused hemorrhaging and increased white pulp in the spleens of the two negative control groups (PBS control and adjuvant control) which were marked with red arrows in the Figures 8D,E; however, these changes were not found in the other groups (Figure 8). found that HTNV antigens were detected in the liver, spleen, and kidney of C57BL/6 mice in both negative control groups (adjuvant control and PBS control; P/N = 10.49, 11.57, and 5.13 in adjuvant control group or 9.89, 11.3, and 4.89 in PBS control group, respectively). However, antigens could not be detected in the liver, spleen or kidney of mice in the inactivated Hantavirus vaccine control group. DISCUSSION Vaccines, collectively a successful strategy for the prevention of infectious diseases, can direct the host’s immune system against pathogens (Ma et al., 2016). Inactivated vaccines for HTNV have been shown to boost the humoral immune response (Kumar et al., 1997), but poor immunogenicity in eliciting neutralizing antibodies and limited cell-mediated immunity following vaccination, as well as concern regarding safety are major obstacles for development (Yang et al., 2005). Therefore, inactivated vaccines are not expected to be able to effectively inhibit HTNV amplification to completely prevent viral infection (Yang et al., 2005). A potent T cell-activating peptide vaccine based on the HTNV structural protein may be a promising strategy for disease control (Rubsamen et al., 2014; Lu et al., 2016). The T cell response is a critical element Meanwhile, the qRT-PCR assay was used to detect HTNV nucleic acids in the tissues of peptide-immunized C57BL/6 mice after challenge with HTNV. HTNV nucleic acids were detected in the liver, spleen, and kidney of C57BL/6 mice in the two negative groups with 11.43-, 13.5,- and 4.63-fold or 10.96-, 13.6,- and 5.57-fold amplification, respectively, compared to the inactivated Hantavirus vaccine group which did not contain viral nucleic acids. Small amounts of HTNV-specific nucleic acids were detected in the same tissues of C57BL/6 mice from the GP6- or NP1-immunized groups, compared to the negative controls (Figures 7C,D,F). Moreover, HTNV-specific nucleic acids could not be detected in other tissues of C57BL/6 mice in all of the groups (Figures 7A,B,E). These results were consistent with the ELISA results. November 2016 | Volume 6 | Article 151 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 9 Identification of the HTNV-GP CTL-Epitope Ma et al. FIGURE 6 | Detection of HTNV-specific antigens from peptide-immunized mice by ELISA. ELISA was used to detect HTNV antigens in the tissues of each group including the cerebrum (A), heart (B), liver (C), spleen (D), lung (E), and kidney (F). (•) represents the GP6 peptide-immunized group; (■) represents the NP1 peptide-immunized group; (▲) represents the inactivated Hantavirus vaccine control group; (◦) represents the PBS control group; (♦) represents the adjuvant control group. Data are expressed as the mean ± SEM (n = 4). *P < 0.05, **P < 0.001. FIGURE 6 | Detection of HTNV-specific antigens from peptide-immunized mice by ELISA. DISCUSSION Taking the complexity of IFN-γ-secreting cells into consideration, the second round of the ELISPOT assay was performed using both CD4+ T cell-depleted splenocytes and CD8+ T cell-depleted splenocytes as effector cells. We tried to use CD4+ T cell-depleted splenocytes as effector cells to recognize the CD8+T cell responsive 8-mer epitopes. On the other hand, CD8+ T cell depletion completely abrogated the IFN-γ response indicating that these peptides were CD8+ T cell epitopes. The data showed that HTNV GP6 induced the largest number of IFN-γ-producing cells (over 450 cells per 106 splenocytes) with CD4 depletion and a small quantity with CD8 depletion (less than 40 cells per 106 splenocytes) among the 15 alternative peptides. These data are consistent with that of NP1, which also induced a significant number of IFN-γ- producing cells (over 470 cells per 106 splenocytes) when CD4+T cells were depleted and <35 SFC when CD8 cells were depleted (Figure 1B). However, other peptides did not induce detectable levels of IFN-γ-producing cells when the CD4+ T cells were depleted. Based on this finding, we speculate that GP6 might be an immunodominant CTL epitope. FIGURE 8 | Histopathological analysis of the spleens from peptide-immunized mice after challenge with HTNV by H&E staining. (A) The GP6 peptide-immunized group. (B) The NP1 peptide-immunized group. (C) The inactivated Hantavirus vaccine group. (D) The PBS control group. (E) The adjuvant control. Red arrows show diffused lymphocyte infiltration, diffused hemorrhaging and increased white pulp in the spleens of two control groups. FIGURE 8 | Histopathological analysis of the spleens from peptide-immunized mice after challenge with HTNV by H&E staining. (A) The GP6 peptide-immunized group. (B) The NP1 peptide-immunized group. (C) The inactivated Hantavirus vaccine group. (D) The PBS control group. (E) The adjuvant control. Red arrows show diffused lymphocyte infiltration, diffused hemorrhaging and increased white pulp in the spleens of two control groups. of naturally acquired immunity to control viral infection, including HTNV infection (Rasmuson et al., 2011). This fact provided the theoretical basis for the development of HTNV peptide vaccines to fight infection by inducing specific T cell responses. In this study, we reported that the GP6 peptide derived from glycoprotein of HTNV could be an immunogenic epitope based on its ability to induce peptide- specific CD8+ T cell responses and on in vivo functional validation. DISCUSSION ELISA was used to detect HTNV antigens in the tissues of each group including the cerebrum (A), heart (B), liver (C), spleen (D), lung (E), and kidney (F). (•) represents the GP6 peptide-immunized group; (■) represents the NP1 peptide-immunized group; (▲) represents the inactivated Hantavirus vaccine control group; (◦) represents the PBS control group; (♦) represents the adjuvant control group. Data are expressed as the mean ± SEM (n = 4). *P < 0.05, **P < 0.001. FIGURE 7 | qRT-PCR results of HTNV-specific nucleic acids in organs of peptide-immunized mice. This method detected HTNV nucleic acid in the tissues of each group including the cerebrum (A), heart (B), liver (C), spleen (D), lung (E), and kidney (F). The viral nucleic acids are distributed mainly in the spleen and liver, but to a lesser degree in the kidney. Data are expressed as the mean ± SEM (n = 6). **P < 0.001, ***P < 0.0001. FIGURE 7 | qRT-PCR results of HTNV-specific nucleic acids in organs of peptide-immunized mice. This method detected HTNV nucleic acid in the tissues of each group including the cerebrum (A), heart (B), liver (C), spleen (D), lung (E), and kidney (F). The viral nucleic acids are distributed mainly in the spleen and liver, but to a lesser degree in the kidney. Data are expressed as the mean ± SEM (n = 6). **P < 0.001, ***P < 0.0001. November 2016 | Volume 6 | Article 151 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 10 Identification of the HTNV-GP CTL-Epitope Ma et al. and Infectious Diseases (Vita et al., 2015). We selected and synthesized 15 peptides and tested their respective antigenicity according to binding score. A higher score means a stronger bind to MHC (Park et al., 2000). This first crucial step increases the probability of successful epitope selection. The high production of antiviral cytokines is a critical characteristic of effective T cell immunity for both suppressing viral replication and eliminating virus-infected host cells. IFN-γ has been implicated in immune regulation and direct antiviral activities (Frese et al., 2002; Gattoni et al., 2006). Therefore, the IFN-γ ELISPOT was conducted for mapping the epitopes of HTNV glycoprotein. In the first round of the ELISPOT assay, six of 15 peptides could induce a strong IFN-γ response, but this datum does not illustrate that the peptides are CTL epitopes. Assays CTL assays were performed to evaluate peptide-specific cytotoxic activity. The data suggested that GP6 elicits a CTL response and also plays a key role in the primary antiviral response during HTNV infection in mice. To examine whether this peptide could enhance peptide-specific cytotoxicity of CTLs, we measured secondary CTL responses after in vitro restimulation. After secondary stimulation with the GP6 peptide, CTL activity against the relevant peptide was increased. At the same time, we also used HTNV-infected macrophages as the endogenous target cells to evaluate whether the peptide-specific CTLs could effectively recognize endogenously processed antigen. The results showed a high level of cytotoxic activity of GP6- specific CTLs even when using the endogenous cells as targets. This suggests that GP6 could be processed and presented by endogenously antigen-presenting system. These results also imply that GP6 can provide restimulation to maintain the cytotoxic activity of primary antiviral CTLs and have the potential to induce T lymphocyte responses during viral infection. DISCUSSION We further provided evidence that immunization with GP6 in C57BL/6 mice successfully induced T cell responses with high production of IFN-γ and potent cytotoxicity, which involved in combatting HTNV infection after HTNV challenge. This reveals that GP6 peptide may be a good candidate for use as a peptide-based vaccine in the design of preclinical studies in vivo. CTL Epitope from HTNV Glycoprotein Provided Significantly Enhanced Immunogenicity over Inactivated HTNV Vaccine HTNV by cellular immune responses, and we found that GP6 could be applied to vaccine design preclinical studies of vaccines that target T cell immunity. HTNV infection in adult mice results in transient viral replication with no symptoms followed by the elimination of virus (Araki et al., 2004). However, HFRS is a severe, life-threatening illness characterized by fever, hemorrhage, and renal failure (Serikawa and Yamada, 1993). Due to limitations of using a model for disease progression, suitable animal models that mimic HFRS are presently unavailable. Our research is based on detecting the viral antigens and nucleic acid to evaluate the protective ability of viral epitopes, and this method can also be used to evaluate each protective therapeutic agent for HTNV. To evaluate the immunogenicity of the identified epitopes from our study ex vivo, the cellular immune response was evaluated at 10 d after the final booster immunization with the specific epitope in C57BL/6 mice. Our results showed that the splenocytes of GP6 group mice always showed stronger T cell responses compared with the other groups. Meanwhile, the splenocytes of the vaccine control group have a weak magnitude of response of T cells, particularly because of the poor immunogenicity of inactivated Hantavirus vaccine to elicit cell-mediated immunity (Kwilas et al., 2014). Our results verified this point again. In summary, one novel CTL epitope peptide GP6 aa456- aa463 (ITSLFSLL) was identified in this study. As far as we know, this is the first report of a CTL epitope on HTNV glycoprotein in mice and we confirmed that the HTNV- specific GP6 peptide is a highly immunogenic epitope restricted by H-2Kb. By challenging C57BL/6 mice with HTNV in vivo, we revealed the immunogenicity of vaccination with the HTNV GP6 peptide and the antiviral efficiency of GP6- specific CTL responses. But our present work only find the CTL epitope on HTNV GP, we should learn more about other epitopes on HTNV GP and NP (such as neutralizing epitope) and the immunological characteristics of their combined application in the next step. Our findings could make some contributions to the establishment of HFRS animal model and research on HFRS multiple peptides vaccine. Identification of the Correct CTL Epitopes within the Glycoprotein of HTNV by ELISPOT Assay Selecting the correct epitope is a priority when designing an effective vaccine. An epitope, which is the portion of an antigen presented on major histocompatibility complex (MHC) molecules that bind the TCR or BCR of T/B lymphocytes, initiates, and evokes T/B cell immune responses (Hoffmeister, 2003). First, we evaluated the sequence of the HTNV GP using an algorithm, provided by IEDB of the National Institute of Allergy November 2016 | Volume 6 | Article 151 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 11 Identification of the HTNV-GP CTL-Epitope Ma et al. The CTL Epitope-Vaccinated Mice Presented Partial Protection Immunity from Challenge with HTNV We further provided evidence of the potential capacity of the identified epitopes to protect mice after HTNV challenge through the animal protection assay. Viral load in each of the major organs of C57BL/6 mice served as parameters to assess protection. Based upon the peak of viral replication, the mice were sacrificed 3 days post-challenge. Using ELISA and qRT- PCR, we found that large amounts of HTNV antigens and nucleic acid could be detected in the spleen, liver, and kidney of C57BL/6 mice in the two negative control groups (PBS control and adjuvant control), but not in the vaccine group. Further comparisons revealed that viral antigens and nucleic acids in the spleen were higher than those in the liver and kidney. It was interesting to note that viral antigens and nucleic acids can only be detected in small amounts in the GP6 and NP1 groups. The data from histopathological analysis by H&E staining also showed some pathological changes in the spleens (Figure 8) of mice belonging to the two negative control groups (PBS control and adjuvant control) but not in vaccine groups after challenge with HTNV. Although, the level of HTNV nucleic acids and antigens in the GP6 group was greater than those in the vaccine group, the potential capacity of GP6, as an 8-mer aa peptide existing on the huge HTNV structure, to protect mice from HTNV infection cannot be ignored. These findings suggests that GP6-specific CTL response could protect animals from HTNV challenge. AUTHOR CONTRIBUTIONS RM and LC are responsible for the contents of main experiments and writing the manuscript. QY and RL are responsible for the flow cytometric analysis. TM, XZ, and ZL are responsible for the animal raising and immunization. LZ and WY are responsible for data collection. FZ and ZX are responsible for experiment instruction. XW and FW are responsible for experimental design. All authors read and approved the final manuscript. An Excellent Method to Evaluate Protective This work was supported by the grants from the Shaanxi Science & Technology Co-ordination & Innovation Project (2013KTCL03-06), the Natural Science Foundation of China (31470890; 31270978). p g Our goal was to identify CTL epitopes on HTNV GP in mice to gain a better understanding of the recognition of Araki, K., Yoshimatsu, K., Lee, B. H., Kariwa, H., Takashima, I., and Arikawa, J. (2004). A new model of Hantaan virus persistence in mice: the balance between HTNV infection and CD8+ T-cell responses. 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HLA-A2 and B35 restricted hantaan virus nucleoprotein CD8+ T-cell epitope-specific immune response correlates with milder disease in hemorrhagic fever with renal syndrome. PLoS Negl. Trop. Dis. 7:e2076. doi: 10.1371/journal.pntd.0002076 Copyright © 2016 Ma, Cheng, Ying, Liu, Ma, Zhang, Liu, Zhang, Ye, Zhang, Xu, Wang and Wu. 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. Ma, Y., Yuan, B., Zhuang, R., Zhang, Y., Liu, B., Zhang, C., et al. (2015). Hantaan virus infection induces both Th1 and ThGranzyme B+ cell immune responses that associated with viral control and clinical outcome in humans. 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Existentialism and Its Influence on Our Understanding of Knowledge, Truth, Morality, Values, and Religion
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Existentialism and Its Influence on Our Understanding of Knowledge, Truth, Morality, Values, and Religion John Michael Sasan Administrative Assistant Research for Innovation, Sustainability and Excellence Office, PAU Excellencia Global Academy Foundation, Inc. Toledo City, Cebu ORCID: https://orcid .org/0000-0001-5987-6937 Johnmichaelsasan27@gmail.com Research for Innovation, Sustainability and Excellence Office, PAU Excellencia Global Academy Foundation, Inc. Toledo City, Cebu ORCID: https://orcid .org/0000-0001-5987-6937 Johnmichaelsasan27@gmail.com Received: Okt 06, 2023; Accepted: Des 24, 2023; Published: Jan 24, 2023 Received: Okt 06, 2023; Accepted: Des 24, 2023; Published: Jan 24, 2023; Abstract: Existentialism is a philosophical movement that has had a significant impact on our understanding of knowledge, truth, morality, values, and religion. This research aims to explore the influence of existentialism on these fundamental aspects of human existence. The research methodology involved an extensive review of literature from prominent existentialist philosopher, Friedrich Nietzsche. The findings of this research highlight that existentialism challenges the traditional view of objective truth and knowledge, emphasizing the subjective and personal experience of individuals. Furthermore, existentialism posits that individuals must create their own values and meaning in life, rather than relying on external sources such as religion or society. In terms of morality, existentialism emphasizes the individual's responsibility for their actions, as opposed to being governed by universal ethical principles. The limitations of this research include the focus on Western existentialist philosophy, neglecting other cultural and philosophical traditions. The contribution of this research is that it sheds light on the influence of existentialism on various aspects of human existence and highlights the significance of subjective experience and personal responsibility. The practical implication of this research is that it provides insight into the importance of individualism and personal agency in various domains, such as education, mental health, and politics. The novelty of this research lies in the comprehensive exploration of existentialism's influence on various aspects of human existence, providing a unique perspective on these topics. Keywords: Existentialism, Ethics, Ontology, Religious studies Keywords: Existentialism, Ethics, Ontology, Religious studies Introduction Existentialism is a well-established philosophical movement that emerged in the late 19th and early 20th century, emphasizing the individual's subjectiveexperience and the search for meaning and purpose in life (Stewart, 2018). Friedrich Nietzsche is widely regarded as a central figure in the development of existentialist thought, having rejected traditional values, religion, and morality in a way that significantly impacted our understanding of knowledge, truth, morality, values, and religion (Crowell, 2013; Kaufmann,1974). Nietzsche's concept of truth challenges the traditional notion of objectivity, instead emphasizing subjectivity and interpretation (D'Amico, 2005). This has led to greater appreciation for diverse perspectives and a recognition that there are multiple ways of interpreting the world around us (Sayers, 2014). 40 40 http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS 40 40 Journal of Learning on History and Social Sciences Volume 1, Issue 1 | 2023 Journal of Learning on History and Social Sciences Volume 1, Issue 1 | 2023 European Journal of Learning on History and Social Sciences European Journal of Learning on History and Social Sciences Nietzsche's ideas have influenced various fields, including philosophy, literature, psychology, and the arts (Magnus & Higgins, 2011). Despite Nietzsche's significant impact, there is still a gap in our understanding ofthe extent of his influence on contemporary understanding of knowledge, truth, morality, values, and religion. While numerous scholars have examined Nietzsche's philosophical ideas, few have explored their practical applications and implications for understanding fundamental aspects of human existence (Crowell, 2013; Kaufmann, 1974). Nietzsche's ideas have influenced various fields, including philosophy, literature, psychology, and the arts (Magnus & Higgins, 2011). Despite Nietzsche's significant impact, there is still a gap in our understanding ofthe extent of his influence on contemporary understanding of knowledge, truth, morality, values, and religion. While numerous scholars have examined Nietzsche's philosophical ideas, few have explored their practical applications and implications for understanding fundamental aspects of human existence (Crowell, 2013; Kaufmann, 1974). p ( Review of Related Literature Existentialism is a philosophical movement that originated in the late 19th and early 20th centuries, with a focus on individual existence, freedom, and choice (Solomon, 2017). Nietzsche, who is regarded as one of the forerunners of existentialism, believed that traditional values, religion, and morality were restrictive and negatively affected human development (Solomon, 2017). Nietzsche's ideas had a significant impact on existentialism and influenced our understanding of knowledge, truth, morality, values, and religion. He rejected the concept of ultimate truth and instead believed that all truth is subjective (Solomon, 2017). Nietzsche proposed that knowledge is a matter of interpretation and judgment and that there is no objective reality (Solomon, 2017). He argued that truth is a result of our senses and language and that our perceptions of reality areshaped by our personal experiences and biases (Solomon, 2017). The existentialist emphasis on individual experience and freedom has influenced contemporary theories of knowledge, truth, and morality. For instance, feminist epistemology recognizes the significance of personal experience in shaping knowledge and truth (Lugones, 1987). Lugones argues that personal experience is a valuable source of knowledge, and knowledge should be assessed based on its relevance to the individual and the community (Lugones, 1987). Existentialism has also had an impact on the study of values and ethics. For example, Beauvoir, a French existentialist, suggested that ethical behavior involves recognizing the impact of our choices on others and taking responsibility for our actions (Beauvoir, 1949). Beauvoir's concept of ethical responsibility highlights the importance of individual choice and freedom in shaping moral behavior. dividual choice and freedom in shaping moral behavior. The existentialist emphasis on individual experience and freedom has also led to a re- evaluation of traditional religious beliefs and practices. Buber, a Jewish existentialist, argued that religious experience is based on the individual's relationship with God and that this relationship should be an authentic and personal experience (Buber, http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS 1923). According to existential learning and teaching, arguing that psychology, indivi duals who a re a uthentic are education should focus on the One of the key concepts in existential psychology is authenticity, which refers to the extent to which individuals are true to themselves and their values, and live a meaningful and existentialist concepts to the study of purposeful life. According to existential learning and teaching, arguing that psychology, indivi duals who a re a uthentic are education should focus on the ychology is authenticity, which refers to the extent to which individuals are true to themselve eir values, and live a meaningful and existentialist concepts to the study of more likely to experience fulfillment and happiness, and to cope better with life's challenges (Batthyany & Guttmann, 2017). Research has shown that authenticity is positively associated with well- being, life satisfaction, and positive emotions (Kernis, 2003). more likely to experience fulfillment and happiness, and to cope better with life's challenges (Batthyany & Guttmann, 2017). Research has shown that authenticity is positively associated with well- being, life satisfaction, and positive emotions (Kernis, 2003). Existentialism has also had an impact on the field of sociology. Sociologists have applied existentialist concepts to the study of social phenomena, including social inequality, social identity, and social change. For example, Berger and Luckmann (1966) applied the concept of social construction to the study of knowledge, arguing that knowledge is not objective or absolute, but is constructed through social processes. They also argued that social identity is constructed through interaction with others, and that individuals have the freedom to shape their social identity through their actions and choices. Existentialist ideas have also been influential in the field of literature. Many writers have been inspired by the themes of existentialism, including the search for meaning and purpose, the struggle for freedom, and the challenge of finding one's place in the world. Writers like Albert Camus, Jean-Paul Sartre, and Franz Kafka have explored these themes in their works, and have contributed to the development of existentialist literature (Mays, 1923). Henceforth, existentialism has played a significant role in our understanding of knowledge, truth, morality, values, and religion. Nietzsche's rejection of traditional values, religion, and morality, and his emphasis on individual experience and freedomhave influenced contemporary theories in these areas. The existentialist emphasis on personal experience and choice has led to a greater appreciation of the diversity of perspectives and a recognition that there are multiple ways of interpreting the world around us. Existentialism is a philosophical movement that has been of interest to many scholars in various fields, including psychology, sociology, literature, and philosophy. The movement emphasizes the individual's subjective experience, freedom, and choice, and encourages the exploration of the meaning and purpose of life. One of the forerunners of existentialism is Friedrich Nietzsche, a prominent German philosopher who rejected traditional values, religion, and morality, and emphasized the importance of 41 41 http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS ropean Journal of Learning on History and Social Sciences Volume 1, Issue 1 ropean Journal of Learning on History and Social Sciences Volume 1, Issue 1 European Journal of Learning on History and Social Sciences European Journal of Learning on History and Social Sciences individual experience and interpretation. Nietzsche's ideas have had a significant impact on our understanding of knowledge, truth,morality,values,and religion,and have influenced many contemporary theories in these areas. One area in which existentialism has had a significant impact is psychology. Existential psychology is a branch of psychology that emphasizes the subjective experience of individuals and the search for meaning and purpose in life. This approach to psychology has been influenced by the ideas of existential philosophers like Nietzsche, and has been applied to various areas of psychology, including psychotherapy, personality psychology, and positive psychology (May, 1958). One of the key concepts in existential One of the key concepts in existential psychology is authenticity, which refers to the extent to which individuals are true to themselves and their values, and live a meaningful and existentialist concepts to the study of purposeful life. According to existential learning and teaching, arguing that One of the key concepts in existential psychology is authenticity, which refers to the extent to which individuals are true to themselves and their values, and live a meaningful and existentialist concepts to the study of purposeful life. 2009). Existentialism has also had an impact on the arts. Many artists have been inspired by existentialist ideas, and have used art as a way to explore the human condition and the search for meaning and purpose. For example, the works of Vincent van Gogh, Edvard Munch, and Jackson Pollock have been interpreted as expressions of existentialist themes, including the struggle for authenticity and the search for meaning (Friedman, 1990). Finally, existentialism has had an impact on education. Educators have ap Finally, existentialism has had an impact on education. Educators have development of the whole person development of the whole person 42 42 http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS an Journal of Learning on History and Social Sciences Volume 1, Issue 1 | 202 an Journal of Learning on History and Social Sciences Volume 1, Issue 1 | 202 European Journal of Learning on History and Social Sciences European Journal of Learning on History and Social Sciences and the exploration of meaning and purpose. According to this approach, education should not simply be a process of acquiring knowledge, but should also involve the exploration of one's values and beliefs, and the development of critical thinking skills (Parker, In a study conducted by Goldstein et al. (2018), the authors explored the relationship between self- compassion and subjective well-being in a sample of adults with chronic pain. The study found that higher levels of self -compassion were associated with greater subjective well-being, as well as lower levels of anxiety and depression. Additionally, the authors found that self- compassion moderated the relationship between pain severity and subjective well- being, indicating that individuals with higher levels of self- compassion were less affected by pain severity in terms of their subjective well-being. Another study by Allen and Leary (2010) Another study by Allen and Leary (2010) examined the relationship between self-compassion and reactions to social comparison among college students. The study found that individuals higher in self- compassion reported less negative affect and lower levels of social comparison following exposure to a negative social comparison scenario. The authors suggest that self-compassion may serve as a protective factor against the negative effects of social comparison. In a study by Neff et al. (2007), the authors investigated the relationship between self- compassion and psychological distress in a sample of undergraduate students. The study found that higher levels of self-compassion were associated with lower levels of anxiety, depression, and perceived stress. 2009). Additionally, the authors found that self-compassion was positively related to emotional intelligence, suggesting that self- compassionate individuals may be more attuned to their own emotional experiences and those of others. In a study by Terry et al. (2013), the authors examined the role of self-compassion in the relationship between mindfulness and stress. The study found that higher levels of self -compassion mediated the relationship between mindfulness and stress, suggesting that individuals who are more self- compassionate may be better able to cope with stress through their mindfulness practice. Finally, in a study by Sirois and Molnar (2013), the authors explored the relationship between self- compassion and health-related quality of life in a sample of individuals with chronic illness. The study found that higher levels of self- compassion were associated with better health- related quality of life, as well as lower levels of depression and anxiety. Overall, these studies provide evidence for Overall, these studies provide evidence for the importance of self-compassion in promoting well-being and resilience in various populations. The findings suggest that individuals who are more self-compassionate may experience greater subjective well-being, lowerlevels of psychological distress, and better health outcomes. Additionally, self-compassion may serve as a protective factor against negative outcomes, such as the negative effects of social comparison and pain severity. These findings have implications for clinical interventions aimed at promoting self-compassion, particularly in populations experiencing chronic pain, chronic illness, or other forms of psychological distress. Methodology The research utilized a literature review approach, which involved reading and analyzing articles and journals related to the topic. The research team identified relevant articles and 43 43 http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS ropean Journal of Learning on History and Social Sciences Volume 1, Issue 1 ropean Journal of Learning on History and Social Sciences Volume 1, Issue 1 European Journal of Learning on History and Social Sciences European Journal of Learning on History and Social Sciences journals from various databases, such as JSTOR, Google Scholar, and EBSCO, using keywords related to existentialism, knowledge, truth, morality, values, and religion. The researcher then carefully read and analyzed the selected articles and journals, using a thematic analysis approach to identify key concepts and ideas. The team used an inductive approach, allowing the themes to emerge from the data, rather than imposing pre-existing categories on the findings. 2013). Firstly, Nietzsche suggested that morality is misguided, as moralists focus too much on judging others, leading to judge mentalism and the urge to punish. Nietzsche claimed that the motives behind moral judgments are ethically corrupt and poison the outcomes (Solomon, 2001). He believed that moral judgments are often made with the intention of elevating the moralist's own position and denigrating that of others. In doing so, the moralist separates themselves from others and reinforces their own sense of superiority (Leiter, 2013). Secondly, Nietzsche argued that morality is 2009). The researcher also used a critical appraisal The researcher also used a critical appraisal process to evaluate the quality of the sources and ensure the reliability and validity of the findings. This process involved assessing the credibility, transferability, dependability, and conformability of the sources. Overall, the research methodology of this study was exploratory and based on a thorough literature review. The research teamused a thematic analysis approach and a critical appraisal process to identify key concepts and ideas associated with existentialism and their impact on our understanding of knowledge, truth, morality, values, and religion. associated with existentialism and their impact on our understanding of knowledge, truth, morality, values, and religion. Self-compassion is an important construct Self-compassion is an important construct that has received increasing attention in the field of psychology. Research suggests that self- compassion is associated with a range of positive outcomes, including greater subjective well- being, lower levels of psychological distress, and better health outcomes. Additionally, self- compassion may serve as a protective factor against negative outcomes, such as the negative effects of social comparison and pain severity. The findings from these studies have important implications for clinical interventions aimed at promoting self- compassion in various populations, and highlight the potential benefits of cultivating self-compassion in promoting resilience and well-being. Findings Findings Morality Morality Nietzsche's challenges traditional values and institutions, proposing new approaches to ethics. Nietzsche argues that morality is misguided, immoral, and mistaken, offering three types of critique to morality. Friedrich Nietzsche was a 19th century German philosopher who challenged traditional values and institutions, offering new approaches to ethics. In his critique of morality, Nietzsche argued that morality is misguided, immoral, and mistaken, offering three types of critique to morality (Leiter, g yp q y ( 2013). 2013). Lastly, Nietzsche traced the origin of morality and introduced embodied morality, which he called the master morality, with virtues of pride, strength, passion, power, and glory. He argued that avoiding slave morality does not mean evil, but it is just unhealthy and self- destructive. Nietzsche believed that slave morality, which is characterized by meekness, humility, and submissiveness, is opposed to human excellence and is a disaster to humanity (Leiter, 2013). The central ethical value in Nietzsche's philosophy is his revaluation of values, which aims to build a standard morality that promotes human excellence. Nietzsche believed that traditional values and institutions are obstacles to human flourishing, and he sought to create a new set of values that were grounded in the pursuit of excellence (Solomon, 2001). Nietzsche's ideas on morality have had a significant impact on shaping our understanding of values and ethics. Nietzsche's revaluation of values challenged traditional moral systems, and his emphasis on the importance of individual freedom and excellence has been influential in modern philosophy. Nietzsche's ideas have also been applied to other areas, such as politics, where his critique of morality has been used to challenge the legitimacy of political systems that rely on moralistic justifications (Leiter, 2013). In conclusion, Nietzsche's critique of In conclusion, Nietzsche's critique of morality challenges traditional values and institutions, proposing new approaches to ethics. Nietzsche argues that morality is misguided, immoral, and mistaken, offering three types of critique to morality. His emphasis on the importance of individual freedom and excellence has been influential in modern philosophy and his revaluation of values has been used to challenge traditional moral systems. Nietzsche's ideas continue to shape our understanding of values and ethics, and his work remains relevant today. Values Leiter (2002) argues that Nietzsche's central ethical claim is that individuals should focus on cultivating and realizing their unique set of virtues. He believes that everyone should strive to achieve their greatest inspirations, aspirations, and dreams in life, and to do so, theymust love their virtueswith the same devotion they give to their god. According to Nietzsche, self-perfection involves giving style to one's character and treating one's life as a work of art. It requires a balance between the two extremes in life and one must maintain proportion and take risks (Leiter, 2002). http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS Secondly, Nietzsche argued that morality is immoral, as it often derives from dubious motives, such as the fear of punishment. Nietzsche believed that morality presupposes freedom, and when morality is used to coerce individuals to http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS 44 44 ropean Journal of Learning on History and Social Sciences Volume 1, Issue 1 ropean Journal of Learning on History and Social Sciences Volume 1, Issue 1 European Journal of Learning on History and Social Sciences European Journal of Learning on History and Social Sciences act in a certain way, it becomes a dominating tyrant. He believed that individuals should be free to act in their own self- interest, rather than being constrained by external moral codes (Solomon, 2001). act in a certain way, it becomes a dominating tyrant. He believed that individuals should be free to act in their own self- interest, rather than being constrained by external moral codes (Solomon, 2001). Lastly, Nietzsche traced the origin of morality and introduced embodied morality, which in life and one must maintain proportion and take risks (Leiter, 2002). Nietzsche emphasizes the importance of steady progress, commitment, and the development of strength to adopt an environment and aim high (ad altiora) (Leiter, 2002). He argues that successful self-cultivation involves continuous self- overcoming and the pursuit of one's distinctive tasks. Nietzsche values specific virtues, such as 2002). He argues that successful self-cultivation involves continuous self- overcoming and the pursuit of one's distinctive tasks. Nietzsche values specific virtues, such as http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS 45 45 European Journal of Learning on History and Social Sciences European Journal of Learning on History and Social Sciences European Journal of Learning on History and Social Sciences Volume 1, Issue European Journal of Learning on History and Social Sciences Volume 1, Issue pursuing a broad range of talents, developing intellectual depth, and maintaining serenity in the midst of harsh challenges. He also values integrity, delicacy, courage, hardness toward oneself, friendship, devotion to the future,truth-seeking, and a willingness to risk everything to progress in life (Leiter, 2002). Regarding the concept of the will to power, Nietzsche's views have been subject to different interpretations. Some interpret it as the power to dominate others, while others argue that it refers to character strength, the ability to control one's actions and succeed in the face of adversity, and to create order in the midst of chaos (Clark, 2015). Nietzsche emphasizes habit transformation, education, and self-understanding, rather than a principle of self- development (Clark, 2015). In summary, Nietzsche believes that individuals should focus on cultivating and realizing their unique set of virtues to achieve self- perfection, which involves giving style to one's character and treating one's life as a work of art. He values specific virtues such as pursuing a broad range of talents, developing intellectual depth, and maintaining serenity in the midst of harsh challenges. Nietzsche's concept of the will to power is subject to different interpretations, but he emphasizes habit transformation, education, and self-understanding to achievecharacter strength and succeed in the face of adversity. Nietzsche's philosophy emphasizes the Nietzsche's philosophy emphasizes the importance of individualism, self -cultivation, and self- perfection, and his concept of the will to power plays a significant role in his ethical theory (Leiter, importance of individualism, self -cultivation, and self- perfection, and his concept of the will to power plays a significant role in his ethical theory (Leiter, 2002). in life and one must maintain proportion and take risks (Leiter, 2002). The will to power is the driving force behind human behavior, and it is the key to understanding human nature (Nietzsche, 1883/2003).According to Nietzsche, the will to power is not a desire to dominate others, but rather a desire to overcome oneself and become the best version of oneself (Nietzsche, 1883/2003). In this sense, the will to power is closely related to self-cultivation and self- perfection. power is closely related to self-cultivation and self- perfection. Nietzsche believes that individuals should focus on cultivating their unique set of virtues, which involves giving style to one's character and treating one's life as a work of art (Leiter, 2002). This requires a balance between the two extremes in life, and one must maintain proportion and take risks (Nietzsche, 1882/1990). Nietzsche defends specific virtues, such as pursuing a broad range of talents, developing intellectual depth, and maintaining serenity in the midst of harsh challenges (Nietzsche, 1883/2003). He also values integrity, delicacy, courage, hardness toward oneself, friendship, devotion to the future, truth- seeking, and a willingness to risk everything to progress in life. Nietzsche's philosophy also emphasizes the importance of steady progress, commitment, and the development of strength in order to adopt an environment and aim high (Leiter, 2002). He argues that successful self-cultivation involves continuous self-overcoming and the pursuit of one's distinctive tasks (Nietzsche, 1882/1990). Nietzsche encourages individuals to adopt a heroic attitude toward life, which involves taking risks, facing challenges, and pushing oneself to the limit (Nietzsche, 1883/2003). He believes that individuals should strive to achieve their greatest inspirations, aspirations, and dreams in life, and to do so, theymust love their virtueswith the same devotion they give to their god. Nietzsche's philosophy has had a significant impact on modern philosophy, particularly in the areas of ethics, existentialism, and postmodernism (Leiter, 2002). His emphasis on individualism and http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS 46 46 Journal of Learning on History and Social Sciences Volume 1, Issue 1 | 2023 Journal of Learning on History and Social Sciences Volume 1, Issue 1 | 2023 European Journal of Learning on History and Social Sciences European Journal of Learning on History and Social Sciences self-cultivation has been influential in the development of existentialist philosophy, which emphasizes the importance of individual freedom and choice (Sartre, 1943/2007). in life and one must maintain proportion and take risks (Leiter, 2002). Nietzsche's critique of traditional morality has also been influential in postmodernist philosophy, which challenges the idea of objective truth and emphasizes the importance of subjective experience and interpretation (Foucault, 1961/1984). However, Nietzsche's philosophy has also been criticized for its elitism and its tendency to romanticize the individual (Young, 1989). Some critics argue that Nietzsche's emphasis on individualism and self-cultivation neglects the importance of social justice and the role of social institutions in shaping human behavior (Fraser & Nicholson, 1990). Others argue that Nietzsche's concept of the will to power is too individualistic and ignores the importance of community and social interaction in human life (Nehamas, 1985). Despite these criticisms, Nietzsche's philosophy remains relevant today, and his emphasis on individualism, self-cultivation, and the will to power continues to inspire people to pursue their dreams and aspirations (Leiter, 2002). Nietzsche's ideas can be seen in the self -help movement, which emphasizes the importance of personal growth and development, as well as in the field of positive psychology, which focuses on Religion Religion Nietzsche's concept of religion is a central theme in his philosophy. He was a harsh critic of traditional religion, which he saw as a repressive force that hindered the development of the individual. In place of traditional religion, Nietzsche advocated for a new type of religion that he called the "religion of life" or the "religion of the future." According to Nietzsche, the religion of the future would be based on the individual's will to power. The will to power is the driving force behind human behavior, and it is the key to understanding human nature.Nietzsche believed that the pursuit of power was the highest goal of human life, and that this pursuit was best achieved through self- overcoming and the cultivation of personal strengths and virtues. Nietzsche saw the traditional Christian religion as a source of weakness and nihilism. He believed that Christianity had denied the value of this life in favor of an afterlife, and that it had glorified self-denial and suffering as virtues. In contrast, Nietzsche saw the religion of the future as a celebration of life and a rejection of nihilism. He believed that the individual's will to power was the source of all value, and that the pursuit of power was the key to a meaningful and fulfilling life. Nietzsche's Nietzsche's concept of religion is "Nietzsche, Will to Power, and the Future of Religion" by Hugo Strandberg and Tom Grimwood (2021). In this study, the authors explore the relationship between Nietzsche's philosophy and contemporary debates in the fieldof religious studies. They argue that Nietzsche's rejection of traditional religion can be seen as a critique of the way religion has been used to control and oppress individuals, and that his concept of the will to power offers a new way of thinking about religious experience and practice. The authors also argue that Nietzsche's philosophy has important implicationsfor thefuture of religion. They suggest that Nietzsche's emphasis on the individual's will to power can be seen as a call for the democratization of religion, in which individuals are free to create their own religious practices and beliefs. They also suggest that Nietzsche's critique of traditional religion can be seen as a call for a new kind of religious tolerance, in which individuals are free to reject traditional religion without being condemned as immoral or irreligious. In conclusion, Nietzsche's concept of religion is a complex and multifaceted aspect of his philosophy. While he was a harsh critic of traditional religion, he recognized the importanceof religious ideas and practices in human culture. He believed that the new religion of the future would be based on the individual's will to power, and that it would be a source of meaning and inspiration for those who rejected traditional religion. Analysis The article discusses the role of existentialism in shaping our understanding of he article discusses the role of existentialism in shaping our understanding of knowledge, truth, morality, values, and religion. Friedrich Nietzsche, a prominent German philosopher, is considered a central figure in the development of existentialist thought. Nietzsche rejected traditional values, religion, and morality and posited that all truth is subjective. He believed that our perceptions of reality are influenced by personal experiences and biases and that knowledge is a matter of interpretation and judgment. The existentialist emphasis on individual experience and freedom has influenced contemporary theories of knowledge, truth, and morality. Feminist epistemology emphasizes the importance of personal experience in shaping knowledge and truth, while Beauvoir's concept of ethical responsibility emphasizes the importance of individual choice and freedom in shaping moral behavior. Existentialism has also had an impact on the study of values and ethics, leading to a re- evaluation of traditional religious practices and beliefs. http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS While Nietzsche rejected traditional religion, While Nietzsche rejected traditional religion, While Nietzsche rejected traditional religion, he recognized the importance of religious ideas and practices in human culture. He believed that the myths and rituals of traditional religion had served important psychological and social functions, and that they could be repurposed for the new religion of the future. Nietzsche believed that the new religion would be created bythe creative individuals who embraced the will to power, and that it would be a source of meaning and inspiration for those who rejected traditional religion. In recent years, scholars have analyzed Nietzsche's concept of religion from a variety of perspectives. Some have emphasized the existentialist and postmodernist aspects of his thought, 47 47 47 47 http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS Journal of Learning on History and Social Sciences Volume 1, Issue 1 | 2023 Journal of Learning on History and Social Sciences Volume 1, Issue 1 | 2023 European Journal of Learning on History and Social Sciences European Journal of Learning on History and Social Sciences seeing his rejection of traditional religion as part of a broader critique of objective truth and universal values. Others have focused on the psychological dimensions of his philosophy, seeing his emphasis on the will to power as a way of understanding the human psyche and the dynamics of power in social relationships. One recent study that examines Overall, Nietzsche's rejection of traditional Overall, Nietzsche's rejection of traditional values and his emphasis on the individual's subjective experience have challenged traditional notions of objectivity and encouraged a greater emphasis on subjectivity and interpretation. This has led to a greater appreciation for the diversity of perspectives and recognition that there are multiple ways of interpreting the world around us. Existentialism has had a significant impact on 48 48 http://e-science.net/index.php/EJLHSS http://e-science.net/index.php/EJLHSS Journal of Learning on History and Social Sciences Volume 1, Issue 1 | 2023 Journal of Learning on History and Social Sciences Volume 1, Issue 1 | 2023 European Journal of Learning on History and Social Sciences European Journal of Learning on History and Social Sciences our understanding of various aspects of human life and continues to be a prominent philosophical movement today. Conclusion In conclusion, existentialism has played a significant role in shaping our understanding of knowledge, truth, morality, values, and religion. Existentialism emphasizes individual freedom, responsibility, and the subjective experience of life, which has influenced many aspects of human thought and culture. Regarding knowledge, existentialism challenges the traditional view of objective truth and emphasizes the importance of subjective interpretation and personal experience. This has led to the recognition of multiple ways of knowing and has paved the way for more nuanced and diverse understandings of knowledge. In terms of truth, existentialism recognizes that truth is subjective and that individuals create their own meaning and purpose in life. This has resulted in a re-evaluation of traditional religious and philosophical systems, and a greater emphasis on individual autonomy and self - determination. Existentialism has also had a significant Existentialism has also had a significant impact on morality, by emphasizing individual responsibility and the importance of personal choice. This has led to a recognition of the complexity of ethical decision-making and the need for empathy, compassion, and understanding in the face of moral ambiguity. In terms of values, existentialism has In terms of values, existentialism has encouraged individuals to explore and create their own values based on their personal experiences and beliefs. This has led to a greater appreciation of diversity and a recognition of the importance of personal authenticity and individualism. Finally, existentialism has had a significant Finally, existentialism has had a significant impact on religion, by challenging traditional beliefs and emphasizing the importance of personal spiritual experiences. Overall, Nietzsche's rejection of traditional This has led to a greater emphasis on personal spirituality and a recognition of the importance of subjective religious experiences. impact on religion, by challenging traditional beliefs and emphasizing the importance of personal spiritual experiences. This has led to a greater emphasis on personal spirituality and a recognition of the importance of subjective religious experiences. Overall, existentialism has played a significant role in shaping our understanding of knowledge, truth, morality, values, and religion by emphasizing the importance of individual freedom, responsibility, and personal experience. Its influence can be seen in many areas of human thought and culture, and its impact is likely to continue to be felt for many years to come. References References 1. Ansell-Pearson, K. (1994). An introduction to Nietzsche as political thinker: The perfect nihilist. Cambridge University Press. 2. Allen, A. B., & Leary, M. R. (2010). 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Social and Personality Psychology Compass, 4(2), 107- http://e-science.net/index.php/EJLHSS | 51 http://e-science.net/index.php/EJLHSS | 51 European Journal of Learning on History and Social Sciences Volume 1, Issue 1 | 2023 European Journal of Learning on History and Social Sciences Volume 1, Issue 1 | 2023 European Journal of Learning on History and Social Sciences Volume 1, Issue European Journal of Learning on History and Social Sciences Volume 1, Issue European Journal of Learning on History and Social Science European Journal of Learning on History and Social Science https://journal.silkroad-science.com/index.php/EJLHSSS | 52
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Ephrin-A2 promotes prostate cancer metastasis by enhancing angiogenesis and promoting EMT
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Ephrin-A2 promotes prostate cancer metastasis by enhancing angiogenesis and promoting EMT Yao Zhao  The second affiliated hospital of soochow university https://orcid.org/0000-0002-1831-5110 Chenchen Cai  medical technology school of xuzhou medical university Lubing Shi  Xuzhou Medical University Jiwei Wang  Xuzhou Medical University Miaomiao Zhang  Xuzhou Medical University Haoliang Zhang  Xuzhou Medical University Ping Ma  (  672443193@qq.com ) Affiliated Hospital of xuzhou medical University Shibao Li  Affiliated Hospital of xuzhou Medical College Research Keywords: Prostate cancer, EphrinA2, Metastasis, Angiogenesis, EMT Posted Date: September 24th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-78710/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License.   Read Full License Version of Record: A version of this preprint was published at Journal of Cancer Research and Clinical Oncology on March 27th, 2021. See the published version at https://doi.org/10.1007/s00432-021-03618 Ephrin-A2 promotes prostate cancer metastasis by enhancing angiogenesis and promoting EMT Yao Zhao  The second affiliated hospital of soochow university https://orcid.org/0000-0002-1831-5110 Chenchen Cai  medical technology school of xuzhou medical university Lubing Shi  Xuzhou Medical University Jiwei Wang  Xuzhou Medical University Miaomiao Zhang  Xuzhou Medical University Haoliang Zhang  Xuzhou Medical University Ping Ma  (  672443193@qq.com ) Affiliated Hospital of xuzhou medical University Shibao Li  Affiliated Hospital of xuzhou Medical College Research Keywords: Prostate cancer, EphrinA2, Metastasis, Angiogenesis, EMT Posted Date: September 24th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-78710/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Journal of Cancer Research and Clinical Oncology on March 27th, 2021. See the published version at https://doi.org/10.1007/s00432-021-03618 Results We revealed that the ectopic expression of ephrin-A2 in prostate cancer cells facilitated cell migration and invasion in vitro and promoted tumor metastasis and angiogenesis in vivo and that the silencing of ephrin-A2 completely reversed this effect. Although ephrin-A2 did not affect tumor cell proliferation in vitro, ephrin-A2 significantly promoted primary tumor growth in vivo. Furthermore, to determine the biological function of ephrin-A2, we assayed the expression of EMT-related markers in stable established cell lines. Results showed that the overexpression of ephrin-A2 in prostate cancer cells down-regulated the expression of epithelial markers (ZO-1, E-cadherin, and claudin-1) and up-regulated the expression of mesenchymal markers (N-cadherin, β-catenin, vimentin, Slug, and Snail), but the knocking out of ephrin- A2 opposed the effects on the expression of EMT markers. Background Ephrin-A2, a member of the Eph receptor subgroup, is used in diagnosing and determining the prognosis of prostate cancer. However, the role of ephrin-A2 in prostate cancer is still unclear. Methods We established stable clones overexpressing or silencing ephrin-A2 from prostate cancer cells. Then, CCK- 8 was used in analyzing the proliferation ability of cells. CD31 staining was used in evaluating angiogenesis. Migration and invasion assay were conducted in vivo and in vitro. The expression of EMT- related markers was evaluated in prostate cancer cells through Western blotting. Conclusions These findings indicate that ephrin-A2 promotes prostate cancer metastasis by enhancing angiogenesis and promoting EMT and may be a potentially therapeutic target in metastatic prostate cancer. Research Version of Record: A version of this preprint was published at Journal of Cancer Research and Clinical Oncology on March 27th, 2021. See the published version at https://doi.org/10.1007/s00432-021-03618- 2. Version of Record: A version of this preprint was published at Journal of Cancer Research and Clinical Oncology on March 27th, 2021. See the published version at https://doi.org/10.1007/s00432-021-03618- 2. Page 1/18 Page 1/18 Cell culture LNCaP, PC-3, and DU145 were purchased from American Type Culture Collection (ATCC). All the cell lines were cultured in DMEM/F-12 media (Gibco, USA), each containing 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, and 100 U/mL streptomycin, at 5% CO2, 95% atmosphere, and 37 °C. Overexpression of ephrin-A2 The full-length cDNA of human ephrin-A2 was cloned into lentiviruses vectors (pLent-U6-GFP-Puro) at certain site. Vectors within ephrin-A2 fragments or control were transfected into 293T cells at 70% confluency for the generation of the lentiviruses. Then, the lentiviruses were transfected into PC-3 cells at appropriate confluency with polybrene for 24 h. Subsequently, infected cells were selected with puromycin at 2 µg/mL for 1 week. Finally, the selected cells were harvested, and ephrin-A2 expression was confirmed through Western blotting. Background Prostate cancer (PCa), one of the most common malignancies, poses threat to the health of men worldwide1. High metastasis and high recurrence rate after castration treatment are the dominant clinical features of PCa. Metastasis has become a constraint to the long-term survival of patients with PCa and is the key to conquering it. Therefore, a major clinical challenge is preventing PCa progression and metastasis. Page 2/18 Page 2/18 As the largest subgroup family of receptor tyrosine kinase, the Eph receptor family contains 14 members in mammals2. Ephrin-A2, belonging to Eph ligands, are widely expressed in normal tissues and has a vital function in tumors3–6 For example, ephrin-A2 is involved in breast carcinoma, liver cancer, and PCa4, 5. In addition, ephrin-A2 promotes the migration of MDA-MB-231 breast cancer cells and reduces E-cadherin expression6. We previously reported that ephrin-A2 expression is dramatically high in the tissues and exosomes derived from patients with PCa7,8. Although ephrin-A2 has an ectopic expression in PCa patients, its function remains poorly investigated. The abnormal activation of signaling pathways plays a significant role in tumor genesis, progression, and metastasis, and blocking key molecules is an approach for antitumor therapy. The Eph family can trigger EMT-related signaling pathways to regulate cancer9–11. EMT and its related markers, including N- cadherin, vimentin, fibronectin, matrix metalloproteinases (MMPs), E-cadherin, β-catenin, Snail, and Slug, are associated with cancer development. When undergoing EMT, epithelial cells show polarity loss, changes in morphology and cytoskeletal organization, and dissolution of direct contacts. Hence, cell deterioration can be prevented by inhibiting the up-regulation of the expression of EMT-related markers, which might be novel anticancer therapeutic targets12. In this study, we reported that ephrin-A2 stimulated tumor growth in vivo and promoted PCa angiogenesis and metastasis. We highlighted a novel method of using ephrin-A2 in altering the expression of EMT- related markers to promote metastasis. Ephrin-A2 may be a potential target for cancer therapy because it regulates EMT-related markers. This study provides novel insights into the role ephrin-A2 in PCa tumorigenesis. RNA interference of ephrin-A2 Page 3/18 Migration assay Modified migration assays were performed as previously described14. Briefly, cells suspended in a 100 μL serum-free medium were added to the up-wells, and the lower chambers contained 10% FBS as a chemoattractant. The plates were incubated for 24 h. Then, the chamber membrane was fixed and stained with 0.1% crystal violet. Finally, the upper non-migrating cells were gently wiped off with cotton swabs. Six fields were counted randomly (×200), and the average of three independent experiments was obtained. Cell proliferation A total of 3000 cells were plated and incubated for 24, 48, 72, and 96 h. CCK-8 reagent (Keygen Biotech, Nanjing, China) was added to the media and cultured for 3 h. The OD450 value was measured with an automatic micro-plate reader every 24 h for 4 days for the generation of a growth curve. Page 3/18 Page 3/18 Ephrin-A2 shRNA (shRNA-1, shRNA-2, and shRNA -3) or control shRNA were used for RNA interference. Briefly, lentivirus shRNA or control were transfected into the 293T cells at 70% confluency to generate lentiviruses. Then, lentiviruses were transfected into LNCaP and DU145 cells at appropriate confluency with polybrene for 24 h. Subsequently, the cells were selected with proper puromycin (LNCaP for 1 µg/mL, DU145 for 4 µg/mL) for 1 week. Finally, the selected cells were harvested, and ephrin-A2 expression was confirmed through Western blotting. Ephrin-A2 shRNA (shRNA-1, shRNA-2, and shRNA -3) or control shRNA were used for RNA interference. Briefly, lentivirus shRNA or control were transfected into the 293T cells at 70% confluency to generate lentiviruses. Then, lentiviruses were transfected into LNCaP and DU145 cells at appropriate confluency with polybrene for 24 h. Subsequently, the cells were selected with proper puromycin (LNCaP for 1 µg/mL, DU145 for 4 µg/mL) for 1 week. Finally, the selected cells were harvested, and ephrin-A2 expression was confirmed through Western blotting. Wound healing assay DU145, LNCaP and PC-3 cell lines were performed for wound healing assay. In brief, a scratch was made with a micropipette tip at 90% confluency. The wounded area was captured immediately after scratching and after 24 h with a microscope. Wound healing abilities were quantified by measuring the average gap area between 0 and 24 h with ImageJ. Evaluation of angiogenesis CD31 staining was used in evaluating microvascular density (MVD)13. Briefly, the slides were immunohistochemical staining against CD31 (Servicebio Technology, Wuhan, China) were identified (×100). Five areas of MVD were selected for counting manually (×200). Brown-stained endothelial cells and their clusters that were clearly separated from adjacent microvessels, tumor cells, and other connective tissues were considered separate countable vessels. The mean score of the five hot spots was set as the level of MVD in each animal. Invasion assay Modified invasion assays were performed as previously described14. Briefly, diluted Matrigel (Corning Costar, USA) with serum-free medium (1:5) was added to the upper chamber, and incubation was performed for 4 h at 37 °C. Cells suspended in a 100 μL serum-free medium were added into the upper Page 4/18 Page 4/18 wells, and the lower chambers contained 10% FBS. The plates were incubated for 24 h. Then, the well membrane was fixed and stained with 0.1% crystal violet. Finally, the upper non-migrating cells were gently wiped off with cotton swabs. Six fields were counted randomly (×200), and the average of three independent experiments was used. Xenograft models Stable established cell lines were cultured in a DMEM/F-12 medium containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin at 5%CO2, 95% atmosphere, and 37 °C. Single cell suspensions with a concentration of 5×107 cells were inoculated into nude mice at one side of the abdomen. Six BALB/C (nu/nu) nude mice aged 4–6 weeks were randomly divided into two groups of three. The inoculation site was observed, and whether the site was infected or not and whether the tumor had subsided naturally were checked daily. The tumor long diameter (a) and short diameter (b) were measured every week, then the tumor volume was calculated according to the formula: volume = length × width2 / 2 for the drawing of a growth curve. After 7 weeks, nude mice were sacrificed, and tumor tissues, forelimb axillary lymph nodes, and lung tissues were obtained, and tumor masses were weighed. The tumor tissues, left axillary lymph nodes, and left lung tissue were subjected to immunohistochemistry and HE staining. Paraffin sections were subjected to staining, and blood vessel density was assayed with Image Plus. PCR-based detection of human Alu sequences Human disseminated tumor cells (DTCs) were detected in lymph nodes and lungs with Alu-qPCR as previously described15 under the following conditions: 95 °C for 10 min, 40 cycles at 95 °C for 5 s, 65 °C for 5 s, and 72 °C for 20 s. Numerical data were determined against a standard curve established using murine blood DNA containing log-fold dilutions of DNA from 1×106 prostate carcinoma cells（PC-3 or DU145）grown in culture. Negative controls were established for each tissue by testing DNA from species with similar sex and age. The independent experiments were repeated at least three times. Immunohistochemistry Immunohistochemical staining was performed according to a standard method. Briefly, primary tumor tissues were fixed in a 10% formalin buffer, embedded in paraffin, and sectioned at 4 μm. Then, the slides were deparaffinized, rehydrated, and microwaved for 30 min (pH 6.0) for the retrieval of antigen epitopes in citrate buffer. The slides were subsequently treated with 3% H2O2 for 10 min for the elimination of endogenous peroxidase. The slides were incubated overnight with a primary antibody (E-cadherin, N- cadherin, CD31). The section were washed with PBS and then incubated with secondary antibody for 30 min at room temperature. 3,3′-Diaminobenzidine (DAB) was used for color detection, and nuclei were counterstained with hematoxylin. Ephrin-A2 expression in stable established PCa cell lines To explore ephrin-A2 exerted function in PCa, we first developed stable clones overexpressing or knocking down ephrin-A2 in PCa cell lines. The expression of ephrin-A2 in established cell lines was confirmed through Western blotting. Ephrin-A2 showed forced expression in overexpressed PC-3 cells. (Fig. 1A). For the purpose of the efficiency of inhibition, we selected three RNA interferences to block ephrin-A2 expression. Compared with shRNA-1 and shRNA-2 vectors, ephrin-A2 showed significantly inhibited expression in the DU145 and LNCaP cells infected with shRNA-3 lentiviral particles (Fig. 1B and C). Therefore, we used cells infected with shRNA-3 lentiviral particles for the next step. Statistical analysis SPSS16.0 (SPSS, Chicago, USA) was used in statistical analyses. Multiple groups were evaluated with one-way analysis of variance (ANOVA), Statistical between groups were determined through t-test. A P value of less than 0.05 was considered statistically significant. Western blotting assay Page 5/18 Page 5/18 Total protein was extracted from cells, and protein concentrations were determined using a BCA protein reagent kit (Beyotime, Nantong, China). Lysates were separated and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, USA) and incubated with ephrin-A2 (Affinity Biosciences, Cincinnati, USA) and EMT primary antibodies (Cell Signaling Technology, Danvers, USA) at 4 °C for overnight. The membranes were washed three times, incubated with secondary antibodies for 2 h, and visualized using an enhanced chemiluminescent substrate (Millipore, Bedford, USA). Finally, the membranes were visualized again using another enhanced chemiluminescent substrate (Bio-Rad Laboratories Inc, California, USA). Total protein was extracted from cells, and protein concentrations were determined using a BCA protein reagent kit (Beyotime, Nantong, China). Lysates were separated and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, USA) and incubated with ephrin-A2 (Affinity Biosciences, Cincinnati, USA) and EMT primary antibodies (Cell Signaling Technology, Danvers, USA) at 4 °C for overnight. The membranes were washed three times, incubated with secondary antibodies for 2 h, and visualized using an enhanced chemiluminescent substrate (Millipore, Bedford, USA). Finally, the membranes were visualized again using another enhanced chemiluminescent substrate (Bio-Rad Laboratories Inc, California, USA). Ephrin-A2 contributes to tumor growth To investigate whether ephrin-A2 functions in tumor growth, we established stable overexpressing ephrin- A2 in PC-3 cells and silencing ephrin-A2 in DU145 and LNCaP, respectively. No significant difference in proliferation between the control and experimental group was observed in vitro (Fig. 2A). To further verify the feature of this effect, a subcutaneous xenograft model in mice was established. Ephrin-A2 overexpression in the PC-3 cells generated xenografts that were larger than those in the control, consistent with the silencing of ephrin-A2 cells (Fig. 2B). This result indicates that ephrin-A2 contributes to tumor growth in vivo, rather than to proliferation in vitro. Ephrin-A2 promotes tumor angiogenesis Ephrin-A2 may affect tumor environments. By determining whether ephrin-A2 plays a role in tumor angiogenesis, we found that the level of CD31, an indicator of angiogenesis, showed a higher increase in the PC-3 group with overexpressed ephrin-A2 than that in the control (Fig. 3). The silencing of ephrin-A2 in Page 6/18 the DU145 group decreased microvascular density. The data indicate that ephrin-A2 may promote tumor angiogenesis to increase tumor growth. Ephrin-A2 led to tumor metastasis To evaluate the metastasis potential of ephrin-A2, we examined the effect of ephrin-A2 on migration in PC-3 cells. The overexpression of ephrin-A2 in the PC-3 cells facilitated migration (Supporting Fig. 1 and Fig. 4A, P < 0.001). Invasion assay showed that the overexpression of ephrin-A2 in PC-3 cells promoted the migration of cells (Fig. 4B, P < 0.001). These data show that ephrin-A2 can promote tumor invasion in PC-3 cells. To further verify whether ephrin-A2 led to invasion in PCa, we performed migration and invasion assays in depletion of ephrin-A2 in LNCaP and DU145 cells. We found that Ephrin-A2 enhanced the capability of migration and invasion in LNCaP cells (Fig. 4A and Fig. 4B, P < 0.001, Supporting Fig.2) and that the loss of ephrin-A2 significantly inhibited DU145 cell invasion (Fig. 4B). Nevertheless, no significantly changes in cell migration ability was observed in the DU145 cells (Supporting Fig.3 and Fig. 4A, P > 0.05). These data suggest that ephrin-A2 promotes PC-3, LNCaP, and DU145 migration and invasion but has no effect on the migration capabilities of DU145. Furthermore, we assessed whether ephrin-A2 can promote metastasis in vivo in a subcutaneous xenograft model using PC-3 and DU145 cells in nude mice. Tissues harvested from overexpressing ephrin-A2 PC-3 groups and ephrin-A2 knock-down DU145 group. HE staining showed that ephrin-A2 significantly increased the incidence of metastases to local draining lymph nodes (LN) and lungs compared with that in the control group (Fig. 4C and Fig. 4D, P < 0.01). Collectively, these observations indicate that ephrin-A2 contributes to tumor metastasis. Discussion In this study, we first developed stable established cell lines with overexpressing and knock-down, and our results demonstrated that ephrin-A2 promotes prostate cancer metastasis and angiogenesis. Furthermore, we provided novel insights into this mechanism, by which ephrin-A2 alters EMT-related markers to promote cancer metastasis. Moreover, ephrin-A2 may be a potential target for cancer metastasis therapy because it regulates EMT-related markers. In our previous reports, we found that ephrin-A2 expression is significantly higher in the tissues and exosomes derived from patients with PCa7,8. However, in the present study, ephrin-A2 overexpression did not affect cell proliferation in PC-3 cell lines in vitro, and the depletion of ephrin-A2 in DU145 and LNCaP confirmed this finding. Ephrin-A2 contributed to tumor growth in vivo, and this result is consistent with the proliferation of liver cancer cells5. These results show that ephrin-A2 may affect cells in tumor environments and promotes tumor growth, rather than in the tumors. Traditionally, tumor growth depends on multiple factors, such as blood vessel development, growth factors, and nutrition supply. Many studies reported that the Eph family is involved in tumor angiogenesis16–18. To further elucidate the biological functions of ephrin-A2, we detected CD31, an indicator of angiogenesis. Our results show that ephrin-A2 contribute to tumor growth in vivo, rather than in vitro, suggesting that tumor proliferation rate is not responsible for tumor growth. Simultaneously, the depletion of ephrin-A2 reduced angiogenesis. Hence, we speculated that ephrin-A2 can induce angiogenesis and promote tumor metastasis. Overall, these observations revealed that ephrin-A2 may promote PCa progression and metastasis by enhancing angiogenesis, at least in part. Typically, an important feature of malignant tumor cells is invasion. In breast tumor cells, the ephrin-A2 gene promotes cell migration. Feng et al5 confirmed that ephrin-A2 can promote liver cancer cell invasion in vivo. Consistent with previous studies, our study also found that ephrin-A2 has a positive action on cell invasion in PCa in vitro and in vivo. Our results show that the activity of ephrin-A2 can enhance cell motility and invasion. Further, ephrin-A2 increased the incidence of metastases in lymph nodes (LN) and lungs in vivo, indicating that ephrin-A2 leads to tumor metastasis. Page 8/18 EMT, an important mechanism of invasion and metastasis, has been observed in many malignant tumors in humans, but the pathogenesis of this disease remains unclear19. Ephrin-A2 regulates EMT related markers Ephrin-A2, as a member of the Eph receptor tyrosine kinase ligand family, regulates a series of downstream molecules. To determine whether the promotion of metastasis by ephrin-A2 is associated with the EMT family, we tested the expression of EMT-related markers (ZO-1, N-cadherin, E-cadherin, β- catenin, vimentin, Slug, Snail, and claudin-1) in stable PCa cells through Western blotting. The results show the down-regulated expression of epithelial markers (ZO-1, E-cadherin, and claudin-1) and up- regulated expression of mesenchymal markers (N-cadherin, β-catenin, vimentin, Slug, and Snail), but the knocking out of ephrin-A2 opposed the effect on the expression of EMT markers (Fig. 5). These data indicate that ephrin-A2 medicated tumor cell metastasis may be associated with the activation of EMT progress. To confirm the hypothesis, subcutaneous xenograft mouse model was used. The mice were sacrificed, and related tissues were harvested for E-cadherin and N-cadherin expression analyses. In the overexpressing ephrin-A2 PC-3 groups, E-cadherin was down-regulated compared with that in the control group, whereas N-cadherin was up-regulated (Fig. 6A and 6B). Similarly, the depletion of ephrin-A2 expression was consistent with a previous result (Fig. 6A and 6B). The results further show that the activation of EMT signaling pathway play a vital role in ephrin-A2 medicated PCa metastasis. Page 7/18 Page 7/18 Conclusion In our study, we revealed that EphrinA2 facilitated cell migration and invasion in vitro and promoted tumor metastasis and angiogenesis in vivo. Furthermore, EphrinA2 could down-regulated the expression of epithelial markers and up-regulated the expression of mesenchymal markers. These above findings indicate that EphrinA2 promotes prostate cancer metastasis by enhancing angiogenesis and promoting EMT and may be a potentially therapeutic target in metastatic prostate cancer. Discussion Furthermore, ephrin-A2 may be a potential target for cancer metastasis therapy because it regulates EMT-related markers. Declarations Availability of data and materials Abbreviations PCa: prostate cancer; EMT:epithelial-mesenchymal transition; MMPs:matrix metalloproteinases; FBS:fetal bovine serum; MVD:microvascular density; LN:lymph nodes. Discussion Zheng et al20 found that after knocking out the CCAT2 gene, cell proliferation, invasion, and migration were significantly enhanced, suggesting that EMT is closely related to PCa invasion and metastasis. In our study, EMT-related markers were evaluated in stable established cell lines through Western blotting. Our studies identified that ephrin- A2 can inhibit E-cadherin, ZO-1, and claudin-1 expression and enhance β-catenin, vimentin, Snail, Slug, and N-cadherin. E-cadherin is involved in the progression of human malignancies progression, and the low expression of E-cadherin is associated with lymphatic vessel invasion10, 21. When EMT occurs, epithelial cells lose polarity and show enhanced ability to migrate and promote cytoskeletal rearrangement. The overexpression of Eph receptor promotes migration and invasion and inhibits E- cadherin-independent ligand-dependent mechanisms through the downstream singling pathways22, 23. Page 8/18 This study reveals that ephrin-A2 down-regulates E-cadherin and loss cell adhesion to promote invasion and metastasis, suggesting that E-cadherin has potential as a tumor suppressor. This study reveals that ephrin-A2 down-regulates E-cadherin and loss cell adhesion to promote invasion and metastasis, suggesting that E-cadherin has potential as a tumor suppressor. In many tumors, N-cadherin is overexpressed and is related to metastasis24, 25. E-cadherin and N-cadherin belong to the cadherin family. E-cadherin loss is always accompanied by the up-regulation of N-cadherin, which indicates poor prognosis26. N-cadherin promotes cancer metastasis to regulate adhesion and angiogenesis27, 28. Owing to the low expression of N-cadherin in DU145, we assayed N-cadherin in the overexpression ephrin-A2 group only. In this study, ephrin-A2 can increase N-cadherin expression but down-regulate E-cadherin. These data indicate that ephrin-A2 promotes PCa metastasis and angiogenesis by regulating EMT-related markers. N-cadherin and vimentin not only promote tumor invasion and are associated with poor prognosis but also are therapeutic targets in cancer11, 29, 30. Our study indicates that ephrin-A2 intensifies the expression of N-cadherin and vimentin, thereby accelerating tumor invasion and resulting in poor prognosis. Thus, ephrin-A2 is a promising target for PCa cancer therapy. Another key factors, Snail and Slug, are linked to the promoter of E-cadherin and directly involved in inhibiting the expression of E-cadherin31, 32. Ephrin-A2 probably interferes with Snail and Slug to down- regulate E-Cadherin. In conclusion, we demonstrated that ephrin-A2 promotes PCa metastasis and angiogenesis by altering EMT-related markers. Furthermore, ephrin-A2 may be a potential target for cancer metastasis therapy because it regulates EMT-related markers. In conclusion, we demonstrated that ephrin-A2 promotes PCa metastasis and angiogenesis by altering EMT-related markers. Funding This study was supported by the Jiangsu Provincial Medical Youth Talent (No. QNRC2016781) and the National Natural Science Foundation of China (No. 81802094) and Postdoctoral Science Foundation of China (No. 2020M670076ZX) Availability of data and materials Page 9/18 The raw data used and analyzed in the current study are available from the corresponding author upon a reasonable request. Authors’ contributions SBL, YZ and CCC contributed to the idea, drafting and editing of the manuscript. LUS and JWW completed statistical analysis. MMZ and HLZ contributed to literature search and feeding animals and dataanalysis. PM and SBL revised the manuscript with critical reviews and comments and all the authors approved the final version. Acknowledgements Not applicable Competing interests The authors declare that they have no competing interests Ethics approval and consent to participate The animals in this study were obtained in accordance with the ethical standards of the ethics committee of the affiliated Hospital of Xuzhou Medical University. Consent for publication Not applicable References 1. Smith RA, Manassaram-Baptiste D, Brooks D, et al. Cancer screening in the United States, 2014: a review of current American Cancer Society guidelines and current issues in cancer screening. CA Cancer J Clin. 2014;64(1):30–51. 1. Smith RA, Manassaram-Baptiste D, Brooks D, et al. Cancer screening in the United States, 2014: a review of current American Cancer Society guidelines and current issues in cancer screening. CA Cancer J Clin. 2014;64(1):30–51. 2. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science. 2002;298(5600):1912–34. 2. 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J Cell Biol. 2000;148(4):779–90. 28. Gerhardt H, Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res. 2003;314(1):15–23. 28. Gerhardt H, Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res. 2003;314(1):15–23. 29. Mariotti A, Perotti A, Sessa C, Rüegg C. N-cadherin as a therapeutic target in cancer. Expert Opin Investig Drugs. 2007;16(4):451–65. 29. Mariotti A, Perotti A, Sessa C, Rüegg C. N-cadherin as a therapeutic target in cancer. Expert Opin Investig Drugs. 2007;16(4):451–65. 30. Satelli A, Li S. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cell Mol Life Sci. 2011;68(18):3033–46. 30. Satelli A, Li S. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cell Mol Life Sci. 2011;68(18):3033–46. 31. Figures Page 12/18 Figure 1 Ephrin-A2 was assessed in established stable cell lines through Western blotting analysis: A. Ephrin-A2 level was detected in PC-3 cell lines (Left panel). The relative expression of ephrin-A2 was normalized to that in PC-3 cells (Right panel). B. Ephrin-A2 level was detected in DU145 cell lines (Left panel). Ephrin-A relative level expression was normalized to that in DU145/NC cells (Right panel) C. Ephrin-A2 level was detected in LNCaP cell lines (Left panel). Ephrin-A2 relative level expression was normalized to that in LNCaP/NC cells (Right panel). *P < 0.05 compared with the control group. Figure 1 Ephrin-A2 was assessed in established stable cell lines through Western blotting analysis: A. Ephrin-A2 level was detected in PC-3 cell lines (Left panel). The relative expression of ephrin-A2 was normalized to that in PC-3 cells (Right panel). B. Ephrin-A2 level was detected in DU145 cell lines (Left panel). Ephrin-A2 relative level expression was normalized to that in DU145/NC cells (Right panel) C. Ephrin-A2 level was detected in LNCaP cell lines (Left panel). Ephrin-A2 relative level expression was normalized to that in LNCaP/NC cells (Right panel). *P < 0.05 compared with the control group. Page 13/18 Figure 2 Ephrin-A2 contributed to tumor growth in stable cell lines in vivo: A. Ephrin-A2 showed no difference in cell proliferation according to the CCK‑8 assay in vitro. B. Ephrin-A2 contributed to tumor growth in vivo. Tumor harvested from overexpressing ephrin-A2 PC-3 groups and ephrin-A2 knock-down DU145 group. Figure 2 Ephrin A2 contributed to tumor growth in stable cell lines in vivo: A Ephrin A2 showed no difference in Figure 2 Ephrin-A2 contributed to tumor growth in stable cell lines in vivo: A. Ephrin-A2 showed no difference in cell proliferation according to the CCK‑8 assay in vitro. B. Ephrin-A2 contributed to tumor growth in vivo. Tumor harvested from overexpressing ephrin-A2 PC-3 groups and ephrin-A2 knock-down DU145 group. Page 14/18 Figure 3 Ephrin-A2 promotes tumor angiogenesis. CD31 was evaluated in the xenograft models through immunohistochemical staining. Tissues harvested from overexpressing ephrin-A2 PC-3 groups and ephrin-A2 knock-down DU145 group. Scale bar, 100 µm. Figure 3 Ephrin-A2 promotes tumor angiogenesis. CD31 was evaluated in the xenograft models through immunohistochemical staining. Tissues harvested from overexpressing ephrin-A2 PC-3 groups and ephrin-A2 knock-down DU145 group. Scale bar, 100 µm. Ephrin-A2 promotes tumor angiogenesis. CD31 was evaluated in the xenograft models through immunohistochemical staining. Tissues harvested from overexpressing ephrin-A2 PC-3 groups and ephrin-A2 knock-down DU145 group. Scale bar, 100 µm. Page 15/18 Page 15/18 Figure 4 Ephrin-A2 led to tumor metastatic. A. The effect of ephrin-A2 on migration in three PCa cell lines. Ephrin- A2 enhanced PC-3 and LNCaP cell migration, but no effect on DU145. B. The effect of ephrin-A2 on invasion in three PCa cell lines. Ephrin-A2 enhanced prostate cell invasion. C&D. Ephrin-A2 promote metastasis in vivo in PC-3 and DU145 group. Tissues were harvested and the existence of metastatic tumor cells in local draining lymph nodes (Left panel) and lungs (Right panel) were assessed by qPCR- based detection of human Alu sequences and HE staining. Scale bar, 100 µm. Scale bar, 100 µm. Figure 4 Ephrin-A2 led to tumor metastatic. A. The effect of ephrin-A2 on migration in three PCa cell lines. Ephrin- A2 enhanced PC-3 and LNCaP cell migration, but no effect on DU145. B. The effect of ephrin-A2 on invasion in three PCa cell lines. Ephrin-A2 enhanced prostate cell invasion. C&D. Ephrin-A2 promote metastasis in vivo in PC-3 and DU145 group. Tissues were harvested and the existence of metastatic tumor cells in local draining lymph nodes (Left panel) and lungs (Right panel) were assessed by qPCR- based detection of human Alu sequences and HE staining. Scale bar, 100 µm. Scale bar, 100 µm. Page 16/18 Page 16/18 Page 16/18 Figure 5 Ephrin-A2 altered the expression of EMT-related markers. A. Western blotting analysis of EMT-related markers protein expression in PC-3 cell lines (Left panel). The relative expression of ephrin-A2 was normalized to that in PC-3 cells (Right panel). B. Western blotting analysis of EMT-related markers prote expression in DU145 cell lines (Left panel). The relative expression of ephrin-A2 was normalized to that ll ( h l) Figure 5 Figure 6 Ephrin-A2 affect the expression of E-cadherin and N-cadherin in xenograft models. A. E-cadherin and N- cadherin were analyzed in xenograft models by immunohistochemical staining. Tissues harvested from overexpressing ephrin-A2 PC-3 groups. Scale bar, 50 µm. B. E-cadherin were analyzed in xenograft models by immunohistochemical staining. Tissues harvested from knock down ephrin-A2 DU145 groups. Scale bar, 50 µm. Figure 5 Ephrin-A2 altered the expression of EMT-related markers. A. Western blotting analysis of EMT-related markers protein expression in PC-3 cell lines (Left panel). The relative expression of ephrin-A2 was normalized to that in PC-3 cells (Right panel). B. Western blotting analysis of EMT-related markers protein expression in DU145 cell lines (Left panel). The relative expression of ephrin-A2 was normalized to that in DU145 cells (Right panel). Page 17/18 Figure 6 Ephrin-A2 affect the expression of E-cadherin and N-cadherin in xenograft models. A. E-cadherin and N- cadherin were analyzed in xenograft models by immunohistochemical staining. Tissues harvested from overexpressing ephrin-A2 PC-3 groups. Scale bar, 50 µm. B. E-cadherin were analyzed in xenograft model by immunohistochemical staining. Tissues harvested from knock down ephrin-A2 DU145 groups. Scale bar, 50 µm. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupportingDOC.docx SupportingDOC.docx SupportingDOC.docx SupportingFig.1.tif SupportingFig.1.tif SupportingFig.2.tif SupportingFig.2.tif SupportingFig.3.tif SupportingFig.3.tif SupportingDOC.docx SupportingDOC.docx SupportingFig.1.tif SupportingFig.1.tif SupportingFig.2.tif SupportingFig.2.tif SupportingFig.3.tif SupportingFig.3.tif SupportingDOC.docx SupportingFig.1.tif SupportingFig.2.tif SupportingFig.3.tif SupportingFig.3.tif Page 18/18
https://openalex.org/W4287592156
https://zenodo.org/records/6676359/files/%D9%85%D9%82%D8%A7%D9%84%D9%87%207.pdf
English
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Gender Differences in Students' Value System, Engagement, Social Competence, and Cognitive Styles
Zenodo (CERN European Organization for Nuclear Research)
2,020
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Gender Differences in Students' Value System, Engagement, Social Competence, and Cognitive Styles Mahboobeh Moosivand Mahboobeh Moosivand Department of Social Science and Development Studies, Women Research Center, AL Zahra University, Tehran, Iran Keywords Value System, Academic Engagement, Social Competence, Cognitive Styles, Students Abstract The value priorities of individuals are influenced by a variety of factors that affect individuals' social behaviors and actions. The present study was conducted to investigate gender differences in students' value systems, Engagement, social competence, and cognitive styles. The statistical population of this study included all undergraduate students at the University of Tehran in the academic year 1397-98. The research sample was 150 male and female students at Tehran University who were selected by stratified random sampling. The value system questionnaire, the engagement of students’ scale belongs to Gunuc & Kuzu, Hart's competence perception, and the carton cognitive styles questionnaire were used to collect information. Data were analyzed using a multivariate analysis of variance. The results showed that male students scored higher on material values, cognitive competence, social competence, and physical competence. In using educational strategies and teaching methods, education professionals need to consider the gender and individual disparities of students based on physical, psychological, cultural, and spiritual aspects. Keywords Value System, Academic Engagement, Social Competence, Cognitive Styles, Students
https://openalex.org/W2070293860
https://ccsenet.org/journal/index.php/ijbm/article/download/3790/3401
English
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Analysis of Utilization of FDI in the Yangtze River Delta
International journal of business and management
2,009
cc-by
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ccounted for 57 percent of the nation s total, said Wei at a forum on the soci ed enterprises. Currently, more than 28 million people work in these enterprises, or abo pulation in cities and towns of the country. China actually used 36.93 billion dollar s of 2007, a growth of 12.92 percent year-on-year, according to the Ministry of Comme spite the large scale of FDI, its development in different regions is uneven. The easter foreign-funde employed po seven month H d foreign-funde employed po seven month H d al economy was largely promoted. China s GDP increases approximately at a rate of 9%, of wh ributed to foreign investment. China's actual use of foreign direct investment (FDI) had cumul lion U.S. dollars by the end of June this year since the beginning of its opening-up policy in y opening up, foreign investment flowed into China. After 199 k of the development of FDI. In 1992, it increased from $5 m to $27 million, a multifold increase. The investment region ext he area expanded from state-owned enterprises to private comp d the infrastructure construction. In 2006, FDI in China deve g g g p on facilities, access to new technology, pro Abstract Since 1990s, FDI are booming in china gradually and become essential impetus in the development of world’s eco and economy in developing countries especially. The Yangtze River Delta is an important region for foreign investment, including two provinces and shanghai city. FDI contributes a lot to the economic development in Ya River Delta. However, there exist many problems, for example, uneven development and scales of FDI in dif cities, imbalanced development in three industries etc. In this paper, I analyze the problems and come up resolutions to improve the FDI environment and its utilization for more economic boost to this region and Chin whole. Keywords: The Yangtze River Delta, FDI Foreign direct investment (FDI) is defined as "A company from one country obtains controlling interest in a (n existing) firm in another country, and then operates that firm as a part of the multinational business of the investing FDI is a momentum for economic growth. It plays an extraordinary and growing role in global business. It can pr skills and financing. Since 1990s FDI developed very fast in China and becoming an important factor in the eco development. According to the United Nations Conference on Trade and Development, since 2002, the inflow of F China has toped to more than US$ 50 billion, overtaking the United States as the largest recipient of FDI inflows world. There is little doubt that FDI has been a significant part of China’s economy especially in the manufac industries. Yangtze River Delta right now is an area with the fastest speed in economic development and gr potential. And FDI is a great contribution to its development. However, a lot of problems occurred in the develo of FDI. I will list some problems and analyze them in this paper for a better utilization of FDI in this region so make a greater contribution to China’s economy and the world’s economy as a whole g g g p on facilities, access to new technology, pro ial responsibili out 10 percent o s in FDI in the erce. rn China is the ial responsibili out 10 percent o s in FDI in the erce. rn China is the nal Journal of Business and Management Septembe ch industry .The development of Yangtze River sides financial aspect, its influence covers large c dus y . e deve op e o g e ve sides financial aspect, its influence covers large r liberalize its service sector, an increasing share of FDI is expected to go into the service sectors in the coming e reason why FDI thrives in Yangtze River Delta ze River Delta is an economic zone with the largest population of 350 million and the most developed economy in . Its importance also lies on its geographic advantage: it is both the hub of water communication and gold coast in with 1000km of coastal line and 600km inland navigation line along which are located Shanghai, Ningbo, han, Nanjing and Zhengjiang etc. From there, vessels can reach the ports of over 160 countries and areas with hai as the center of the modernized network. The main agriculture products cover the half of the national products e markets are quite potential. The network system in this region is also in the leading place in China. What’s more, Yangtze River Delta has a relatively stronger cluster of higher education institutions turning out a greater local y of quality workers prevailing as the base of R&D in the hi-tech industry The development of Yangtze River ple; second, some government officials only t instead of the quality There are too many ple; second, some government officials only instead of the quality There are too many buted. The population in Shanghai and Suzhou cities only account for 25% of the total, the land area only 15%. he accumulative FDI of these tow cities has been up to 58%. They are the only two that have accumulative FDI ssing 30 billion US dollars. The main cities that attracted high FDI are Wuxi, Nnajing, Ningbo, Hangzhou, gzhou, Zhenjiang, Nantong and the rest shared relatively low ratio. In 2004, the utilized FDI reached 24 billion s of which Jiangsu province, Shanghai city, Zhejiang province shared respectively 47.5%, 27%, 25.5%. The bed FDI of Shanghai exceeds that of Zhejiang province and FDI of Jiangsu province almost twice of that in ang. There exists large gap between different cities in Yangtze River Delta. gap was generated by two reasons: first, due to the differences of city scale, geographic situation, and relative including policies, the way the organizations act. While Shanghai has more developed financial and trading es, its leading role is irreplaceable. me new problems oblems occurred in the development Uneven development in different cities of this region o the distinction of investment environment and competitive edge, FDI in Yangtze River Delta is unevenly Analysis of Utilization of FDI in the Yangtze River Delta Zhiwei Xiong School of Intercultural Studies, Jiangxi Normal University 99 Zi yang Street, Nanchang 330022, China Tel: 86-791-6013-946 E-mail: lightinair@163.com International Journal of Business and Management n about the political record and pursue the quantity of investment instead of the quality. There are too many ps in the so called “pillar industry”. Many cities are eager to bring in more investment on electronic, chemical ering, and medicine industry and ignore the different situations of these cities. For example, there are 11 cities g automobile industry and 8 cities choosing petrifaction industry. This kind of disorderly development will lead to ndous waste of resources and imbalanced investment proportion. er factor that caused the gap is that different cities adopt different economic developing models. For example, ng used to spur its economy in nongovernmental economic model Most part of its GDP is from private dquarters here, such as Siemens, AMD and General engaged in manufacturing activities including the ghai, Nanjing, Wuxi, Changzhou, Nantong, Yangzhou, Zhenjiang, Taizhou, Hangzhou, Ningbo, Jiaxing, Huzhou, xing, Zhoushan 15 cities. Since 1990s, FDI in Yangtze River Delta developed very fast. Take Shanghai for example, tually absorbed foreign investment was $177million and in 2006 it amounted to $7.107billion, almost 71 times of n 1990.( See Table one). The investment distribution in Yangtze River Delta appears like that: Shanghai is in the r and FDI descend through the cities along Huning railway and Huhang railway. The absorbed foreign investment anghai, Suzhou, Wuxi cities accounted 55% of that in the whole region and Shanghai played a leading role in it. In shanghai attracted $6.541 billion FDI which is number one in the 15 cities. r foreign investors in the Yangtze River Delta include General Motors, Shell, Matsushita, Exxon, Sony, Siemens, opment. In the first half of 2007, $28.5 billion flew into this part, accounting nearly 90% of the total in the whole ry. The west area absorbed $1.5 billion from foreign investors, a 52% increase of the same period of last year. In iddle area, the number reached $1.9 billion which increase 6% compared to last year. In the future, there will be and more investment from multinationals instead of from small and media size companies of HK, Macao and n. evelopment of FDI in the Yangtze River Delta ze River Delta is an economic region compromised by Shanghai, Jiangsu and Zhejiang province, including , No. 9 nte national Jou nal of usiness and anagement n foreign investors choose, the volume of the FDI in the west area is relatively low with a high speed of opment In the first half of 2007 $28 5 billion flew into this part accounting nearly 90% of the total in the whole y , p , p g p 2.2.2 Approval system needs to be improved In administration, some departments have not held the belief to serve the people and barricade too much app procedure which made it very complicated to approve a program. When they passed through the approval procedure, all the business opportunities have gone. There existed management problems. ratio of investment in this area asc len. investment of tertiary industry ce ratio of investment in this area asc llen. dquarters here, such as Siemens, AMD and General engaged in manufacturing activities including the The departments that have the power will fight with each other for the programs that are in p g p 2.2.2 Approval system needs to be improved In administration, some departments have not held the belief to serve the people and barricade too much app procedure which made it very complicated to approve a program. When they passed through the approval procedure, all the business opportunities have gone. There existed management problems. The departments that have the power will fight with each other for the programs that are in ources is the bottleneck for China to transform it econ asset” In a knowledge economy we need to attract and hold on to people who know a lot. In such cases, attra talented people to work in this region in a way means attracting more foreign investment. Although the supply of resources surpasses the demand in the job market in China, and we have the advantage of cheap labor, but obvious are lack of talents. Despite the relatively advanced technology and education in Yangtze River Delta., it is still h meet the demand of the highly developing economy in China, especially the demand for technology talents. B same token, the rapid speed of the development of FDI requires a large number of professional technology talents school education, however, can not meet the requirement of labor market for different talents, not even the dema f i i t Th f th l ti f l b i th b ttl k f Chi t t f it shortage will speed up the shift of foreign capitals. 2.2 Problems in all these places 2.2.1 “People problem” We’re now faced with an era in which globalization deepens with each passing day. And the competition be nations has been the competition among talented people. Lots of managers announced that “our people are our b ent plan. Experts said, the For instance, in Wuxi, there was a shortage of technicians totaled over ten thousand. A survey covering more tha foreign companies and joint ventures gave this result. Resources shortages significantly influence the investment environment of Yangtze River Delta and had som ff i lik i h fid f f i i ’ i l E id h Land shortage. More and more development zone to some extend spurred the economic development but on the side lead to the decreasing arable land. Lack of Technicians. Human resources especially the lack of advanced technicians was the problem facing this r F i t i W i th h t f t h i i t t l d t th d A i th which is almost the number of that in 2002. That set a new historical record and companies had to cut produ because of this. L d h t M d d l t t t d d th i d l t b t th growth mode featured high input, large consumption, high growth and low benefit is no longer suitable for to society. A shortage of electricity, land, water, and technicians has ringed the alarm to this region. Electricity shortage. Since 2003, the amount of the limited electricity power used have reached 5.6 billion kil hi h i l t th b f th t i 2002 Th t t hi t i l d d i h d t t d g y y y y 2.1.3 Resource shortages The lack of resources made Yangtze River Delta not that desirable and attractive. Right now, the extensive econ growth mode featured high input large consumption high growth and low benefit is no longer suitable for to th pattern, optimizing the economic nvironment At the same time the th pattern, optimizing the economic nvironment At the same time the s not only an innovation of FDI development model but also a model for economic development. Through ing foreign capitals with their local resources, we can combine the two markets together and boost this kind of economy. tract more investment to the first and tertiary industry overnment should lead the investment orientation and promote construction of rural area. Although our country great attention to the rural area construction, the fixed asset investment in Yangtze River Delta has not been the in investors’ hand. Hence, the agriculture industry needs more capital to develop. Jiangsu and Zhejiang provinces arge land areas and it will be significant to boost agriculture industry. nment should encourage the development of the service industry because the service industry together with the acturing industry now serves as an engine for the region's overall economic growth. Yangtze River Delta should h t it f t l i Chi d t f th i th tt ti i i th i ent of tertiary industry ce t t i thi If we can reduce the phenomenon of ent of tertiary industry ce t t i thi y g y 2.1.2 Imbalanced development in different industries As changed taking place in global industries, the tendency of FDI is turning from labor intensive industry into c and technology intensive industry, from manufacturing industry into tertiary industry that mainly rely on servic new and high-tech industry as well as infrastructure construction. Apparently, investment in Yangtze River Delta on the second industry, and especially manufacturing industry, of which the electronic and communication equi manufactures are the number one. It accounted for 15.4% of the total manufacturing industry that foreign inves aimed at. From these statistics, the focal point of investment is on the capital and technology intensive ind especially processing and packing industry in the labor intensive industry. The ratio of agriculture industry account little in the whole investment. But under the encouragement of our co there is much increase in utilization of agriculture and infrastructure construction. This demonstrates that some f hejiang province started attracting foreign inves overseas market expansion as their top development target, while 8% said their top priority was listing. As the de caused by economic development and increasing international economic confliction, the disadvantage of purely r i i h d i d il b i Ri h Zh ji i d i f i i International Journal of Business and Management September, enterprises Jiangsu and Zhejiang have the largest number of private enterprises in China, and according to a surv Jiangsu private enterprises conducted by HKTDC in 2003, 56% of the responding private enterprises cons rain, seemingly, the people problem will be reduced What’s more, bringing in more technology is also crucial. We should associate technology with innovation and ish an open, fair and orderly labor market to meet the need of the fast development of Yangtze River Delta. During ast 30 years since China’s reform and opening up, there are an increasing number of students’ studies aboard. nts leaving this region for west countries accounts for a large proportion. If we can reduce the phenomenon of What’s more, bringing in more technology is also crucial. We should associate technology with innovation and ish an open, fair and orderly labor market to meet the need of the fast development of Yangtze River Delta. During st 30 years since China’s reform and opening up, there are an increasing number of students’ studies aboard. nts leaving this region for west countries accounts for a large proportion. g g provide training course to different kinds of y. This solution does not only help to create a desirable environment for FDI development, but also do good to our ry’s economic growth. Apparently, our government has concerned about energy problems, but there is still a long o go to achieve this. ultivate professionals in different areas an increasing number of foreign enterprises making their way into Chinese market the “people issue”, especially anagement of local and foreign staff, have been brought to the forefront more than ever before. For most foreign anies operating in China, it is not surprising that they would like to acquire professional services that align with ational practice and their parent companies' standards. These foreign companies would also appreciate a service der who is familiar with the China market. As a result, there is a need for a large amount of talents that are ient in different areas. They may be specialists in laws, finances, English, international trading etc or people who professional knowledge in several areas Cultivating such kind of talents is now the task facing this region for its another round of FDI development. What s more, pay uld improve the energy efficiency and use renewable nother round of FDI development. What s more, pay uld improve the energy efficiency and use renewable ive one. The Chinese leadership has realized that China s past development norm is physically unsustainable, and cally and socially undesirable. The basic features of Intensive development model are increasing economic growth gh improving the quality of material and work efficiency with a relatively low consumption and cost. The ormation of economic growth model will at the same time cushion us from the effect of resources shortage. Easing gent problems of the resources shortage in turn will spur another round of FDI development What’s more pay g g y y , ment structure of second industry need to be adjusted. To achieve this, we should strengthen the innovation and ore value to the product. Moreover, Shanghai city and the two provinces should avoid the overlaps of the industry ure and shape differentiation competition so that to be competitive in the world market. ansform the economic growth model roblem of resources shortages that mention above alarms us that we need to change our economic growth model better utilization of energy resources. In 2006, China started to alter its old extensive growth model to a more i Th Chi l d hi h li d th t Chi ’ t d l t i h i ll t i bl d , g ( ) p g g f j g Graphic Communication, 10, pp. 44-48. NG fung Yee Linda and TUAN Chyan. (2002). FDI Facilitated by Agglomeration Economies: Evidence Manufacturing and Services joint Ventures in China. Journal of Asia Economics, Vol.13 No.6, pp. 749-765. improve the investment environment and strength the whole region’s competitiveness, balancing the interreg development problems as well as handling the bumpy thing on the road of approval. Only through this way, w reach the goal of developing FDI in Yangtze River Delta coordinating social and ecology problems and at last ac References Edward M. Graham, & Erika Wada. (2001). FDI in China: Effects on Growth and Economic Performance. O University Press g p g g , g gy p the sustainable development of our nation. g , pp y g p p p improve the investment environment and strength the whole region’s competitiveness, balancing the interre development problems as well as handling the bumpy thing on the road of approval. Only through this way, w reach the goal of developing FDI in Yangtze River Delta, coordinating social and ecology problems and at last ac the sustainable development of our nation. References Edward M. Graham, & Erika Wada. (2001). FDI in China: Effects on Growth and Economic Performance. O University Press. Q g , p n more FDI through private companies. 4, No. 9 International Journal of Business and Management ow efficiency, and companies have more challenges to conquer. All of these lead to bad situation for investment tion and promotion and make it even harder to develop FDI in Yangtze River Delta. Hence, the entry barriers of us service sectors must to be lowered for foreign investors and we should improve the approval system. utions for the problems mentioned above alancing the interregional development of FDI t interregional Economic Structure to balance the development of FDI in different cities. Suzhou, Shanghai Wuxi d stick to the opening up policy, taking the advantage of foreign capitals to promote the capital-intensive and ology- intensive industry. In Shanghai Pudong new area, Suzhou Industrial Park as well as all kinds of national mic & development area, the environment and equipment should be improved. We also need to expand our ment scale to improve the software and hardware environment. The rest of the place is better to make full use of ocal resources and advantage, promoting innovation to attract more foreign capitals. Private enterprises need to be ative in systems and management as well as in technology said Zong Qinghou chairman of Wahaha Group
https://openalex.org/W2800820544
http://library.oapen.org/bitstream/20.500.12657/26578/1/1003494.pdf
German
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Jacob Grimm und Vuk Karadžić
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1,977
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Verlag Otto Sagner München ∙ Berlin ∙ Washington D.C. Digitalisiert im Rahmen der Kooperation mit dem DFG-Projekt „Digi20“ der Bayerischen Staatsbibliothek, München. OCR-Bearbeitung und Erstellung des eBooks durch den Verlag Otto Sagner: Vera Bojić Jacob Grimm und Vuk Karadžić Ein Vergleich ihrer Sprachauffassungen und ihre Zusammenarbeit auf dem Gebiet der serbischen Grammatik Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access http://verlag.kubon-sagner.de © bei Verlag Otto Sagner. Eine Verwertung oder Weitergabe der Texte und Abbildungen, insbesondere durch Vervielfältigung, ist ohne vorherige schriftliche Genehmigung des Verlages unzulässig. S l a v i s t i c h e B e i t r ä g e BEGRIJNDĘT VON ALOIS SCHMAUS HERAUSGEGEBEN VON JOHANNES HOLTHUSEN UND JOSEF SCHRENK REDAKTION: PETER REHDER Band 106 00051004 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Bayerische Staatsbibliothek München ISBN 3-87690-127-8 Copyright by Verlag Otto Sagner, München 1977 Abteilung der Firma Kubon & Sagner, München Druck: Alexander Grossmann Fäustlestr. 1, D -8000 München 2 Vera Boji - 9783954793044 Bayerische Staatsbibliothek München Bayerische Staatsbibliothek München Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ISBN 3-87690-127-8 Copyright by Verlag Otto Sagner, München 1977 Abteilung der Firma Kubon & Sagner, München Druck: Alexander Grossmann Fäustlestr. 1, D -8000 München 2 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vorwort Die vorliegende Arbeit wurde im Sommersemester 1976 von der Philosophischen Fakultät der Universität Bonn als Dissertation angenommen* Herrn Prof. Dr. Hans Rothe bin ich für sein wohlwollendes Interesse und zahlreiche Anregungen zu großem Dank verpflichtet. Mein herzlicher Dank g ilt Herrn Prof. Dr. Pavle Ivic, Belgrad, der mir wichtige Hinweise und Ratschläge e rte ilt hat. Ebenso danke ich der Serbischen Akademie der Wissen- schäften in Belgrad und dem Brüder Grimm-Museum in Kassel für die Überlassung der Archivmaterialien. Herrn Prof. Dr. Johannes Holthusen und Herrn Prof. Dr. Josef Schrenk danke ich für die Aufnahme der Arbeit in die Reihe "Slavistische Beiträge". Bonn, im Januar 1977 Vera Bojic - 6 - Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access INHALTSVERZEICHNIS Vorwort ........................................................................................................................................................ 5 EINLEITUNG ............................................................................................................................................... 9 A. JACOB GRIMMS SPRACHAUFFASSUNG ....................................................................... 13 I. Grimms Ansichten über den Ursprung und die Entwicklung der Sprache .............................................................................. 13 1. Der Gegensatz zwischen der alten und der neuen Sprache ..................................................................................................... 14 2. Die Entwicklung der Sprache als Vervollkommnung ........................................................................................................... 17 3. Sprache als Form........................................................................................................ 1Ѳ 4. Sprache als Geschichte .................................................................................... 20 5. Der sprachliche Organismus ....................................................................... 21 II* Die nationale Schriftsprache ........................................................................ 22 1. Die Entstehung der Schriftsprache ................................................. 22 a. Schriftsprache und Mundarten ....................................................... 23 b. Das Verhältnis von Schriftsprache, Volkssprache und Dichtersprache ............................................. 24 2 Ѣ Die Schriftsprache als konstitutiver Faktor der Nation ..................................................................................................... 28 3• Der nationale Charakter der Sprache und das Problem der Übersetzbarkeit ....................................................... 29 I I I . Die Aufgabe der Sprachwissenschaft ................................................. 31 1. Die Diachronie als Vorstufe der Synchronie .................... 32 2. Grimms Versuch der Orthographiereform .................................... 34 B* VUK KARADZICS AUFFASSUNG DER SPRACHE ................................................. 38 I. Die sprachliche Situation bei den Serben zu Beginn von Vuks Reformbemühungen .................................................... 38 II.Vuks Beziehung zur zeitgenössischen Sprachwissenschaft ............................................................................................................ 42 1. Vuk und Kopitar ............................................................................................................ 42 2. Vuk und Herder ............................................................................................................... 47 3* Vuk und J. Ch. Adelung ..................................................................................... 51 III. Die Entwicklung von Vuks Ansichten über die Schriftsprache ............................................................................................................ 54 1. Die Rolle der Dichter und des Volkes bei der Bildung der Schriftsprache ........................................................... 54 a. Die Begründung der Schriftsprache ........................................ 55 b. Die Erweiterung der Ausdrucksmittel ................................. 63 c. Vuks philologische Methode .............................................................. 6 5 2. Die Beziehung der serbischen Schriftsprache zur kirchenslavischen Sprache .............................................................. 67 3. Die Schriftsprache und die Dialekte ........................................... 87 a. Vuks Weg zum synthetischen Dialekt .................................... 87 b. Diachronie im Dienste der Synchronie bei Vuk ............................................................................................................................ 94 c. Von der Dorfsprache zur Stadtsprache .............................. 98 d. Die einheitliche Schriftsprache .............................................. lOO 4. Vuks Ansichten über den čakavischen und den kajkavischen Dialekt ............................................................................... 104 C. VUKS UND GRIMMS ZUSAMMENARBEIT AUF DEM GEBIET DER SERBISCHEN GRAMMATIK ............................................................................................... 110 I. Die deutsche Übersetzung von Vuks serbischer Grammatik ............................................................................................................ 110 1. Die Abweichungen der Übersetzung vom Original ...................................................................................................................... 115 a. Die Erweiterung der Grammatik ..................................................... 115 b. Die von Vuk und Grimm gemeinsam geplanten Ergänzungen ............................................................................... 115 c. Grimms Ergänzungen der Grammatik ........................................... 125 d. Grimms Kürzungen, Umstellungen und Umformulierungen des Originaltextes ................................. 134 e. Mißverständnisse des Originaltextes und Übersetzungsfehler ............................................................................ 142 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" ............................................................................................................................ 145 3. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access INHALTSVERZEICHNIS "Wuk's Stephenowitsch kleine serbische Grammatik verdeutscht und mit einer Vorrede von Jacob Grimm" - ein gemeinsames Werk der beiden Autoren ............................................................................................................... 162 II. Grimms und Vuks Plan einer erweiterten Auflage der serbischen Grammatik ...............................................................................................164 1. Vuks Vorbereitungen für die erweiterte Auflage seiner Grammatik ......................................................................................165 a. Vuks Arbeit zur Wortbildung der Substantive und Adjektive .........................................................................165 b. Vuks Arbeit über die Verben .............................................................168 2. Jacob Grimms Materialien in seinem Handexemplar der "Kleinen serbischen Grammmatik ......................................................172 a. Zur Siedlungs- und Kulturgeschichte .........................................174 b. Zur Lautlehre ...............................................................................................................176 c. Zur Deklination der Substantive ......................................................179 d. Zum Adjektiv ..................................................................................................................180 e. Zu den Zahlwörtern, Pronomina und Partikeln ............................................................................................................................181 f. Zu den Verben ...............................................................................................................182 g. Der Zeitraum von Grimms Beschäftigung mit der serbischen Sprache ......................................................................185 h. Der Umfang von Grimms Kenntnissen der der serbischen Sprache ..................................................................................188 D. SCHLUSSBETRACHTUNG ....................................................................................................................191 E. ANHANG I. Verzeichnis der Abweichungen der deutschen Fassung von Vuks Original der serbischen Grammmatik ........................................203 II. Zusammenstellung der Notizen aus Grimms Handexemplar mit Quellenangaben ..................................................................2 30 LITERATURVERZEICHNIS ........................................................................................................................247 NAMENREGISTER ..............................................................................................................................................255 ABKÜRZUNGESVERZEICHNIS ..................................................................................................................257 1 Die Genese von Vuks Orthographie wird ausführlich von Lj. Stojano- vie, Život i rad Vuka Stef. Karadzièa, 1924, S. 132-145, darge- s te llt. Unter den zahlreichen Arbeiten, die sich mit dieser Proble matik beschäftigen, sei hier auf folgende neuere Untersuchungen hingewiesen: P. Ivic, 0 Vukovom Rječniku iz 1818 godine, 1966, S. 29ff, 64ff; M. Kravar, Drei Fragen der serbokroatischen Phonologie In: Osteuropastudien... Hessen, R. I I I , Bd. 8, 1966, S. 47-50; A. Mladenovic, Slovo ä pre Vuka. In: Zbornik FL VI, 1963, S. 158-162; Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM i f EINLEITUNG Die Zusammenarbeit von Vuk Karadžic und Jacob Grimm war in der Zeit, als Vuk um die Neubegründung der serbischen Schriftsprache kämpfte, von großer Bedeutung, Grimm hat mit seinen Rezensionen der serbischen Volksdichtung wesentlich zu deren Aufnahme und Verbrei- tung in Europa beigetragen. Es war vor allem Grimms Verdienst, daß die Bedeutung von Vuks Bemühungen zuerst in Deutschland gewürdigt wurde; diese Anerkennung im Ausland trug bei den Serben zum Durch- bruch von Vuks Reformen bei. Grimms Verdienste um Vuk wurden in der Forschung oft hervorgeho- ben. Allerdings stand dabei Grimms hohe Einschätzung der serbischen Volksdichtung im Vordergrund. Grimm ging in seinen Rezensionen aber auch auf die sprachliche Situation bei den Serben ein. Deshalb is t es erforderlich, die Beziehung zwischen Vuk und Grimm auch im Zusam- menhang mit verschiedenen sprachlichen Problemen zu untersuchen. Das Ziel der vorliegenden Arbeit is t es, Grimms und Vuks Ansich- ten Über die Sprache einander gegenüberzustellen und ihre Zusammen- arbeit auf dem Gebiet der serbischen Grammatik zu untersuchen. Es sollen Gemeinsamkeiten und Unterschiede in ihren theoretischen Auf- fassungen festgestellt und gegenseitige Einflüsse aufgedeckt werden, die sich auch in ihrer empirischen Arbeit ausgewirkt haben. Die Darstellung von Grimms Sprachauffassung wird an den Anfang gesetzt (A). Grimm war bereits ein anerkannter Wissenschaftler, als Vuks selbständige Entwicklung begann, so daß Einflüsse zunächst von seiten Grimms ausgingen. Auch Grimms Beurteilung der serbischen Spra- che und seine Behandlung verschiedener sprachlicher Probleme können nur im Zusammenhang mit seiner Sprachauffassung richtig verstanden werden. In diesem Kapitel wird auch auf Anregungen hingewiesen, die Grimm aus seiner Zusammenarbeit mit Vuk erhalten hat. Vollständigkeit in der Darstellung von Grimms Sprachauffassung wird in dieser Arbeit nicht angestrebt, weil der Schwerpunkt auf denjenigen Problemen liegt, die auch bei Vuk auftreten. Das sind vor allem die Fragen, die im Zusammenhang mit der Begründung und Ent- wicklmg der Schriftsprache stehen. Eine Ausnahme bilden Grimms An- sichten über die Entwicklung der Sprache im allgemeinen, deren Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Darstellung u.E. zum Verständnis von Grimms Beurteilung der serbi- sehen Sprache erforderlich is t. Darstellung u.E. zum Verständnis von Grimms Beurteilung der serbi- sehen Sprache erforderlich is t. Im zweiten Teil der Arbeit (B) wird die Entwicklung von Vuks Ansichten über die Schriftsprache dargestellt. Seine Beziehung zu Grimm wird dabei ständig im Auge behalten#um Einflüsse festzustel- len. EINLEITUNG Es werden sowohl Gemeinsamkeiten als auch Unterschiede zwischer Vuks und Grimms Ansichten hervorgehoben und ihre Hintergründe aufge- zeigt. In diesem Zusammenhang werden auch Vuks Beziehungen zu Kopi- tar, Herder und Adelung herangezogen. Vuks Reform des serbischen Alphabets und der Orthographie wird im Rahmen dieses Themas nicht behandelt. Die Entwicklung von Vuks Orthographie is t in der Forschung ausführlich erörtert worden und kann als geklärt angesehen werden. Die Ergebnisse der Wissenschaft- liehen Untersuchungen stimmen im allgemeinen darin überein, daß Vuks Graphie phonologisch und die Orthographie phonetisch-phonologisch is t1. Der dritte Teil der Arbeit (C) befaßt sich mit Vuks und Grimms Zusammenarbeit auf dem Gebiet der serbischen Grammatik. Zunächst wird die deutsche Fassung von Vuks serbischer Grammatik, als deren Übersetzer Grimm g ilt, untersucht (I). Es wird ein Vergleich der Übersetzung mit dem Original unternommen. Die festgestellten Abwei- chungen werden sprachlich und hinsichtlich der Grammatikkonzeption untersucht, um ihren Urheber festzustellen und um die Frage nach dem Übersetzer der Grammatik zu beantworten; sie werden zugleich in bezug auf ihren wissenschaftlichen Wert innerhalb der Grammatik ge- prüft. Anschließend werden Vuks und Grimms Materialien untersucht (II)! die vermutlich zu dem von Grimm angeregten Plan einer erweiterten Ausgabe der serbischen Grantnatik gehören. Dabei handelt es sich um zwei Arbeiten von Vuk, von denen eine bisher unveröffentlicht ge- blieben is t (II,b). Grimms Materialien sind in Form von handschrift- schriftlichen Eintragungen in seinem Handexemplar der "Kleinen ser- bischen Grammatik" enthalten. Es sind Notizen in Stichworten, zu deren Auswertung und Verständnis die Ermittlung von Grimms Quellen erforderlich war. schriftlichen Eintragungen in seinem Handexemplar der "Kleinen ser- bischen Grammatik" enthalten. Es sind Notizen in Stichworten, zu deren Auswertung und Verständnis die Ermittlung von Grimms Quellen erforderlich war. Die Untersuchung dieses Materials versucht, über Grimms und Vuks Grammatikkonzeption und ihre in diesen Materialien enthalte- nen Forschungsergebnisse Auskunft zu geben. Dabei wird die Frage gestellt, inwiefern ihre Zusammenarbeit weiterführende Ergebnisse für beide Seiten gebracht hat. Auch Grimms germanistische Arbeiten werden in diesem Zusammenhang herangezogen. Die Grimm-Forschung hat L. Denecke treffend charakterisiert, wenn er sagt, der Beurteilung durch Zeitgenossen, die "achtungsvoll, aber freimütig und kritisch wertend" war, sei bei der 100־jährigen Geburtstagfeier eine Wende gefolgt: die neue Generation fühlte sich in ihrem nationalen Denken durch Grimms Liebe zu den deutschen Men- sehen bestätigt. Im 20. Jahrhundert folgte die "Romantisierung" der Gebrüder Grimm, in dem Sinne, daß sie vorbehaltlos in die Romantik eingeordnet wurden1. ders.. Znak j и perspektivi predvukovskog ciriličkog pravopisa. In: Godišnjak FF и Novom Sadu VII, 1962/63, S. 45-52; ders., Priloq pro učavanju porekla slova dž. In: Anali FF 5, 1965, S. 125-157. 1 Vgl. L. Denecke, Jacob Grimm und sein Bruder Wilhelm, 1971, S. 180. 2 Vgl.die Aufsätze von W. Boehlich und R. Gruenter in: Der Monat, H. 154 (1961), 196 (1965), 197 (1965), 217 (1966). 3 Vgl. darüber H. Kolb, Karl Marx und Jacob Grimm. In: Archiv für das Studium der neueren Sprachen und Literaturen, Bd. 2 0 6 , 1969-70, S. 96-114. ( ) ( ) ( ) ( ) 3 Vgl. darüber H. Kolb, Karl Marx und Jacob Grimm. In: Archiv für das Studium der neueren Sprachen und Literaturen, Bd. 2 0 6 , 1969-70, S. 96-114. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access EINLEITUNG Dazu hat auch die bisher ausführlichste Jacob Grimm-Biographie von W. Scherer beigetragen. Die Verfremdung des Bildes der Gebrüder Grimm wurde in der Nach- kriegszeit im Prozeß der "Vergangenheitsbewältigung" fortgesetzt. Es war eine unhistorische Betrachtungsweise, die den nationalen Ge- danken von Grimm aus der Zeit der Zerstückelung Deutschlands in viele kleine Fürstentümer mit dem expansiven Nationalismus des 20. 2 Jahrhunderts in Verbindung brachte . Aus dieser Haltung heraus er- klärt sich auch die Stagnation der Grimm-Forschung bis in die 60-er 3 Jahre hinein . Seit dem Gedenkjahr 1963 zeichnet sich eine Wende ab. Es is t ei- ne Reihe von Sammelbänden mit Aufsätzen erschienen, in denen ver- 3 Vgl. darüber H. Kolb, Karl Marx und Jacob Grimm. In: Archiv für das Studium der neueren Sprachen und Literaturen, Bd. 2 0 6 , 1969-70, S. 96-114. 3 Vgl. darüber H. Kolb, Karl Marx und Jacob Grimm. In: Archiv für das Studium der neueren Sprachen und Literaturen, Bd. 2 0 6 , 1969-70, S. 96-114. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access schiedene Probleme im Zusammenhang mit Grimms Werk aus heutiger Sicht behandelt werden. Unter den neueren Veröffentlichungen leistet die Arbeit von L. Denecke, die bio-bibliographische Ziele übertrifft auch der künftigen Forschung eine wertvolle Hilfe. Es is t als ein Versäumnis der Grimm-Forschung anzusehen, daß es bisher keine k ri ־ tische Grimm-Ausgabe gibt, die das gesaunte handschriftliche Material aus den Handexemplaren berücksichtigt und den vollständigen Brief- Wechsel enthält. Daraus erklärt sich z.T. auch das Fehlen einer um- fassenden wissenschaftlichen Biographie. schiedene Probleme im Zusammenhang mit Grimms Werk aus heutiger Sicht behandelt werden. Unter den neueren Veröffentlichungen leistet die Arbeit von L. Denecke, die bio-bibliographische Ziele übertrifft auch der künftigen Forschung eine wertvolle Hilfe. Es is t als ein Versäumnis der Grimm-Forschung anzusehen, daß es bisher keine k ri ־ tische Grimm-Ausgabe gibt, die das gesaunte handschriftliche Material aus den Handexemplaren berücksichtigt und den vollständigen Brief- Wechsel enthält. Daraus erklärt sich z.T. auch das Fehlen einer um- fassenden wissenschaftlichen Biographie. Die Literatur über Vuk KaradŹic is t sehr umfangreich. Abgesehen von den zahlreichen Aufsätzen, die jährlich anläßlich der Vuk-Feier erscheinen, existiert eine Reihe von wissenschaftlichen Darstellunge über ihn. Zu den wertvollen Untersuchungen von A. Belic sind an- läßlich der 100-jährigen Todesfeier mehrere Sammelbände mit Aufsätze erschienen. Es wurde auch eine kritische Ausgabe begonnen, die neber Vuks Schriften auch Untersuchungen einzelner Werke aus heutiger Sicht enthält, wie z.B. 1 Vgl. bes. die Aufsätze von M. Mojasevic. 2 Vuk Stefanovic Karadžic, Kleine serbische Grammatik, übersetzt une mit einer Vorrede von Jacob Grimm (1824). Neu hrsg. und eingel. von M. Mojaševic und P. Rehder, München/Belgrad 1974. EINLEITUNG P. Ivics Arbeit über das Wörterbuch von 1816 An Stelle einer gewissen "Vuk-Romantik" t r i t t allmählich eine nüch- terne, kritische Forschung. Die neuen Forschungsergebnisse werden auch eine neue wissenschaftliche Biographie von Vuk erforderlich machen, die die an Informationswert bisher unübertroffene Darstel- lung von Lj. Stojanovic von 1924 ablösen würde. In der jugoslawischen Vuk-Forschung wurde auch der Vuk-Grimm- Beziehung1 besonderes Interesse gewidmet. 1974 wurde diesem Thema im Rahmen der jährlichen internationalen Slavistenversammlung in Belgrad ein zweitägiges Symposium gewidmet. Zu diesem Anlaß er- schien auch die Faksimileausgabe von Grimms Handexemplar der "Klei- 2 nen serbischen Grammatik" von Vuk Karadzic . 1 Vgl. Denecke, a.a.O. S. 40. 2 Vgl. H. Arens, Sprachwissenschaft, 1955, S. 177. 3 Vgl. B. Benes, Wilhelm von Humboldt, Jacob Grimm, August Schlei- eher. Ein Vergleich ihrer SprachaufFassungen, 1958, S. 56ff. 4 Vgl. L. Hermann, Jacob Grimm und die sprachtheoretischen Konzep- tionen der französischen Aufklärung. In: Wiss. Z.Humboldt-Univ. Berlin, Ges.-Sprachw.R.XIV (1965), S. 447-453. 5 In seiner Auseinandersetzung mit A. von Arnim von 1811/12 hat sich Grimm zu dieser Theorie und dem goldenen Zeitalter bekannt. ״ Vgl. Achim von Arnim und die ihm Nahestehenden, hg. von R. Steig und H. Grimm, Bd. I I I , S. 139ff. 1 Vgl. bes. die Aufsätze von M. Mojasevic. 2 Vuk Stefanovic Karadžic, Kleine serbische Grammatik, übersetzt une mit einer Vorrede von Jacob Grimm (1824). Neu hrsg. und eingel. von M. Mojaševic und P. Rehder, München/Belgrad 1974. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access A. JACOB GRIMMS SPRACHAUFFASSUNG Das umfangreiche Werk von Jacob Grimm enthält eine V ielfa lt von Ideen, die seine Zuordnung zu einer bestimmten Epoche und gei- stigen Strömung erschwert• L. Denecke hat ihn einen jener Einzel- gänger genannt, *1die nur sich selbst gleich sind"1. Die Untersuchun- gen des Werkes von Grimm haben zu entgegengesetzten Urteilen über ihn geführt: seiner Sprachauffassung wurden einerseits romanti- 2 3 scher und magischer Charakter, andererseits Verwandtschaft mit der Aufklärung4 zugeschrieben. Die unterschiedlichen Urteile erklä- ren sich u.a. daraus, daß einzelne Probleme in Grimms Werk is o lie rt und ohne Beachtung ihrer Entwicklung betrachtet wurden. Zu diesen Problemen gehören auch seine Ansichten von dem Entwicklungsgang der Sprache. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access I. Grimms Ansichten über den Ursprung und die Entwicklung der Sprache In der frühen Forschungsphase von Grimm, die mit dem Beginn der Arbeit an der "Deutschen Grammatik" zu Ende war, sind viele Ge- meinsamkeiten mit der Romantik festellbar, vor allem mit dem Mythen- forscher A. Kanne, der eine Vereinigung von Mythologie und Etymolo- gie anstrebte. Für Grimms Sprachauffassung zu dieser Zeit war die Theorie von der göttlichen Offenbarung der Sprache bedeutend. Die- se Theorie beinhaltet u.a. eine Urvollendung der Sprache und be- greift das Wort als den Namen des Dinges, der dem Menschen im golde- nen Zeitalter verständlich war5. Wie Kanne hat auch Grimm spekula- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access tive Etymologien betrieben1.In der Gesamtheit seines Werkes erschei- nen sie als ,1vorwissenschaftliche" Phase: zu seinen Wissenschaft- liehen Ergebnissen und seinem Ruhm is t Grimm durch empirische For- schung gelangt• In der Einleitung zum ersten Teil der"Deutschen Grammatik" von 1819 hat Grimm seine Ansichten über den Entwicklungsgang der Spra- che dargelegt. In seiner Sprachauffassung zeigen sich Veränderungen, auf die wir im folgenden hinweisen möchten. Grimms Ansichten aus dieser Zeit sind auch in bezug auf seine Beurteilung der serbischen Sprache bedeutend. 1 Vgl. u.a. seine Abhandlung "Irmenstrasze und Irmensäule" von 1815 jetzt in; Jacob Grimm, Kleinere Schriften (im weiteren Text GKS), Bd. V III, S. 471-504). - 1819 meinte Grimm: "Kanne hat mich früher angezogen, aber ich bin völlig davon zurückgekommen. An dem Ein- zelnen, was ich genauer gelernt habe, hat sich mir die Nichtig- keit des Vielen, was er zusammenfassen w ill, bewährt..." Vgl. Leitzmann, Bd. I, S. 22. 1. Der Gegensatz zwischen der alten und neuen Sprache Grimms Vorliebe für ältere Sprachzustände is t bekannt; sie wur- 2 de als ein romantischer Zug in seiner Sprachauffassung gedeutet . In der Einleitung zu seiner Grammatik von 1819 nennt Grimm die alte Sprache "reiner, unbewuster, dem himmlischen ursprung noch näher", als die neue, die "unter den menschenhänden arm und ver- wickelt geworden" sei (GKS VIII, S. 47). Er sieht die Entwicklung der Sprache und des menschlichen Geistes als einen Gegensatz: "wie die eine seite steigt, sinkt die andere." Er geht von der "urvoll- endung" der Sprache aus (ebd.,S.46). "die alte spräche is t leiblich, sinnlich, voll unschuld; die neue arbeitet darauf hin, geistiger, abgezogener zu werden... jene hat groszen reichtum an Wörtern und drückt blosze Wendungen mit an- dern wurzeln aus, alle ihre wurzeln halben glieder und gelenke, die der mannigfaltigsten bewegung gehorchen, durch ihre zusammensetzun- 3 gen dringt noch der innere sinn ... diese gibt eine wurzel nach der ändern hin, ihr ausdruck wird schärfer, bewuster, bestimmter und 1 Vgl. u.a. seine Abhandlung "Irmenstrasze und Irmensäule" von 1815 jetzt in; Jacob Grimm, Kleinere Schriften (im weiteren Text GKS), Bd. V III, S. 471-504). - 1819 meinte Grimm: "Kanne hat mich früher angezogen, aber ich bin völlig davon zurückgekommen. An dem Ein- zelnen, was ich genauer gelernt habe, hat sich mir die Nichtig- keit des Vielen, was er zusammenfassen w ill, bewährt..." Vgl. Leitzmann, Bd. I, S. 22. 2 Vgl. Arens, a.a.O. S. 177f. 3 Grimms Anm. dazu: "die eigennamen der leute, Örter, thiere, pflan- zen haben noch bedeutung, die später ausstirbt... selbst die hülfs- Wörter bedeuten etwas..." g , 3 Grimms Anm. dazu: "die eigennamen der leute, Örter, thiere, pflan- zen haben noch bedeutung, die später ausstirbt... selbst die hülfs- Wörter bedeuten etwas..." Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 000Б1004 - 15 - ihre mittel erscheinen von auszen, sie setzt lieber zusammen, um- schreibt... die poesie vergeht und die prosa... wird uns angemes- sener." (ebd.,S. 46). ihre mittel erscheinen von auszen, sie setzt lieber zusammen, um- schreibt... die poesie vergeht und die prosa... wird uns angemes- sener." (ebd.,S. 46). Da Grimm die Sprache immer in bezug auf die Dichtung betrach- tet, erscheinen der Verlust des Poetischen und die allmähliche Pro- saisierung als die wichtigsten Komponenten des von ihm beschriebe- nen Gegensatzes zwischen der alten und der neuen Sprache. 1 In der magischen Sprachauffassung gewinnt das Ding die Existenz durch seinen Namen. Die Sprache erscheint schöpferisch und darin der Poesie gleich. - Vgl. E. Fiesel, Die Sprachphilosophie der deutschen Romantik, 1927, S. 68f., 73f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1. Der Gegensatz zwischen der alten und neuen Sprache Wenn man in Grimms metaphorischer Ausdrucksweise seine Ansicht von dem poeti- sehen Gehalt in der alten Sprache festzustellen versucht, so ergibt sich, daß er der alten Sprache einen "leiblichen" und "sinnlichen", der neuen dagegen einen abstrakten Charakter zuschreibt. Mit dem "Leiblichen" meint Grimm die Lautung, den Reichtum an Wurzeln und Flexionsformen. Sein Begriff von der "Sinnlichkeit" der alten Spra- che is t unscharf. Er spricht zugleich von dem"inneren sinn", der "bedeutung", der "deutlichkeit, d.h. der sinnlichen" (ebd.,S.49) und verbindet mit diesen Eigenschaften den poetischen Gehalt der Sprache. Das is t besonders deutlich an seiner Gegenüberstellung von w Í G o n t e s - s t e i g a und У і е в е п а Ь е ід zu sehen:"des poetischen gehalts dieser namen habe ich bereits gedacht, den meisten stehn noch die äugen offen; die heutigen sind blind oder schielen, z.b• bei Wie- sensteig denkt man sich etwas falsches, nicht dasz der bergort von einem pfad des wilden thieres wisunt geheiszen worden." (ebd.,S.48). Grimm beurteilt hier den poetischen Gehalt der Sprache nicht mit den innersprachlichen Kriterien; sein Kriterium is t das hinter dem Wort stehende Ding. Diese Verbindung des poetischen Gehalts der Spra- che mit der Dingwelt erinnert an die romantische Auffassung des Wor- tes als Name1. Dennoch kann Grimms Verfahren nicht mit der romanti- sehen Sprachauffassung erklärt werden. Er hat nämlich mit der "Deut- sehen Grammatik" die Erforschung der Wörter als Stellvertreter für Sachen begonnen, mit dem Glauben, daß man durch die Sprache in die 00051004 ־ 16 ־ Urgeschichte der Menschheit Vordringen kann1. Mit der "Sinnlichkeit" der Sprache meint Grimm den Reichtum an Wörtern für sinnliche Vor- Stellungen. Seine Beurteilung der älteren Sprachformen als poetisch muß auch im Zusammenhang mit seinem Interesse an den durch Wörter bezeichneten Sachen gesehen werden• Der ”leibliche" und "sinnliche" Charakter der alten Sprache, d.h der Reichtum an Flexionen und Wörtern für sinnliche Vorstellungen stellen für Grimm die Vollendung der Sprache dar. Da die Entwicklung der Sprache über die Reduzierung der Flexionen zu einer immer höhe- ren Abstraktionsstufe verläuft, erscheint sie in seiner Sicht als ein allmähliches Sinken der ursprünglichen Vollendung. Diese Komponente von Grimms Sprachauffassung finden wir auch in seiner Beurteilung der serbischen Sprache• Er nennt sie "rein" und "edel", vermutet in ihr viele "echt slavische wurzeln.., die ihre einfache urbedeutung" bewahrt haben; er nennt u.a. 1 In der Vorrede schrieb er: "die todten stehen gleichsam auf, um die abkunft... unseres volks zu bezeugen•" (GKS VIII, S.41). W. von Humboldts Forderung nach der Verbindung von Sprach- und Geschichtsstudium hat vermutlich einen Einfluß auf Grimm ausge- übt. - Vgl. W. Schoof, Jacob Grimms Deutsche Grammatik in zeitge- nössischer Beurteilung. In: ZfdPh 82, 1963, S. 363-377. 1 Vgl. dazu auch M. Vasmer, Bausteine I, 1939, S. V llff. 2 In der Grimm-Forschung fehlt bisher eine umfassende Untersuchung dieser Arbeit. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1. Der Gegensatz zwischen der alten und neuen Sprache "das einfache wort um (mens), davon den Polen und Böhmen nur ableitungen blieben", und bewundert "die innere Vollkommenheit so vieler serbischen flexic nen, zumal bei verbum", wobei er insbesondere den Aorist hervor- hebt (GKS IV, S. 103f.? 1819). Man muß Grimms Ansichten über die Sprachvollendung kennen, um z.B. seine Beurteilung der serbischen Sprache im Vergleich mit der dänischen richtig zu verstehen: "und die serbische spräche is t un- gleich vollkommener geblieben, als die dänische, wenn man sie mit dem alterthum vergleichen wollte, ja sie übertrifft an grammati- scher fülle im ganzen betrachtet die böhmische und polnische•.." (ebd., S. 105; 1819). Mit diesen Kriterien konnte Grimm nur die äußere Form der Sprache erfassen; vom Standpunkt der Funktionalität aus gesehen, befand sich die serbische Sprache erst im Stadium des Entstehens einer Schriftsprache und der Entwicklung der dazu erfor- derlichen Ausdrucksformen. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 17 ־ Unter den slavischen Sprachen hat Grimm die serbische am besten beherrscht. Er hat auch die čechische, polnische und russische Spra- che zumindest theoretisch gut gekannt1• Das wird durch seine Heran- Ziehung dieser Sprachen sowohl in der Vorrede zu Vuks Grammatik und in den germanistischen Arbeiten als auch durch seine Eintragungen im Handexemplar der serbischen Grammatik bestätigt. 2. Die Entwicklung der Sprache als Vervollkommnung die älteste spräche war melodisch aber weitschweifig und haltlos, die mittlere voll gedrungener poetischer kraft, die neue spräche sucht den abgang an Schönheit durch harmonie des ganzen sicher ein- zubringen, und vermag mit geringeren mitteln dennoch mehr-"(GKS X, S. 282, 284). Grimm hat seine Vorliebe für flektierende Sprache bis zum Ende seines Lebens beibehalten. Die Sprachvollendung und die poetische Kraft der Sprache sind für ihn aber nicht mehr vom Flexionsreich- tum abhängig. Der Wandel in seinen Ansichten is t besonders an der Be urteilung der englischen Sprache deutlich zu sehen1: "keine unter allen neueren sprachen hat gerade durch das aufgeben und zerrütten alter lautgesetze, durch den wegfall beinahe sämtlicher flexionen eine gröszere kraft und stärke empfangen als die englische..." Sie habe eine "gewalt des ausdrucks... wie sie villeicht noch nie einer ändern menschlichen zunge zu geböte stand". Der größte Dichter der Neuzeit, Shakespeare, sei aus ihr emporgewachsen (ebd.,S. 293). Die Entwicklung der Sprache sieht Grimm jetzt im Einklang mit der Entwicklung des menschlichen Geistes als "wachsende vollkommen- heit", "fortschritt und Zuwachs ihrer inneren kraft"; "ihre reichste frucht wird sie erst einmal in der zukunft darreichen." (ebd., S. 278, 290, 293). In der Darstellung des sprachlichen Entwicklungsganges zeigt Grimm eine energetische Sprachauffassung, die derjenigen von W. von Humboldt nahesteht. 2. Die Entwicklung der Sprache als Vervollkommnung 2. Die Entwicklung der Sprache als Vervollkommnung Grimms Ansichten über die Sprachvollendung zeigen in den 50er Jahren eine wesentliche Veränderung. Unter den späten Arbeiten is t zum Verständnis seiner Sprachauffassung die Akademie-Abhandlung 2 "Uber den ursprung der spräche" von 1852 von besonderer Bedeutung • Eines der Ziele, die Grimm mit seiner Schrift verfolgt, is t der Be- weis des menschlichen Ursprungs der Sprache• Bereits daran zeigt sich eine Veränderung in seiner Sprachauffassung, da er in der frü- hen Phase vom göttlichen Ursprung der Sprache ausging. Vor Grimm hatte Herder den Ursprung der Sprache mit der mensch- liehen Fähigkeit zu denken erklärt. Auch Grimm sieht Sprache und Denken als eine untrennbare Einheit. Es is t aber ein wichtiger Un- terschied zu Herder feststellbar, indem Grimm die kommunikative Funk- tion der Sprache zur Erklärung ihres Ursprungs heranzieht: "Denken und reden sind bedingungen des menschengeschlechts, Ursache seiner freiheit, quelle aller sprachen, der mensch redet weil er denkt, und denkt auch wenn er schweigt- das geredete drang aus seiner seele, er würde stumm geblieben sein, hätte er sich nicht an einen ändern richten können, dem er es anheim gäbe, theilnehmender antwort ge- wärtig." (GKS I I I , S. 236). Grimm unternimmt auch den Versuch, den Entwicklungsgang der Spra che vom ersten Stadium der Wortfindung an über die Ausbildung der Flexionen bis zu deren Verfall nachzuzeichnen. Er schildert die Ent- Wicklung der Sprache in drei Phasen: die "des schaffens, gleichsam Wachsens und sich aufstellens der wurzeln und Wörter, die andere des emporblühens einer vollendeten flexion, die dritte aber des triebs zum gedanken, wobei die flexion als noch nicht befriedigend wieder fahren gelassen... wird, es sind laub, blüte und reifende frucht... 1 Vgl. dazu auch M. Vasmer, Bausteine I, 1939, S. V llff. 2 In der Grimm-Forschung fehlt bisher eine umfassende Untersuchung dieser Arbeit. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. dazu auch M. Vasmer, Bausteine I, 1939, S. V llff. 2 In der Grimm-Forschung fehlt bisher eine umfassende Untersuchung dieser Arbeit. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access die älteste spräche war melodisch aber weitschweifig und haltlos, die mittlere voll gedrungener poetischer kraft, die neue spräche sucht den abgang an Schönheit durch harmonie des ganzen sicher ein- zubringen, und vermag mit geringeren mitteln dennoch mehr-"(GKS X, S. 282, 284). 1 1819 nannte er die englische Sprache als das Beispiel des fortge- schrittenen Formenzerfalls (GKS VIII, 51). 3. Sprache als Form Grimm gebrauchtden Begriff der Form nur in bezug auf die äußere Form der Sprache. Es is t aber aus seinen Ausführungen deutlich, daß er die Sprache nicht nur als Ergon betrachtet. Er nennt sie "eine fortschreitende arbeit", "werk und that der menschen" und spricht von "der alle gedanken deutlich fassenden, bestimmt greifenden, ge- gliederten spräche", "ohne spräche... würde die beste kraft der menschheit sich verzehrt haben und ermattet sein (GKS I, S.276, 294, 296, 278). Es is t bereits hier deutlich zu sehen, daß Grimm, 1 1819 nannte er die englische Sprache als das Beispiel des fortge- schrittenen Formenzerfalls (GKS VIII, 51). V B ji 9783954793044 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ähnlich wie Humboldt und die spätere Sprachwissenschaft1, die Spra- che als eine formende, geistige Kraft ansieht; die formlose Sphäre der menschlichen geistigen Tätigkeit wird durch die Sprache zu Be- griffen und Gedanken, indem sie im Lautlichen eine Form erhält. Grimms Auffassung der Sprache als Form kommt in seiner Darstel- lung der Entwicklung und des Zerfalls der Flexion besonders deut- lieh zum Ausdruck. In der ersten Phase entwickelt die Sprache zwar einen Reichtum an Wörtern zu jedem Begriff, sie drückt aber die Ge- danken durch eine bloße Reihung von Wörtern aus, d.h. sie besitzt keine Grammatik,"ihre gedanken haben nichts bleibendes, stätiges..." (GKS I, S. 290). In der zweiten Phase vollzieht sich eine Umgrup- pierung des Wörtervorrats, wobei "auf die nebenbegriffe schwächeres gewicht" f ä llt "und sie der hauptvorstellung als mitbestimmende theile sich anfügen, die flexion entspringt aus dem einwuchs lenken- der und bewegender bestimmwörter, die... aus ihrer ursprünglich auch sinnlichen bedeutung in eine abgezogne übergegangen sind... zuletzt hat sich auch die flexion abgenutzt und zum bloszen ungefühl- ten Zeichen verengt..." (ebd., S. 2 83). Durch die Ausbildung der grammatischen Formen läßt die Sprache "das wesentliche aus dem zu- fälligen, das waltende aus dem untergeordneten vortreten.. . " (ebd., S. 291). Es is t die Wirkung der Sprache auf das Denken: sie unter- stützt die Entwicklung und Festigung des Denkens. 1 Vgl. W.von Humboldt, Gesammelte Schriften (Akademie-Ausg.), Bd. 7,2, S. 45ff. - Vgl. u.a. F.de Saussure, Grundfragen der allge- meinen Sprachwissenschaft, 2.Ausg. 1967, S. 132ff. 3. Sprache als Form Grimm erklärt den Zerfall der sprachlichen Formen durch die gei- stige Entwicklung des Menschen: "Da nun aber die ganze natur des menschen, folglich auch die spräche dennoch in ewigem, unaufhalt- barem aufschwung begriffen sind, konnte das gesetz dieser zweiten periode der Sprachentwicklung nicht für immer genügen, sondern muste dem streben nach einer noch gröszeren ungebundenheit des ge- dankens weichen, welchem sogar durch die anmut und macht einer voll- endeten form fessel angelegt schien." (ebd.,S. 291f.) Da der ent- wickelte Geist die Flexionsformen als Gedankenstütze nicht mehr be- nötigte, gab die Sprache sie auf, um das Denken von den überflüssi- gen Fesseln zu befreien. Ähnlich hat W. von Humboldt den Grund für den Verfall der Flexion "in der steigenden Zuversicht" des "gereiften Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 20 - menschlichen Geistes gesehen1• g , , , , 2 Vgl. J. Grimm, Geschichte der deutschen Sprache, 4.Aufl. 1880, S. 4 1 Vgl. W.v.Humboldt, a.a.O., Bd. 7,2, S• 239f. 2 Vgl. J. Grimm, Geschichte der deutschen Sprache, 4.Aufl. 1880, S. 4 1 Vgl. W.v.Humboldt, a.a.O., Bd. 7,2, S• 239f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 4• Sprache als Geschichte In Grimms Darstellung der Sprachentwicklung is t auch seine spe- zifische Auffassung des Zusammenhangs zwischen der Sprache und dem Denken, der Sprache und dem Volk sichtbar* ,*Diese spräche, dies den- ken steht aber nicht abgesondert da für einzelne menschen, sondern alle sprachen sind eine in die geschichte gegangene gemeinschaft und knüpfen die weit aneinander• ihre mannigfaltigkeit eben is t be- stimmt, den ideengang zu vervielfachen und zu beleben, von dem sich ewig erneuernden, wechselnden menschengeschlecht wird der köstliche allen dargebotne erwerb auf die nachkommen übertragen und vererbt, ein gut das die nachweit zu erhalten, zu verwalten und zu mehren an- gewiesen is t• ” (GKS I, S. 277)• Grimm betont den gesellschaftlichen Charakter der Sprache und bringt ihre Geschichte in den engsten Zusammenhang mit der Geschieh- te ihres Trägers. Die Sprache wird "sowol durch blühende kraft der Völker gefördert als durch deren barbarei niedergehalten..." (ebd., S. 294). Die Sprach- und Volksbildung sieht Grimm als abhängig von einander an. Aus der Entwicklung der Sprache, den in ihr stattge- fundenen Veränderungen, lasse sich die Geschichte des Volkes er- schließen; aus dem Vergleich der Sprachen untereinander können Rückschlüsse auf die Urgeschichte der Menschheit gezogen werden (ebd S• 280f.) Diese Auffassungen waren ausschlaggebend für Grimms Be- mühung, durch die Wörter zu den Sachen vorzudringen, und für seine Forderung an die damalige historische Sprachforschung, nicht nur die Lautgestalt, sondern auch "ursprung, fo rtsch ritt und Übergang der begriffe" zu berücksichtigen (ebd., S. 123f•)• "Es gibt ein le- bendigeres zeugnis über die Völker als knochen, waffen und gräber, 2 und das sind ihre Sprachen•" Grimm erhob die Sprachforschung zur Geschichtsforschung, denn: "Unsere spräche is t auch unsere geschichte." (GKS I, S. 290). 1 Vgl. W.v.Humboldt, a.a.O., Bd. 7,2, S• 239f. 2 Vgl. J. Grimm, Geschichte der deutschen Sprache, 4.Aufl. 1880, S. 4 g S. 4 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Grimms Verbindung zur naturwissenschaftlichen Schule wurde bisher nicht eingehend untersucht. Vgl. Denecke, a.a.O., S. 98. 2 Dieser Gedanke von Grimm wurde als "seine irrationale Tendenz" ge- deutet. - Vgl. Arens, a.a.O., S. 178f. 3 Vgl. auch E. Arndt, Diachronie und Synchronie in Jacob Grimms "Deutscher Grammatik". Ausnahme und Regel in neuer Sicht. In: Wiss.Z. Humboldt-Univ. Berlin, Ges.-Sprachwiss. R. XIV, 1965, S. 481. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access II. Die nationale Schriftsprache II. Die nationale Schriftsprache Grimm hat die Sprache eine "errungenschaft der menschen" genannt "wodurch sie zugleich getrennt und geeint werden1( ״GKS I, S. 277). Die Sprache als Phänomen existiert nur in Form von Einzelsprachen. Nach Grimms Auffassung sind in der Bildung von Einzelsprachen zwei Faktoren wirksam: "... raum und zeit sind anlasz aller Veränderungen der menschensprache, aus ihnen allein läszt sich die mannigfaltigkei* und abweichung der einem quell entstammenden Völker begreifen." (ebd S. 265). Außer den in Raum und Zeit zentrifugal wirkenden Kräften sieht Grimm auch eine einigende Kraft in der geistigen Entwicklung der Menschen: unter den "im weiten raum zerarbeiteten" Sprachen wer- den "endlich nur solche des feides meister.., die nährende geistes- frucht gebracht und geboren haben" (ebd., S. 2 9 4 ) Eine solche Eini- gung vollzieht sich nach seiner Auffassung im Prozeß der Bildung von Schriftsprachen. 1 Er entwickelt als Gegenstück zur "babylonischen Sprachverwirrung" eine Zukunftsvision von einer Weltsprache, die "manches edle in sich aufnehmen" wird, "was jetzt in den sprachen verwilderter Stämme wie zertrümmert liegt" (ebd.) - Diese Utopie läßt die Vor- würfe eines engen nationalen Denkens bei ihm als absurd erscheinen, Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 5• Der sprachliche Organismus In Grimms Abhandlung über den Ursprung der Sprache is t eine ge- wisse Nähe zur naturwissenschaftlichen Schule feststellbar: in seiner Darstellung des Unterschieds zwischen den tierischen und menschlichen Lauten weist Grimm zur Begründung auf den Bau der menschlichen A rti- kulationsorgane hin1 (GKS I, S. 266f.). Er verwendet auch den da- mals allgemein verbreiteten Terminus "Organismus" der Sprache (ebd., S. 258, 282). Aber gerade in seinen Ansichten vom sprachlichen Orga- nismus is t der Unterschied zwischen seiner und der naturwissenschaft- liehen Sprachauffassung deutlich erkennbar. Bereits in der Vorrede zur "Deutschen Grammatik" hat Grimm her- vorgehoben, "dasz es kaum eine regel gibt, die sich steif überall durchführen läszt... es is t ein groszes gesetz der natur, das auch in der spräche anomalien und mängel neben den uns erkennbaren regeln bestehen lassen w ill..." (GKS VIII, S. 35). Er sprach sogar vom 2 "organismus der anomalien" (ebd., S. 44) . Diese Ansichten v e rtritt Grimm auch in den 50er Jahren: "alle grammatischen ausnahmen scheinen mir nachzügler alter regeln, die noch hier und da zucken, oder Vorboten neuer regeln, die über kurz oder lang einbrechen werden." (GKS I, S. 329). Die Sprache ist"nicht starr und ewig wirkendem naturgesetz... anheim gefallen.., sondern menschlicher freiheit in die warme hand gegeben... ihre gleichförmig- keit wäre undenkbar, da dem neu hinzutretenden und nachwachsenden ein Spielraum offen stehen muste, dessen nur das ruhig fortbestehen- de nicht bedarf." (ebd., S. 294). Der Dynamismus in GrimmsSprachauffassung steht der Ansicht von der Ausnahmslosigkeit der Gesetze entgegen. Nach seiner Auffassung is t der sprachliche Organismus keine von einem Gesetz bestimmte Ein- heit, sondern er enthält auf jeder Stufe Reste des Gewesenen und 3 Keime des Werdenden . Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 22 - 1. Die Entstehung der Schriftsprache Grimm hat seine Ansichten über die Schriftsprache am Beispiel der deutschen Sprachgeschichte und der vor seinen Augen entstehenden serbischen Schriftsprache entwickelt. In der Vorrede zur "Deutschen Grammatik" versucht er, die Ent- Wicklung der deutschen Schriftsprache rückblickend nachzuzeichnen: "In der frühen zeit gelten viele dialecte gleichansehnlich neben- einander, ihre grenzen laufen mit denen der einzelnen Stämme; so- bald herrschaft und bildung einem volke vorgewicht geben, fängt sei- ne mundart an sich über benachbarte, abhängige auszubreiten, d.h. von deren edlem theile angenommen zu werden, während die einheimi ־ sehe mundart unter den volkshaufen flüchtet. Die stärkere mundart steigt, die schwächere sinkt und wird gemein, doch selbst die herr- sehende muß durch ihre wachsende ausdehnung unvermerkt eigenheiten der ändern Stämme an sich ziehen, folglich dem ungebildeten theile 1 Er entwickelt als Gegenstück zur "babylonischen Sprachverwirrung" eine Zukunftsvision von einer Weltsprache, die "manches edle in sich aufnehmen" wird, "was jetzt in den sprachen verwilderter Stämme wie zertrümmert liegt" (ebd.) - Diese Utopie läßt die Vor- würfe eines engen nationalen Denkens bei ihm als absurd erscheinen, Vera Boji - 9783954793044 00051004 - 23 ־ des stainmes, von dem sie ausgieng, gleichfalls entrückt werden... Das resultat wird daher dieses seyn: ein dialect ist so alt und eben־ bürtig, als der andere, ehmahls aber sprach der gemeine mann wie der edle, heute ist die aus Verschmelzung der Völkerschaften errungene spräche eigenthum des gebildeten theils, also jedem erwerbbar; der ungebildete theil bleibt bei der angestammten mundart und pflanzt sie fort, sie hat lebenswärme, bildungswärme geht ihr ab." des stainmes, von dem sie ausgieng, gleichfalls entrückt werden... Das resultat wird daher dieses seyn: ein dialect ist so alt und eben ־ bürtig, als der andere, ehmahls aber sprach der gemeine mann wie der edle, heute ist die aus Verschmelzung der Völkerschaften errungene spräche eigenthum des gebildeten theils, also jedem erwerbbar; der ungebildete theil bleibt bei der angestammten mundart und pflanzt sie fort, sie hat lebenswärme, bildungswärme geht ihr ab." Nach Grimms Auffassung verläuft die Bildung der Schriftsprache über die Reduzierung der Dialekte, wobei sich der "stärkste" zu ei- ner Art synthetischen Dialekt entwickelt.Die Übermacht eines Dia- lekts beruht auf der kulturellen und wohl politisch-ökonomischen Stär- ke ("herrschaft") seines Trägers. Im Prozeß der Entwicklung der Schriftsprache spielen die kulturellen Faktoren die entscheidende Rol־ le: die Vereinheitlichung der mundartlichen Züge in dem synthetischen Dialekt vollzieht sich nach Grimms Meinung in der Literatur. Er sieht bereits in der mhd. 1 Vgl. J. Grimm, Deutsche Grammatik 1.Т., 2.Aufl. 1822, S. X llf. 2 Vgl. ebd., S. XIII. 3 W. Bondzio, Jacob Grimm und die neuhochdeutsche Schriftsprache. In: Wiss. Z. Humboldt-Univ. Berlin, Ges.-Sprachwiss. R. XIV, 1965, S. 474. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1. Die Entstehung der Schriftsprache Dichtung "eine allgemeine spräche, deren sich al- le dichter bedienen", in der die Mundarten "verschwommen und aufge- löst" sind. Im 16. Jh. ist schon "die neuhochdeutsche Schriftsprache durch das gesammte reich herrschend, alle abzeichen früherer stammes- Verschiedenheit [sind] gewichen, freiheiten, die sich noch mittel- 2 hochd. dichter genommen, unedel und unerlaubt." Es is t sicher richtig, daß in der Zeit des Fehlens einer Sprach- norm die Literatur eine vereinheitlichende Funktion ausgeübt hat. Die Dichter haben bewußt die Provinzialismen vermieden, um von einem breiteren Publikum verstanden zu werden. Die Dichtung allein schafft aber nicht die einheitliche schriftsprachliche Norm. a. Schriftsprache und Mundarten 1 Hervorhebung von V.B. 2 Vgl. "Deutsche Grammatik" 1. T., 2. Aufl., S. X lllff. a. Schriftsprache und Mundarten a. Schriftsprache und Mundarten Grimms Ausführungen über die Mundarten sind u.E. auch in der neuesten Zeit mißverstanden worden, wenn man meint, daß er die Mund- arten "unhistorisch als bloßen Gegensatz zur Schriftsprache auffaß- 3 te" und daß er sie als "verderbte Sprache" ansah . Grimms Betrachtung der Mundarten bezieht sich hauptsächlich: 1. auf die Zeit vor der Entstehung der Schriftsprache und 2. auf das Verhältnis zwischen Schriftsprache und Mundarten, Er betont, daß vor der Entwicklung der Schriftsprache alle Mundarten als gleichwertig und "edel” galten. Nachdem sich eine Mundart als Schriftsprache durchgesetzt hat, is t sie als einzige im kulturel- len Leben erlaubt. Die Schriftsprache wird zum Zeichen der Bildung, während die Mundarten vom ungebildeten Volk gesprochen werden. Grimms Formulierungen über das "Sinken" der übrigen Mundarten im Vergleich mit der einen, zur Schriftsprache "gestiegenen" Mund- art meinen eigentlich die soziale Schichtung der Sprache. Grimm hat diese Ansichten auch in bezug auf die damalige sprach liehe Situation bei den Serben vorgetragen, wo es noch keine a ll- gemein anerkannte Schriftsprache gab: " es gibt in den serbischen ländern keine gemeine, pöbelhafte volksmundart..♦ die abwesenheit des rohen, gemeinen elements in der serbischen spräche darf uns aber nicht wundern, vor tausend jahren und später verhielt es sich in Deutschland eben so... so schien damals... jedem seine landes- mundart edel, aber h e r r e n u n d k n e c h t e 1 pflagen ihrer ohne unter- schied, allmählich wurde... die ausbildung auf wenige mundarten eingeschränkt, bis zuletzt nur eine Schriftsprache allein die höhe h ie lt, volksdialecte das gleichgewicht verloren und in gemeinheit und trübe versanken.*1 (GKS IV, S. 199f.). Grimm sieht die Schriftsprache im Verhältnis zu den Mundarten als eine höhere Stufe der Sprachentwicklung an: die Schriftsprache als Trägerin des geistigen Lebens wird weiterentwickelt, sie hat ״bildungswärme"; die Mundarten dagegen bleiben "ohne belebende l i - teratur", ihre Entwicklung is t langsamer. Grimm betonte die Bedeu- 2 tung der Mundarten für die Sprachforschung . b. Das Verhältnis von Schriftsprache, Volkssprache und Dichterspräche 1 Vgl. Bondzio, a.a.O., S. 475f. 2 Das hat Grimm in der Vorrede zum "Deutschen Wörterbuch" erläu- tert (GKS V III, S• 321). - Vgl. auch Bondzio, a.a.O., S. 476. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. den Brief an K. Lachmann vom 24.8.1838; Leitzmann Bd. II, S. 690. 2 Vgl. "Deutsches Wörterbuch" Sp. 358: a n g r ü n e m wie gehts leben? "es grünt wieder an" antwortete ein dienstmädchen. 3 Vgl. darüber Bondzio, a.a.O. S. 476f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access b. Das Verhältnis von Schriftsprache, Volkssprache und Dichterspräche Grimm sieht die Schriftsprache als eine einheitliche Norm, die gegenüber den Mundarten abgegrenzt und durch Schulbildung jedem er­ 2 Vgl. "Deutsche Grammatik" 1. T., 2. Aufl., S. X lllff. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 25 - werbbar is t. Durch eine Ausdehnung der Bildung auf das ganze Volk wäre sie also die über den Mundarten stehende "Volkssprache1״• Grimm geht von der Existenz einer solchen "Normalspräche" bei den Deut- sehen aus, die etwa mit dem heutigen Begriff der Umgangssprache faßbar wäre1. Nach seiner Meinung sollte es zwischen der Schrift- spräche und ihrer gesprochenen Variante nicht den Unterschied geben, über den er klagt: "wir sind dann genöthigt doppelter spräche zu pflegen, einer für das buch, einer ändern im leben, und können die gröszere wärme des lebens nicht unmittelbar dem ausdruck des buchs lassen angedeihen." (GKS I, S. 332). Diese Ansicht entspricht sei- ner Auffassung der Norm, die einen Spielraum neben den Regeln for- dert. Nach Grimms Auffassung is t für die Entwicklung der Schriftspra- che ihre lebendige Pflege in der Sprachgemeinschaft wichtig. Er be- ruft sich auf Goethes Worte in "Kunst und Alterthum" 3, 51: "poesie und leidenschaftliche rede sind die einzigen quellen, aus denen dieses leben hervordringt, und sollte sie in ihrer heftigkeit auch etwas bergschutt mitführen, er setzt sich zu boden und die reine welle flieszt darüber her." (GKS V III, S. 36, Anm.). Grimm schreibt den Dichtern sowohl im Prozeß der Entstehung als auch der Fortbildung der Schriftsprache eine außerordentlich wichti- ge Rolle zu. Wenn die Sprache stagniert oder Verluste erleidet, dann sind es die Dichter, die "unsichtbares walten... des unermüd- lieh schaffenden sprachgeistes•.. vernehmen... in der begeisterung und bewegung durch ihr gefühl" (ebd., S. 36). Er betont oft die Rolle von Luther, der die deutsche Sprache verjüngt habe, und von Goethe und Schiller, die sie erneut zur Höhe gebracht haben (GKS I, S. 375ff.). Nur die Dichter haben das Recht, die Grenze zwischen der Schriftsprache und den Mundarten zu überschreiten, allerdings nur auf dem lexikalischen Gebiet und unter der Wahrung der einheit- 2 liehen phonetisch-morphologischen Norm . Es is t sicher eine unrealistische Vorstellung, daß die Dichtung in der Lage sei, die Fehlentwicklungen in der Sprache zu korrigierer wie Grimm hinsichtlich der "bezeichnung der person in der anrede" (Ihr, Sie an Stelle von Du) meint (GKS I, S. 333). 1 Seine Nachfolger haben dieses Wort in der der Schriftsprache ange- paßten Form (n e h m a re c h ) in das "Deutsche Wörterbuch" aufgenommen. 2 Er bat Vuk uni Auskunft über etwaige serb. Entsprechung zum dt. k a l t e r b a u e r (prepiska II, S. 64f.). b. Das Verhältnis von Schriftsprache, Volkssprache und Dichterspräche Grimms Betonung der schöpferischen Rolle der Dichter in der Sprachentwicklung is t offenbar von der romantischen Dichter- und Poesieauffassung beein- fluBt. (Ihr, Sie an Stelle von Du) meint (GKS I, S. 333). Grimms Betonung der schöpferischen Rolle der Dichter in der Sprachentwicklung is t offenbar von der romantischen Dichter- und Poesieauffassung beein- fluBt. Es is t eine wichtige Komponente in Grimms Sprachauffassung, daß er Sprache und Poesie als Einheit betrachtet. Die Sprache is t das Medium der Dichtung; die Dichtung festigt und bewahrt die Spra- che. "bei Völkern, die keine oder eine dürftige literatur erzeugten musten sprachformen, Wörter und ausdrucksweisen... in Vergessenheit sinken..." (ebd., VIII, S. 316). Die Kraft der Sprache zeigt sich in der Poesie; die Poesie wiederum is t der mächtigste "hebel zur erhöhung des menschengeschlechts" (ebd., I, S. 375). Grimm sieht die dichterische Sprache als die höchste Stufe der Sprachverwirklichung an; sie wirkt auf die Sprachbildung im allge- meinen, die Sprach- und Volksbildung sind eng miteinander verbunden Diesen Zusammenhang hat er auch in bezug auf Vuk Karadzics Bemühun- gen um die serbische Schriftsprache hervorgehoben (s.u. S. 153). In Grimms Auffassung der Schriftsprache sind Volk und Dichter Träger ihrer Entwicklung. Diese Auffassung kommt auch im "Deutschen Wörterbuch" zum Ausdruck. Die Konzeption seines Werkes hat er fol- gendermaßen formuliert: "Das werk soll in sich begreifen alles was die hochdeutsche spräche vermag nach der ausprägung, die ihr in drei jahrhunderten durch dichter und tüchtige schriftsteller widerfahren is t." 1 Neben den Belegen aus der Literatur zieht Grimm aber auch die lebendige, gesprochene Volkssprache heran, für die er Zitate aus Berliner Zeitungen und aus dem Munde von Dienstmäd- 2 chen anführt . Sein Verfahren wurde von seinen Zeitgenossen als "in einem Wörterbuch noch nicht dagewesen" kritisiert"*. Obwohl die Einbeziehung der gesprochenen Sprache im Einklang mit Grimms Ansichten über die Schriftsprache steht, haben u.E. seine Erfahrungen aus der Zusammenarbeit mit Vuk Karadziè in der Konzeption des "Deutschen Wörterbuchs" ihren Niederschlag gefunden. Das b e trifft in erster Linie die Einführung der Sprache des ein- 476f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access fachen Volkes und der sog. "anstöszigen Wörter". fachen Volkes und der sog. "anstöszigen Wörter". Das Wörterbuch von Vuk Karadzic aus dem Jahre 1818 is t auf der Sprache des einfachen Volkes begründet. Vuk führt in der Wortdeu- tung, neben den von ihm gesammelten Volksliedern, nur die lebendi- ge Volkssprache als Quelle an. Es entsprach der Konzeption seines Wörterbuchs, bei den einzelnen Belegen auch ihre Herkunft und den Zusammenhang mit verschiedenen Volkssitten zu erläutern. Grimm hat ein besonderes Interesse für dieses ethnographische Material in Vuks Wörterbuch gezeigt (GKS IV, S. 103). Obwohl Grimms Wörterbuch als ein historisches Wörterbuch der neuhochdeutschen Schriftsprache sich von Vuks Werk im wesentlichen unterscheidet, hat man stellenweise den Eindruck, als lese man Vuks Wörterbuch, z.B. Sp. 296 bei Vuk unter K a n o v c i: "Po nekim se ־ lima и Bijogradskoj naiji imaju ljudi običaj te govore gotovo uza svaku rije č kanom , kanom b a t o . Na Vračaru 1804 godine prozvali su i (Bijograflani i drugi becari kojekakvi) zato к а п о ѵ с г т а i podsmije- vali su im se koješta, i toliko puta malo se nijesu iz pušaka tu kli zato." Und Sp. 564f. bei Grimm: "... in der Wetterau nennt man g e b a r a c h und nommarach einen der gern gibt und gern geschenk- tes nimmt." Grimm wendet das Verfahren von Vuk an und indem er, der Volkssprache folgend, das mundartliche nomm- an Stelle von nehm - aufnimmt. Überschreitet er die Grenze der einheitlichen schriftsprachlichen Norm1. Vuk hat in seinem Wörterbuch auch die sog. obszönen Wörter verzeichnet, was seinen Gegnern Anlaß zur Kritik geboten hat. In der zweiten Auflage von 1852 hat er diese Wörter aus taktischen Gründen ausgelassen. Auch Grimm hat diese Sprachschicht in sein Wörterbuch aufgenommen und während der Arbeit auch Vuks Werk zu Rate gezogen. Am 22. Dez. 1852 schrieb er an Vuk: "Beim gebrauch Ihrer zweiten ausgabe habe ich ausser trefflichem Zuwachs doch auch einigen abgang wahrgenommen, namentlich sind die obscoenen Wörter weggeblieben... an sich aber sind alle Wörter gut und un- 2 schuldig, reinen ohren alles rein." Mit ähnlichen Gedanken hat 1 Seine Nachfolger haben dieses Wort in der der Schriftsprache ange- paßten Form (n e h m a re c h ) in das "Deutsche Wörterbuch" aufgenommen. 2 Er bat Vuk uni Auskunft über etwaige serb. Entsprechung zum dt. k a l t e r b a u e r (prepiska II, S. 64f.). 1 Vgl. "Deutsche Grammatik" 1. T., 2.Aufl., S. XIII Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access fachen Volkes und der sog. "anstöszigen Wörter". Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 28 ־ Grimm sein Verfahren in der Vorrede zum "Deutschen Wörterbuch" ge- rechtfertigt (GKS V III, S. 337f.). Dieser kleine Exkurs zeigt, daß bei der Betrachtung der Bezie- hung zwischen Grimm und Vuk die Anregungen in beiden Richtungen zu suchen sind. Auch das slavische Sprachmaterial in Grirams Wörter- buch is t ein Resultat seiner über Jahrzehnte hinaus andauernden Be- schäftigung mit slavistischen Problemen, wozu er durch Vuk immer wieder angeregt wurde. 1 Vgl. J. Grimm, Deutsche Grammatik 1. T., 2. Aufl., S. X III. 2 B. BeneŠ, a.a.O., S. 62f., hat bemerkt, daß Weisgerber das Gedan- kengut von J. Grimm weitergeführt hat. 3 Vgl. J. G. Herder, Sämtliche Werke, hg. B. Suphan, 1877, Bd. II, S. 24ff. 2. Die Schriftsprache als ein konstitutiver Faktor der Nation 2. Die Schriftsprache als ein konstitutiver Faktor der Nation Der von Herder hervorgehobene Zusammenhang zwischen der Sprache und dem Volk, das sie spricht, erscheint in Grimms Sprachauffassung in verschiedenen Formen. Nach Grimms Meinung übt die Sprache in Bezug auf das Volk eine einigende Funktion aus: "die kraft der spräche bildet Völker und hält sie zusammen, ohne solches band würden sie sich versprengen, der gedankenreichtum bei jedem volk is t es hauptsächlich was seine Weltherrschaft festigt." (GKS I, S. 276). Dieses Band is t die Schriftsprache. Er sah die Zersplitterung in viele Mundarten als eine "Versprengung" der geistigen Kräfte eines Volkes an: "Mich dünkt, die entwicklung eines volks fordert auch für die spräche,., wenn sie nicht verkümmern soll, erweiterte äuszere grenzen."1 Er betrachtete die Bildung der Schriftsprache, die eine größere Sprach* gemeinschaft umfaßt, als einen Vorteil für die Sprachentwicklung: "seit Luther is t die herrschaft des hochdeutschen dialects unab- änderlich festgestellt und w illig entsagen alle theile Deutsch- lands einzelnen vortheilen, die jede vertrauliche mundart mitführt, wenn dadurch kraft und stärke der aus ihnen allen aufsteigenden gemeinschaftlichen Schriftsprache gehoben wird, jeder verlust is t für ein glück zu achten, der höhere gewinne zu wege bringt." (GKS VII, S. 558). Andererseits wird nach Grimms Auffassung die so entstandene Volksgemeinschaft durch die Schriftsprache zusammengehalten: "erst kraft der Schriftsprache fühlen wir Deutsche lebendig das band un- serer herkunft und gemeinschaft und solchen vortheil kann kein stamm glauben zu theuer gekauft zu haben oder um irgend einen preis hergeben wollen.”1 Grimm sah die über den Mundarten stehende und sie einigende Schriftsprache als diejenige Kraft an, die eine Volks- gemeinschaft zur Nation bildet. Diesen Gedanken finden wir auch in seiner Vorrede zu Vuks serbischer Grammatik, wo er sich für ei- nen einheitlichen Volksnamen für die e i n e Sprache sprechenden "Stäm ־ me" einsetzt (s. u. S.150). Er meinte: "ein volk is t Inbegriff von menschen, welche dieselbe spräche reden.” (GKS V III, S. 557). Aus einer solchen Auffassung der Sprache ergibt sich auch Grimms Mei- nung, durch Bemühung um die einheitliche Schriftsprache zur natio- nalen Einigung beitragen zu können. Seine Arbeit am "Deutschen Wörterbuch" is t auch von dieser Hoffnung getragen. stamm glauben zu theuer gekauft zu haben oder um irgend einen preis hergeben wollen.”1 Grimm sah die über den Mundarten stehende und sie einigende Schriftsprache als diejenige Kraft an, die eine Volks- gemeinschaft zur Nation bildet. 2. Die Schriftsprache als ein konstitutiver Faktor der Nation Diesen Gedanken finden wir auch in seiner Vorrede zu Vuks serbischer Grammatik, wo er sich für ei- nen einheitlichen Volksnamen für die e i n e Sprache sprechenden "Stäm ־ me" einsetzt (s. u. S.150). Er meinte: "ein volk is t Inbegriff von menschen, welche dieselbe spräche reden.” (GKS V III, S. 557). Aus einer solchen Auffassung der Sprache ergibt sich auch Grimms Mei- nung, durch Bemühung um die einheitliche Schriftsprache zur natio- nalen Einigung beitragen zu können. Seine Arbeit am "Deutschen Wörterbuch" is t auch von dieser Hoffnung getragen. Grimm hat der Sprache die wichtigste Stelle unter den Phänome- nen des menschlichen Geistes zugewiesen. Diese Uberbetonung der Macht der Sprache läßt sie als die "geschichtsbildende" Kraft er- 2 scheinen, wie L. Weisgerber sie später definiert hat . Aus der heu- tigen Sicht erübrigt sich die Diskussion darüber, ob die Sprache nur eines der Merkmale der Nation is t. Zur damaligen Zeit stand Grimm mit seinen Ansichten nicht alleine. Auch bei den Südslaven haben die Vertreter der "Illyrischen Bewegung" und Vuk Karadzic ge- hofft, über die Sprache die nationale Einigung erreichen zu können. 3. Der nationale Charakter der Sprache und das Problem der Übersetzbarkeit 3. Der nationale Charakter der Sprache und das Problem der Übersetzbarkeit Seit Herder die Sprache ein "Spiegelbild der Nation", "ein In- halt ihrer Gedankenwelt und eine Form ihrer Art"^ genannt hatte, is t der Zusammenhang zwischen der Sprache und ihrem Träger zum Ge- genstand der Sprachwissenschaft geworden. Auch Grimm meint, daß "sprachformen und denkungsart der Völker unsichtbar in einander greifen" (GKS VII, S. 558) und daß "einzelne züge der spräche von der sitte und gewohnheit des volks unzertrennbar scheinen" (GKS I, S. 332). Die Auffassung der Sprache als Weltbild gewinnt bei Grimm eine eigentümliche Prägung, die an seiner Ubersetzungstheorie besonders deutlich zu sehen is t1• Sie is t für uns auch deshalb wichtig, weil Grimm serbische Volkslieder übersetzt hat. Grimm hielt das Gelingen einer Übersetzung für nicht möglich, weil "sich form und gehalt der Wörter in zwei sprachen niemals ge- паи decken können" (GKS I, S. 330) und weil ״*Inhalt und form einer wahren dichtung so wesentlich verbunden sind, dasz sie nicht aus- einander gerissen werden können" (GKS VIII, S. 27). 1 über damalige Übersetzungstheorien, an denen auch A.W. Schlegel mitwirkte, vgl. E. Fiesel, a.a.O., S. 39ff. 1 Vgl. Beneš, a.a.aO., S. 62f.; vgl. L. Weisgerber, Von den Kräf- ten der deutschen Sprache, Bd. 1-4, 2. Aufl. 1954-59. zustecken.11 (GKS V III, S. 28f.). Diese Ansichten von Grimm erklären auch seine Unzufriedenheit mit den Übersetzungen der serbischen Volkslieder von Talvj und seine Äußerung, daß die serbischen Lieder unübersetzbar sind (GKS IV, S. 419ff.). Grimm hat am Beispiel der serbischen Volkslieder gezeigt, wie er sich die Vermittlung zwischen zwei Sprachen auf unterschiedlichen Entwicklungsstufen vorgestellt hat. Er meinte in bezug auf Talvjs Übersetzung der epischen Formel "grad gradili" als "eine fest' erbauten" folgendes: "bürgten bürg übersetzt folg- lieh getreuer, zumal das verbum kein neubackenes deutsch und schon von Notker 106, 36 gebraucht is t." Er schlug ebenso "jagd jaget" für "lövi lova" ,'1ernte erntete" für "žetvu zela" vor (ebd., S. 420). Da die geistige, d.h. abstrakte Bildung der deutschen Sprache fortgeschrittener is t, als die der serbischen, sollte beim übersetzen auf die ältere Sprachstufe zurückgegriffen werden. Die Übersetzung soll Form und Gehalt des Originals zu erreichen suchen und darf "etwas undeutsches an sich haben" (ebd.). Grimm b illig te nur solche Übersetzungen, die für das ganze Volk gedacht waren, und diejenigen aus "einer fern abgelegenen spräche", für die keine Hilfsmittel zur Verfügung standen. Deshalb hat er die "unübersetzbaren'1 serbischen Lieder selbst übersetzt. Von den Gebildeten verlangte er, die Dichtung in der Originalspra- che zu lesen (GKS V III, S. 28). III. Die Aufgabe der Sprachwissenschaft 2. Die Schriftsprache als ein konstitutiver Faktor der Nation "während also die freien Übersetzungen blosz den gedanken erreichen wollen und die Schönheit des gewandes daran geben, mühen sich die strengen das gewand nachzuweben pedantisch ab und bleiben hinter dem urtext stehn, dessen form und inhalt ungesucht und natürlich zusammen- stimmen." (GKS I, S. 330f•). Grimm verbindet die Ansicht von der nationalen Eigenart der Sprache mit der historischen Sicht seiner Sprachbetrachtung: "zu einer idealisch treuen Übersetzung würde erforderlich sein, dasz unsere spräche, mit der wir übersetzen wollen, der fremden, woraus wir übersetzen wollen, das gleichgewicht halte, nicht blosz in der form, sondern auch in der geistigen entwicklung. das t r i f f t jedoch practisch niemals zusammen, jemehr annäherungen aber dazu vorhanden sind, desto besser kann die Übertragung geraten... zu- gleich folgt hieraus, dasz sich ein altes epos, sei es ein noch so kleines Volkslied, gar nicht übersetzen lasse, denn weil man nur in einer gebildeten zeit übersetzt, so is t der abstand zwischen ihr und der natürlichen zeit, worin diese dichtungen entstanden und lebten, zu bedeutend und die beiderseitigen mittel liegen ein- ander zu fern, als dasz das werk gelingen könnte." (GKS V III, S. 28). Deshalb lehnte Grimm das übersetzen der alten deutschen Dichtung in die neuhochdeutsche Sprache ab: "fremde werke bleiben gleichwol von der deutschen bearbeitung unabhängig fortbestehen; denkmäler unserer vorzeit hingegen, weil sie uns näher sind und die Verstimmung zwischen verwandten tönen schreiender is t, als zwischen solchen, die weit auseinander liegen, empfinden es desto schlimmer, wenn man sie zwingt, die färbe der heutigen weit auf- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 31 ־ zustecken.11 (GKS V III, S. 28f.). zustecken.11 (GKS V III, S. 28f.). III. Die Aufgabe der Sprachwissenschaft Die Aufgaben, die Grimm der Sprachwissenschaft s te llt, sind von seiner Sprachauffassung bestimmt. Es is t bereits bemerkt wor- den, daß seine Wortforschung in der "Deutschen Grammatik" mit den von ihm aufgestellten "Wortfeldern" einen Versuch darstellt, das "Weltbild"1der deutschen Sprache aufzudecken. Grimms Auffassung der Sprachwissenschaft enthält den Gedanken, daß die Sprache im Zusammenhang mit der Geschichte, die sprachli­ 1 Vgl. Beneš, a.a.aO., S. 62f.; vgl. L. Weisgerber, Von den Kräf- ten der deutschen Sprache, Bd. 1-4, 2. Aufl. 1954-59. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access chen Veränderungen nicht is o lie rt von Menschen und den Veränderun- gen in der Gesellschaft erforscht werden sollen1. Im Einklang mit der Auffassung der Sprache als Geschichte erscheint Grimms Sprach- forschung nicht als Selbstzweck, sondern als ein Teil der Geschichts forschung. Er selbst hat sie folgendermaßen definiert; "Sprachfor- schung der ich anhänge und von der ich ausgehe, hat mich doch nie in der weise befriedigen können, dasz ich nicht immer gern von den Wörtern zu den sachen gelangt wäre? ich wollte nicht blosz häuser bauen, sondern auch darin wohnen, mir kam es versuchenswerth vor, ob nicht der geschichte unsers volks das bett von der spräche her stärker aufgeschüttelt werden könnte und... auch die geschichte aus dem unschuldigeren standpunct der spräche gewinn entnehmen so llte .2״ chen Veränderungen nicht is o lie rt von Menschen und den Veränderun- gen in der Gesellschaft erforscht werden sollen1. Im Einklang mit der Auffassung der Sprache als Geschichte erscheint Grimms Sprach- forschung nicht als Selbstzweck, sondern als ein Teil der Geschichts forschung. Er selbst hat sie folgendermaßen definiert; "Sprachfor- schung der ich anhänge und von der ich ausgehe, hat mich doch nie in der weise befriedigen können, dasz ich nicht immer gern von den Wörtern zu den sachen gelangt wäre? ich wollte nicht blosz häuser bauen, sondern auch darin wohnen, mir kam es versuchenswerth vor, ob nicht der geschichte unsers volks das bett von der spräche her stärker aufgeschüttelt werden könnte und... auch die geschichte aus dem unschuldigeren standpunct der spräche gewinn entnehmen so llte .2״ Grimm is t in die Geschichte der Sprachwissenschaft vor allem als der Begründer der historisch-vergleichenden Grammatik der deut- sehen Sprache eingegangen. III. Die Aufgabe der Sprachwissenschaft Obwohl er hauptsächlich historische Sprachforschung trieb, war die Sprachgeschichte nicht das Endziel seiner Forschung. 1 Es muß auch auf Grimms Irrtümer hingewiesen werden, wie z.B. sei- ne Erklärung der beiden Lautverschiebungen durch den deutschen Nationalcharakter. - Vgl. Geschichte der deutschen Sprache, a.a.O S. 292. 2 Vgl. ebd., S. XI. 3 Diese Termini von F. de Saussure werden hier aus praktischen Gründen eingeführt. 1 Es muß auch auf Grimms Irrtümer hingewiesen werden, wie z.B. sei- ne Erklärung der beiden Lautverschiebungen durch den deutschen Nationalcharakter. - Vgl. Geschichte der deutschen Sprache, a.a.O S 292 1. Die Diachronie als Vorstufe der Synchronie^ Grimm hat der damaligen normativen Grammatik, die er ”pedantisch nannte, "die völlige Unzulänglichkeit der bisher ausgeklügelten re- geln" vorgeworfen und sie als "täuschung und irrthum" abgelehnt (GKS V III, S. 31). "die pedantische ansicht der grammatik schaut über die schranke der sie befangenden gegenwart weder zurück, noch hinaus, mit gleich verstockter beharrlichkeit lehnt sie sich auf wider alles in der spräche veraltende, das sie nicht länger faszt, und wider die keime einer künftigen entfaltung, die sie in ihrer seichten gewohnheit stören.” (GKS I, S. 329). Wir haben gesehen, wie Grimm den "Organismus" der Sprache ge- Wir haben gesehen, wie Grimm den "Organismus" der Sprache ge- 1 Es muß auch auf Grimms Irrtümer hingewiesen werden, wie z.B. sei- ne Erklärung der beiden Lautverschiebungen durch den deutschen Nationalcharakter. - Vgl. Geschichte der deutschen Sprache, a.a.O. S. 292. S. 292. 2 Vgl. ebd., S. XI. 3 Diese Termini von F. de Saussure werden hier aus praktischen Gründen eingeführt. 1 Vgl. W. Scherer, Jacob Grimm, 2. Aufl. 1885, S. 159ff. 2 Diese Forderung Grimms wurde als Verbot jeden Eingriffs in das "natürliche Wachstum" der Sprache mißverstanden. - Vgl. Arens, a.a.O., S. 177. 3 Vgl. Geschichte der deutschen Sprache, a.a.O., S. XII; diesen Wunsch hat er auch während der Arbeit am Wörterbuch geäußert.- Vgl. dazu Arndt, a.a.O. S. 484f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 2 Vgl. ebd., S. XI. 3 Diese Termini von F. de Saussure werden hier aus praktischen Gründen eingeführt. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access sehen hat; daraus erklärt sich seine K ritik der Grammatiker, die die aufgestellten Regeln "zum gesetz für die Zukunft zu erheben be- müht waren" (GKS V III, S. 37) und "die hinter vorgedrungenen aus- nahmen s t ill blickende regel gar nicht ahnen" (GKS I, S. 328). sehen hat; daraus erklärt sich seine K ritik der Grammatiker, die die aufgestellten Regeln "zum gesetz für die Zukunft zu erheben be- müht waren" (GKS V III, S. 37) und "die hinter vorgedrungenen aus- nahmen s t ill blickende regel gar nicht ahnen" (GKS I, S. 328). Grimms Äußerungen müssen im Zusammenhang mit der zeitgenössi- sehen Tätigkeit der Puristen gesehen werden1, die er entschieden 2 abgelehnt hat. Er warnte vor jedem Eingriff in die Sprache , weil ihm "eine tiefe forschung vorhergehen müste". "auf jeden fa ll is t soviel einleuchtend, wenn man beabsichtigte, das gebiet der jetzt vorhandenen Wörter und formen zu erweitern, dasz die gründlichste, durchdringendste kenntnis aller eigenschaften und triebe der spra- che vorausgesetzt werden müste, um die vermeintlichen lücken und schwächen von nicht blosz einer seite zu beleuchten und die vorge- schlagene ergänzung oder besserung vernünftig zu berechnen." (GKS V III, S. 34, 35). Um die "eigenschaften und triebe" der Sprache zu begreifen, is t nach Grimms Auffassung die historische Sprachforschung unentbehrlich Er meinte, daß "die heutigen formen unverständlich seien, wo man nicht bis zu den vorigen, alten und ältesten hinaufsteige, dasz folglich die gegenwärtige grammatische struetur nur geschichtlich aufgestellt werden dürfe..." (ebd., S. 44). Grimm hat die Mängel der damaligen Grammatik gesehen, die sich aus der mechanischen An- wendung der Regeln ergaben. Obwohl Grimm die historische Grammatik die "wahre" genannt hat (GKS I, S. 407), verstand er sie eigentlich als eine unentbehrliche Vorarbeit für eine gelungene Darstellung der Gegenwartssprache, die er selbst unternehmen wollte. "Es war längst mein vorsatz, die regel neuchhochdeutscher, d.h. 1 Vgl. Deutsche Grammatik 1. T., 2.Aufl. 1822, S. XVIII. - Seine Erklärung des Ursprungs der "deutschen" Schrift und der Groß- Schreibung is t von der späteren paläographischen Forschung im Wesentlichen bestätigt worden. - Vgl. K. Hiehle, Jakob Grimm als Wegbereiter einer lautrichtigen Rechtschreibung. In: Zs.f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 2 Vgl. ebd., S. XI. 3 Diese Termini von F. de Saussure werden hier aus praktischen Gründen eingeführt. ganz in unsre gegenwart gerückten spräche vollständig und überall auf die geschichte gestützt hinzu- stellen, ich weisz aber nicht, ob es mir vergönnt sein wird..."^ - 34 ־ Nach Grimms Auffassung sollten sich die diachronische und syn- chronische Betrachtungsweise gegenseitig ergänzen.Diese Meinung hat er auch in der Rezension des serbischen Wörterbuchs von Vuk ausge- sprochen: es "wird sich aus diesem serbischen für das altslavische lernen lassen* dasz aus der falschen (unhistorischen) gelehrsamkeit gerade die ärgsten irrthümer entspringen können, beweisen die slave־ nischen grammatiker selbst; Maximow und Ewgeni glaubten z.b* den dual I. budewa II. budeta I I I . budeta gewaltig zu verbessern, wenn sie I. budema II. budewa I I I . budeta aufstellen, damit unterschei- den sich zwar I und II, aber die historische Wahrheit, dasz II und I I I im dualis zusammenfallen, wird verbaut, aus dem bodeva, bodeta, bodeta der crainischen Volkssprache (Kopitar s. 317) hätten diese verbesserer lernen können, dasz Smotrisky den dual ganz richtig ge- faszt hatte.” (GKS IV, S. 105). Grimms Ansicht, daß bei der Aufstellung der schriftsprachlichen Norm die Erkenntnisse der historischen Sprachforschung berücksich- tig t werden sollten, hat in seinem Versuch der Orthographiereform einen Niederschlag gefunden. 2. Grimms Versuch der Orthographiereform 2. Grimms Versuch der Orthographiereform Grimms Ansichten über die Probleme der deutschen Orthographie zeigen viele Parallelen zur Orthographiereform von Vuk Karadzic. Einige Tatsachen sprechen dafür, daß Grimms Reformversuch durch Vuk angeregt und beeinflußt wurde. Grimm hat den ersten Schritt zur Reform der deutschen Orthogra- phie in der zweiten Auflage des ersten Bandes seiner Grammatik von 1822 unternommen. Er hat an Stelle der sog. "deutschen"die lateini- sehe Schrift und die Kleinschreibung eingeführt. Die Abschaffung der Großschreibung hat er folgendermaßen begründet: "Für sie spricht kein einziger innerer grund, wider sie der beständige frü- here gebrauch unserer spräche bis ins sechzehnte, siebzehnte jahr- hundert, ja der noch währende aller übrigen Völker, um nicht die erschwerung des schreibens, die verscherzte einfachheit der schrift anzuschlagen-"1 Grimm stützte sein Argument, daß die Großschreibung nicht zum Wesen der deutschen Sprache gehört, auf die Erkenntnisse aus der Sprachgeschichte. nicht zum Wesen der deutschen Sprache gehört, auf die Erkenntnisse aus der Sprachgeschichte. Grimm hat in der Zeit zwischen der ersten und zweiten Auflage seiner Grammatik die Orthographie von Vuk Karadziè kennengelernt und ihre Vorteile in der Rezension des serbischen Wörterbuchs und in der Vorrede zur deutschen Fassung von Vuks Grammatik gelobt. Unter Grimms Äußerungen über Vuks Orthographie sind zwei von besonderer Bedeutung, weil sie auch in seinen Ansichten über die deutsche Orthographie erscheinen, nämlich: 1. Grimm hebt die ein- fachen kyrillischen Zeichen von Vuk gegenüber den traditionellen hervor, weil die "einzelnen, anders angenommenen Zeichen... die eigenthümlichkeit der laute zu erfassen" vermögen (GKS IV, S. 102); 2. er lobt den phonetischen Charakter von Vuks Orthographie, weil der "Wohllaut der serbischen Buchstabenverhältnisse"1 sich in der Schrift widerspiegelt: "wir würden die feinen Modificationen der Consonanten weniger erkennen, wenn der Verfasser mehr die etymolo- gische Wurzel festgehalten,als die Abstufungen der wahren Ausspra ־ che beachtet hätte. Das Problem der deutschen Orthographie ist für Grimm erst mit dem Beginn der Arbeit am Wörterbuch akut geworden. Er geht in sei- nen Schriften aus dieser Zeit wiederholt auf dieses Problem ein. In seiner Kritik der deutschen Schreibweise und in seinen Vorstel- lungen über eine gute Orthographie erscheinen die Gedanken aus sei- ner Beurteilung der Orthographie von Vuk: "was in jeder guten schrift stattfindet, die annahme einfacher Zeichen für beliebte consonantenverbindungen, wie bei uns CH und SCH sind, is t gänzlich vermieden und dadurch der anschein schleppender breite hervorge- bracht... 1 In Grimms Terminologie werden die Begriffe "Buchstabe" und "Laut" nicht von einander abgegrenzt. 2 Vgl. Grimms Vorrede zur "Kleinen serbischen Grammatik", a.a.O., S. XXII. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. Srpska gramatika S. XXXII. 2 Vgl. ebd., S. XXXIIf. 3 Hervorhebung von V. B. 4 Grimm hat zu dieser Zeit in Vuks Grammatik und Wörterbuch ge- lesen; das wird sowohl durch den Briefwechsel als auch durch Eintragungen in Grimms Handexemplar bestätigt. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM i f 2. Grimms Versuch der Orthographiereform Auf den wollaut und das gesetz aller ändern sprachen, dasz inlautend buchstab vor buchstab schwinden müsse, wenn er nicht mehr auszusprechen is t, wird herkömmlich nicht geachtet..." (GKS I, S. 348f., 1847). Grimms Argumentation gegen die etymologische Rechtschreibung stimmt stellenweise fast wörtlich mit derjenigen von Vuk über- ein. Vuk hat in seiner serbischen Grammatik geschrieben: " ... da su slova samo zato izmisfjena, da se njima zapisuju rije č i onako kao sto se izgovaraju ... a Sekoji bi ljudi celi da im slova po- kazuju i kako se rije č i izgovaraju, i šta znače, i od kuda su po- stale!1 ״Auch Grimm meint: ״der Schreibung, die ihre volle pflicht thut, wenn sie alle wirklichen laute zu erreichen sucht, kann nicht das unmögliche aufgebürdet werden, zugleich die geschichte einzelner Wörter darzustellen.( ״GKS I, S. 350, 1847). Vuk hat die Forderung nach etymologischer Rechtschreibung durch Konstru- ierung von Beispielen wie "knež" zu "kneže" ad absurdum geführt und die Vokalgemination als Längebezeichnung (z. B. "raana") ein F l i c k e n der serbischen Sprache nach dem Vorbild der deutschen 2 genannt . Auch Grimm formuliert ähnlich mit konstruierten Beispie- len: "sollte die schrift alle vocale nachholen, die allmählich zwischen den buchstaben unserer Wörter ausgefallen sind; sie hät- te nichts zu thun als zu h ä k e l n und wer würde setzen mögen Eng'land, men'sch, wün'schen, hör'en?" (GKS I, S. 350). Diese Übereinstimmung von Grimms und Vuks Formulierungen und die Verbindung zwischen Grimms Äußerungen über die deutsche Orthographie und seiner Beurteilung der Orthographie von Vuk wei- sen darauf hin, daß Vuks Orthographiereform einen Einfluß auf 4 Grimms Ansichten und seinen Reformversuch ausgeübt hat . Grimm konnte mit seinem Reformvorschlag nicht so weit gehen wie Vuk. Der Macht der Gewohnheit, die solchen Reformen widerstrebt, stand auch, anders als bei den Serben, eine starke schriftsprachliche Tradition zur Seite. Grimms Versuch der Orthographiereform, den er in seinem Wör- terbuch zur Diskussion gestellt hat, betraf, wie bei Vuk, sowohl Graphie als auch Orthographie. Im Zusammenhang mit der Graphie deuten seine Ausführungen über f , v : w darauf hin, daß seine 1 Vgl. Srpska gramatika S. XXXII. 2 Vgl. ebd., S. XXXIIf. 3 Hervorhebung von V. B. 4 Grimm hat zu dieser Zeit in Vuks Grammatik und Wörterbuch ge- lesen; das wird sowohl durch den Briefwechsel als auch durch Eintragungen in Grimms Handexemplar bestätigt. 1 Vgl. Hiehle, a.a.O. S. 312. - Grimm hat seine Wahl des Zeichens 8z mit dem Hinweis auf die polnische, litauische und ungarische Sprache begründet (GKS V III, S. 371). 2 Vgl. darüber H. Moser, Groß- oder Kleinschreibung? Ein Haupt- problem der Rechtschreibreform, 1958 (= Duden-Beiträge H. 1); ders.. Vermehrte Großschreibung ־ein Weg zur Vereinfachung der Rechtschreibung?, 1963 (= Dudenbeiträge H. 16). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 2. Grimms Versuch der Orthographiereform Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vorstellung von den 1*wirklichen" Lauten dem Begriff des Phonems nahesteht. Da in der deutschen Sprache die Aussprache von f und v Zusammenfalle, sollte der Laut mit f bezeichnet werden, dem dann das טan Stelle von w als Opposition treten würde. Grimm wies darauf hin, daß diese Zeichen im Mittelhochdeutschen bereits so verwendet wurden (GKS V III, S. 373). "Bei seinen konfusen s, ss, sz-Schreibungen, die besonderen Anstoß erregten, erlag Grimm allerdings einer Autosuggestion, bei den verschiedenen s־Lauten ihrer etymologischen Herkunft ent- sprechende Lautverschiedenheiten noch heraushören zu können."1 Auf dem Gebiet der Orthographie wollte Grimm, außer der Groß- Schreibung, die inkonsequente Bezeichnung der Vokallänge, (die entweder auf dreifache Weise: Gemination, Einschub eines e oder eines h geschieht, oder unterbleibt), abschaffen. Die von Grimm hervorgehobenen Probleme sind Gegenstand der bis in die heutige Zeit andauernden Diskussion über die Notwen־ 2 digkeit einer Reform der deutschen Orthographie . 1• Die sprachliche Situation bei den Serben zu Beginn von Vuks Reformbemühungen 1• Die sprachliche Situation bei den Serben zu Beginn von Vuks Reformbemühungen Vuk begann seine Tätigkeit in der Blütezeit der sog. slaveno- serbischen Periode1, als sich ihr bekanntester Vertreter Milovan Vidakoviò gerade auf der Höhe seines Ruhmes befand. Die Sprache von Vidakovic und seinen Zeitgenossen wird "slavenoserbisch" ge- nannt. I. Grickat hat recht, wenn sie meint, daß vom linguisti- sehen Standpunkt aus nicht von der Sprache im üblichen Sinne die Rede sein kann . Der Anteil der serbischen und russischen Sprach- elemente war von Fall zu Fall unterschiedlich und keinen Regeln unterworfen; es gab keine allgemeingültige Norm.Die Werke der ein- zelnen Schriftsteller, sogar die desselben Autors, zeigen Unter- schiede auf allen Gebieten der Sprache, der Phonetik, Morphologie, Syntax und Lexik. Das slavenoserbische Sprachgebilde hat sich im Laufe der Zeit auf der Grundlage der russ.-ksl. Sprache entwickelt, die bekannt- lieh zu Beginn des 18. Jhs. in die serbische Kirche3 und dann 1 Zur Terminologie: Vuks Reformbemühungen betrafen zunächst nur die sprachlichen Probleme bei den Serben (Ablösung der slaveno- serbischen Sprache, Reform des kyrillischen Alphabets); deshalb wird bei der Erörterung dieser Problematik in bezug auf die Sprache der Terminus s e r b i s c h verwendet. Vuk hat in seine Dia- lektforschung, außer den serbischen, auch k r o a t i s c h e Dialekte einbezogen. Alle diese Dialekte sind im linguistischen Sinne Teile ein und derselben Sprache auch in der fraglichen Zeit Vuks; der Terminus S e r b o k r o a t i s c h ( K r o a t o s e r b i s c h ) wird jedoch nicht auf die Zeit vor 1850 angewandt. Aktuelle Probleme der bei den heutigen Varianten der serbokroatischen Sprache können hier nicht berücksichtigt werden. g 2 Vgl. I. Grickat, U сети je značaj i kakve su specifienosti slave nosrpskog perioda и razvoju srpskohrvatskog jezika. In: Zbornik FL IX, 1966, S. 64. 2 Vgl. I. Grickat, U сети je značaj i kakve su specifienosti slave nosrpskog perioda и razvoju srpskohrvatskog jezika. In: Zbornik FL IX, 1966, S. 64. 3 Uber historische Voraussetzungen dazu vgl. A. Belic, Vukova bor- ba za narodni i književni jezik, 1948, bes. S. 7-24. 3 Uber historische Voraussetzungen dazu vgl. A. Belic, Vukova bor- ba za narodni i književni jezik, 1948, bes. S. 7-24. 1 Zur Terminologie: Vuks Reformbemühungen betrafen zunächst nur die sprachlichen Probleme bei den Serben (Ablösung der slaveno- serbischen Sprache, Reform des kyrillischen Alphabets); deshalb wird bei der Erörterung dieser Problematik in bezug auf die Sprache der Terminus s e r b i s c h verwendet. Vuk hat in seine Dia- lektforschung, außer den serbischen, auch k r o a t i s c h e Dialekte einbezogen. Alle diese Dialekte sind im linguistischen Sinne Teile ein und derselben Sprache auch in der fraglichen Zeit Vuks; der Terminus S e r b o k r o a t i s c h ( K r o a t o s e r b i s c h ) wird jedoch nicht auf die Zeit vor 1850 angewandt. Aktuelle Probleme der bei den heutigen Varianten der serbokroatischen Sprache können hier nicht berücksichtigt werden. 1 Die Einführung des Russ.-Ksl. hat die Kontinuität in der Entwick- lung der serbischen Schriftsprache gebrochen. Die neuesten Unter- suchungen der serbischen Literatur des 17. und 18. Jhs. und die Teilausgabe des Nachlasses von Venclovic ( um 1680-1747), der außer Predigten auch Lyrik in einer ausdrucksvollen Sprache auf der volkssprachlichen Grundlage geschrieben hat, lassen vermuten, in welcher Richtung sich die serbische Schriftsprache entwickelt hätte. - Vgl. M. Paviå, Istorija srpske književnosti baroknog doba (XVII i XVIII vek), 1970; ders., Gavril Stefanovic Venclo- vie, 1972; ders. (Hg.), Gavril Stefanovic Venclovic, Crni bivo и sreu. Legende,besede, pesme, 1966. Teilausgabe des Nachlasses von Venclovic ( um 1680 1747), der außer Predigten auch Lyrik in einer ausdrucksvollen Sprache auf der volkssprachlichen Grundlage geschrieben hat, lassen vermuten, in welcher Richtung sich die serbische Schriftsprache entwickelt hätte. - Vgl. M. Paviå, Istorija srpske književnosti baroknog doba (XVII i XVIII vek), 1970; ders., Gavril Stefanovic Venclo- vie, 1972; ders. (Hg.), Gavril Stefanovic Venclovic, Crni bivo и sreu. Legende,besede, pesme, 1966. 2 Vgl. u.a. J. Rajiò, Isto rija raźnych slavenskich narodov.., 1795, I, S. 25 - 32. 3 Vgl A Ivic Teodor Jankovic Mirijevski и odbranu c irilic e In: и sreu. Legende,besede, pesme, 1966. 2 Vgl. u.a. J. Rajiò, Isto rija raźnych slavenskich narodov.., 1795, I, S. 25 - 32. 1• Die sprachliche Situation bei den Serben zu Beginn von Vuks Reformbemühungen Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access auch in die Schulen eingeführt wurde1. Diese Entwicklung wurde ei- nerseits durch die ungenügende Kenntnis der russischen Kirchenspra- che, andererseits durch die Bemühungen der serbischen Schriftstel- 1er, für das Volk in der ihm verständlichen Sprache zu schreiben, verursacht. Bereits Z. Orfelin (1726 - 1785) und J. Rajiò (1726 ־ 1801) haben, neben den in russ.-ksl. Sprache verfaßten Werken, ihre Schriften für das Volk in einer Art von Volkssprache geschrieben. Orfelin g ilt auch als der Begründer der slavenoserbischen Schule. Die serbischen Gebildeten vor Dositej und Vuk vertraten die Auffassung, daß man für gehobenere Ansprüche eine gebildetere Spra- che als die des einfachen Volkes brauche. Sie strebten eine Annähe-־ rung an die russische Sprache an, die man für die ursprüngliche sia- 2 vische und somit auch serbische h ie lt . Die sprachliche Situation bei den Serben erscheint am deutlichsten in dem 1781 verfaßten ”Memoár" von Teodor Jankoviè Mirijevski ("de Mirijevo"), dem Her- ausgeber der zweiten Auflage des St. Petersburger vergleichenden Wörterbuchs. Er führt drei Sprachen an: neben der Kirchensprache für den kirchlichen Bereich sollte in der "Civilsprache" für die Gebildeten und in der Volkssprache für das Volk geschrieben wer- den3. Mirijevskis "Civilsprache" entsprach dem "mittleren S til" (srednji slog), den Vuks Zeitgenossen vertreten haben und den Vuk, besonders in seinen Polemiken mit Vidakovic, bekämpft hat. Die Vorstellung von dem "mittleren S til" beruhte auf dem Irr- tum der damaligen Wissenschaft, daß die serbische Volkssprache im Vergleich mit der altkirchenslavischen einen "gesunkenen" Dialekt g p 2 Vgl. u.a. J. Rajiò, Isto rija raźnych slavenskich narodov.., 1795, I, S. 25 - 32. 3 Vgl. A. Ivic, Teodor Jankovic Mirijevski и odbranu c irilic e . In: JF XI, 1931, S. 197-216. - Einen Auszug aus dem "Memoár" gibt auch Belic, Vukova borba.., S. 21f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access darstelle . Sogar der **Vater der Slavistik", Joseph Dobrovskÿ, den Vidakoviò zum Richter im Streit mit Vuk gewählt hat, vertrat in seinem Brief an Vidakovic die Ansicht: **Mir aber, wenn ich mir ei- ne Stimme anmaßen dürfte, w ill es gar nicht behagen, daß sich die Serbier zur Dorfsprache herablassen sollen. 1 Vgl. Skupljeni gramatički i polemicki spisi Vuka Stef. Karadžica (im weiteren Text: GPS) , Bd. I - I I I , 1894; I, S. US). 2 Vgl. Pis'ma Dobrovskago i Kopitara, hg. V. Jagic, 1885, S. 428f. (im weiteren Text: Pis'ma). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1• Die sprachliche Situation bei den Serben zu Beginn von Vuks Reformbemühungen Es muß doch auch eine edlere Sprache für erhabenere Gegenstände geben. Es dürfte ja ein Stylus medius (Srednji slog) ein Mittelweg ausgemittelt werden, der sich der alten liturgischen, und zum *Theil wieder der Umgangs- spräche der Nation näherte. Denn das prosto muß doch nicht bis auf die fehlerhafte Syntaksis der praeposition ausgedehnt werden.1*״ Seine Meinung über die Fehlentwicklungen in der serbischen Sprache hat er in einem Brief an Kopitar mitgeteilt, da er, offenbar aus Rücksicht auf den Freund, gegen Vuk nicht öffentlich auftreten wollte. ״*Was offenbar Corruption is t, wie sunze, vuk, suza, vidio, dafür wünschte ich, dass man since, vlk, slza, v id il schreibe.״* Auch die neuštok. Endung -ma im Lok. PI. sollte abgeschafft und die ksl. Endung -ch wieder hergestellt werden. Der stilus medius sollte diese Korrekturen enthalten und eine Auswahl der Elemente aus den beiden Sprachbereichen darstellen, die nach festen, allgemeingülti- gen Prinzipien durchgeführt werden müßte. Hinsichtlich der sprach- liehen Situation bei den Serben hat er aber an der Realisierbar- 2 keit seiner Vorstellungen vom ”mittleren S til" gezweifelt . Der stilus medius in der Praxis der serbischen Schriftsteller bedeutete eine willkürliche Mischung der serbischen und russ.-ksl• Sprache. In der Bemühung, die serbische Sprache nach dem Vorbild der russischen zu verbessern, hatten die serbischen Schriftsteller keine Regeln, weshalb ihre Sprachverwendung inkonsequent war. Die- se Inkonsequenzen boten Vuk ein schlagkräftiges Argument in seinem Kampf gegen die Ansichten, daß außer in der Volkssprache auch in einer gebildeteren Sprache geschrieben werden sollte. Vuk hat die- sen Kampf nach seinen Polemiken mit Vidakovic bereits zu Beginn der 20er Jahre gewonnen. Vor Vuk hatte sich Dositej als erster gegen die Ansichten von der Notwendigkeit zweier Schriftsprachen gewandt und eine einheit- liehe Schriftsprache auf der volkssprachlichen Grundlage verlangt; somit war er der theoretische Vorläufer von Vuk. Dositej hat aber weder die Volkssprache ausreichend gekannt, noch die praktischen und taktischen Fähigkeiten besessen, um seine Theorie in der Praxis durchführen zu können. somit war er der theoretische Vorläufer von Vuk. Dositej hat aber weder die Volkssprache ausreichend gekannt, noch die praktischen und taktischen Fähigkeiten besessen, um seine Theorie in der Praxis durchführen zu können. 1 Wie bereits erwähnt wurde,schrieben auch Venclovic, Rajic und Dositej in der Volkssprache. 2 Zur Diskussion darüber vgl. M. Selimovic, Za i protiv Vuka, 1967. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access I I . Vuks Beziehung zur zeitgenössischen Sprachwissenschaft I I . Vuks Beziehung zur zeitgenössischen Sprachwissenschaft Vuk war kein Theoretiker, sondern vor allem ein Empiriker. In seinen Schriften und in seiner Praxis is t dennoch eine vielfache Verbindung zur zeitgenössischen Sprachwissenschaft feststellbar. Seine theoretischen Ansichten sind, insbesondere in der Anfangs- phase, durch B. Kopitar entscheidend geprägt. 1 Z it. nach Lj. Stojanovic, Život i rad Vuka Stef. Karadžica, 1924, S. 92. 2 Vgl. N. M. Petrovskij, Pervye gody deja te l1 nosti В. Kopitarja, 1906, S. 176. 3 Vgl. F. Petrê, Zojs, Kopitar i Karadzic. In: Naucni sastanak, Bd. 3, 1973, S. 234. 4 Vgl. Petrovskij, a.a.O., S. 734. 5 Vgl. GPS I I I , S. 66? Prepiska I, S. 163f.? P. Ivic, Srpski narod i njegov jezik, 1971, S. 259. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1• Die sprachliche Situation bei den Serben zu Beginn von Vuks Reformbemühungen Vuk, der mit Recht als der Begründer der serbischen Schriftspra- che g ilt, war also weder der erste, der in der Volkssprache ge- schrieben hat1, noch hat er als erster das Problem der Schriftspra- che zur Diskussion gebracht • Die moderne serbokroatische Schrift- spräche is t aber von Vuks Ansichten stark geprägt. Der Streit, den sie seinerzeit ausgelöst haben, wird in unterschiedlichen Formen bis heute geführt. Vuk wird immer wieder, oft in entgegengesetzten Argumentationen, als die oberste Autorität z itie rt; auf der ande- ren Seite wiederum werden Zweifel geäußert, ob sein Weg der beste und einzig mögliche war. Das beweist, daß Vuks Lösung des Problems der Schriftsprache von Anfang an Keime für neue Probleme enthielt, die in der nachfolgenden Zeit nicht bewältigt werden konnten. Die Feststellung dieser Tatsache ist kein Votum ”für" oder 2 "gegen" Vuk ; denn, sein Auftreten in der serbischen Kulturgeschich te und seine Reform waren die dringenden Forderungen der Zeit. Ver- schiedene Einzelheiten seiner Sprachkonzeption, die wir kritisch beleuchten möchten, waren zweifellos nicht die besten. Das wesent- liehe Resultat seiner Bemühungen aber, die Einführung und Festi- gung der Volkssprache in der Literatur, war eine unaufschiebbare Notwendigkeit und die einzige Möglichkeit, das Hemmnis der kultu- rellen Entwicklung bei den Serben, das in der damaligen sprachli- chen Situation lag, aus dem Wege zu räumen. Der Begriff "Volkssprache" is t ungenau und muß definiert wer- den. Ein Volk is t keine homogene Einheit; diese Tatsache spiegelt sich auch in der Sprache wider. Die Sprache der städtischen Be- völkerung der Vojvodina zu Vuks Zeiten z.B. unterschied sich von der Dorfsprache durch viele Russismen und Germanismen für kultu- relie und Zivilisationsbegriffe. Auf der anderen Seite ist die un- genormte Volkssprache ein Mosaik von Dialekten mit größeren oder kleineren Unterschieden. Vuks Begriff der Volkssprache macht im Laufe der Zeit eine Entwicklung durch, auf die wir in der Darstel- lung seiner Ansichten über die Schriftsprache hinweisen möchten. 00051004 - 42 - 1. Vuk und Kopitar 1. Vuk und Kopitar Vuks Tätigkeit is t mit dem Namen von B. Kopitar unzertrennlich verbunden. Kopitars Einfluß auf Vuks Werdegang zum Reformer der serbischen Sprache und Orthographie is t unbestreitbar. Der Brief- Wechsel zwischen den beiden Freunden is t ein bleibendes Zeugnis ihrer Zusammenarbeit und Kopitars Einsatz für Vuk. Vuk hat ihm ei- ne große Anerkennung mit den Worten ausgesprochen: "U ostalom Sto sam ja postao knjiž evnik... ja sam obavezan samo Kopitaru."1 Die Beziehung zwischen Vuk und Kopitar is t viel erörtert wor- den. Die ersten Werke von Vuk, zwei Liederbände, die beiden Gramma- tiken und das Wörterbuch, die in einem kurzen Zeitraum von vier Jahren erschienen, lenkten die Aufmerksamkeit der Forscher auf Ko- pitars Rolle in Vuks Tätigkeit. Es wurde festgestellt, daß Kopitar sich auch vor seiner Bekanntschaft mit Vuk für Volkspoesie inter- 2 essiert hat und selbst ein Wörterbuch und eine Grammatik der ser- bischen Sprache ausarbeiten wollte3. Vuks erste Werke sind zwei- 4 5 fellos nach Kopitars Plänen und mit seiner Hilfe entstanden . Eine kurze Darstellung von Kopitars Sprachauffassung erleich- te rt den Überblick über seinen Einfluß auf Vuk. 1. Kopitar vertrat die Auffassung von der Notwendigkeit des brauchs der Volkssprache als Schriftsprache:"So lange nicht alle über alles in der Muttersprache zu schreiben anfangen, kann kein 00051004 - 43 - Volk auf Cuitur Anspruch machen."1 Wie Herder gebraucht Kopitar mit Vorliebe den Terminus "Muttersprache". Auch die Formulierung: "Wehe dem Volke, dessen Gelehrte sich schämen in der Mutterspra- che berühmt zu werden*1, weist deutlich auf die Verbindung zu Her- der hin^ • 2. Kopitar verstand unter der Volkssprache die lebendige, ge- sprochene Sprache des Volkes und die der Volkslieder3, die der 4 Grammatiker, wie ein Statistiker, aufnehmen und beschreiben soll . "Wie auch Herder erschien ihm die Sprache aus dem Munde des Volkes und das Sprachkunstschaffen des Volkes als Inkarnation des rein- sten Volksgenies. Deshalb suchte er immer die Bauernsprache und die 'Bauern1 ־oder ' Hajdúkén' -Poesie. Mit dieser Sprach- und Poesieauffassung geriet Kopitar in Konflikt mit Preserœund der jungen slovenischen Dichtergeneration, die ihr Vorbild nicht in der Volkspoesie, sondern in der europäischen Kunstpoesie suchten. 3. Die Volkssprache in der Literatur bedeutet bei Kopitar den Gebrauch aller Dialekte. 1 Vgl. B. Kopitars Kleinere Schriften I, 1857 (im weiteren Text: KKS) , S. 236. ־Eine ähnliche Formulierung finden wir auch bei A. L. Schlözer, Nestor.., 1802, S. 46: "Cultur fängt unter den Völkern nicht eher an, als bis sie in ihren eigenen Sprachen schreiben.. . ” 1. Vuk und Kopitar *1Nach meiner Überzeugung soll jeder Dia- lekt sein volles Recht haben und üben, als wäre er der einzige in seiner Familie."^ Seine Ansichten über die Dialekte sind im Grunde die konsequenteste Verwirklichung von Herders Forderung nach der Rückkehr zur Volkssprache. Hinzu kommt sein sprachwissenschaftli- ches Interesse für die Mundarten als den "Schatz für die Geschieh- te der ... Sprache"^. Die sprachliche Situation bei den Südslaven verglich Kopitar mit derjenigen bei den alten Griechen, "die ihre verschiedenen Dia- lekte nicht nur sprachen, sondern auch schrieben... Weit entfernt 1 Vgl. B. Kopitars Kleinere Schriften I, 1857 (im weiteren Text: KKS) , S. 236. ־Eine ähnliche Formulierung finden wir auch bei A. L. Schlözer, Nestor.., 1802, S. 46: "Cultur fängt unter den Völkern nicht eher an, als bis sie in ihren eigenen Sprachen schreiben.. . ” 2 Vgl. Pis'ma, S. 278. - Herder meinte, daß kein Genie sich seiner Muttersprache schämen oder sich über sie beklagen darf,״weil über haupt für einen jeden vortrefflichen Schriftsteller die Gedanken Söhne des Himmels, die Worte Töchter der Erde sind". - Vgl. Her- der I, S. 240. 3 Vgl. KKS I, S. 262; vgl. Lj. Stojanovic, a.a.O. S. 35. 4 Vgl KKS I S 236 4 Vgl. KKS I, S. 236. 5 Vgl. F. Petrë, Kopitar und Herder. In: Slawisch-deutsche Wechsel- beziehungen, 1969, S. 570. g , 5 Vgl. F. Petrë, Kopitar und Herder. In: Slawisch-deutsche Wechsel- beziehungen, 1969, S. 570. g 6 Vgl. Arkiv za povjestnicu jugoslavensku XII, 1875, S. 96; vgl. auch KKS I, S. 40. Vera Boji - 9783954793044 7 Vgl. KKS I, S. 268; vgl. auch Herder XXV, S. 5. j Downloaded from PubFactory at 01/10/2 7 Vgl. KKS I, S. 268; vgl. auch Herder XXV, S. 5. Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access den Zeitpunct, da, wie am Ende bei den Griechen, ein Schriftdia- lekt alle übrigen verschlingt, vor der Zeit herbei zu wünschen, freut sich der philosophische Sprachfreund des frohnachbarlichen gemeinsamen Lebens aller auch in Schriften..."1 den Zeitpunct, da, wie am Ende bei den Griechen, ein Schriftdia- lekt alle übrigen verschlingt, vor der Zeit herbei zu wünschen, freut sich der philosophische Sprachfreund des frohnachbarlichen gemeinsamen Lebens aller auch in Schriften..."1 4. Kopitar wandte sich entschieden gegen jede Sprachmischung, 2 die er "Macaronismus" nannte . 1. Vuk und Kopitar Er war noch vor der Bekanntschaft mit Vuk über die Sprachsituation bei den Serben unterrichtet3 und beschäftigte sich mit Dositej, der als erster "die slavische todte Büchersprache mit dem lebendigen Dialekt seiner Landsleute ver- 4 tauscht hat" . Er verlangte die Trennung "der alten todten Kirchen- spräche" von der "neuen lebendigen Volkssprache", die "im Munde al- 1er lebenden Serben" zu suchen sei5. Die Kirchensprache, wie auch die lateinische, soll der Gelehrte "zur Ergründung und sogar zur Be- reicherung seiner Muttersprache" studieren, nachdem er in seiner Muttersprache eine "allgemeine Bürgerbildung erhalten hat"6. Kopitars Ausführungen lehnen sich an Herders Gedanken an über die Vorteile der Muttersprache gegenüber der toten Sprache und über die Notwendigkeit, daß der Geist die Bildung in der Muttersprache erhält7. 5. Kopitar war ein enthusiastischer Anhänger der phonetischen Orthographie. Er k ritis ie rte die deutsche und die französische Ortho graphie und meinte, daß die "göttliche Schreibkunst" in den Händen О der Abendländer entstellt sei . Er forderte Schaffung neuer Buchsta- ben an Stelle der Anpassung des lateinischen Alphabets mit Hilfe о der diakritischen und diagraphischen Zeichen . Er wollte D o b r o v s k ÿ überreden, als "lateinischer K y rill" ein einheitliches Alphabet für die lateinisch schreibenden Slaven anzufertigen10. Kopitars ortho- graphische Prinzipien hat Vuk verwirklicht. 5. Kopitar war ein enthusiastischer Anhänger der phonetischen Orthographie. Er k ritis ie rte die deutsche und die französische Ortho graphie und meinte, daß die "göttliche Schreibkunst" in den Händen О der Abendländer entstellt sei . Er forderte Schaffung neuer Buchsta- ben an Stelle der Anpassung des lateinischen Alphabets mit Hilfe о der diakritischen und diagraphischen Zeichen . Er wollte D o b r o v s k ÿ überreden, als "lateinischer K y rill" ein einheitliches Alphabet für die lateinisch schreibenden Slaven anzufertigen10. Kopitars ortho- graphische Prinzipien hat Vuk verwirklicht. 1 Vgl. KKS I, S. 40. 2 Vgl. KKS I, S. 263. 3 Vgl. Lj. Stojanovic, a.a.O., S. 16ff. 4 Vgl. KKS I, S. 120. 5 Vgl. KKS I, S. 262. 6 Vgl. KKS I, S. 262. 7 Vgl. Herder I, S. 400f. 8 Vgl. KKS I, S. 24l f f ., 252, 258. 9 In seinen Briefen an Dobrovsky, Grimm und Vuk benutzte er oft Vuks kyrillische Zeichen an Stelle der angepaßten lateinischen. 10 Vgl. Pis'ma, S. 84. 1 Vgl. KKS I, S. 40. 2 Vgl. KKS I, S. 263. 3 Vgl. Lj. Stojanovic, a.a.O., S. 1 Vgl. dazu S. 150f. dieser Arbeit. 2 Vgl. Jerneja Kopitarją spisov I I . del.., 1. knjiga, 1944 (im wei- teren Text: Spisi), S. 201• 3 Vgl. Pis'ma, S• 237; vgl• auch KKS I, S. 23f. - Eigentlich hat zu- erst Dobrovskÿ das Kroatische und Slovenische als eine Sprache in Verbindung gebracht, indem er den ״illyrischen Dialekt" von dem "kroatischen" trennte und dem letzteren auch den "Windischen in Steyermark, Kärnten, Krain" zuwies. - Vgl. DobrovskJ, Geschichte der böhmischen Sprache. In Spisy a projevi, Bd. VII, 1936, S. 5. 4 Vgl• Spisi II, 1, S. 201f . 5 Vgl• ebd., S. 202. 6 Bereits A. Linhart, Versuch einer Geschichte von Krain, Kärnten ui Steyermark, II , 1791, Vorr. S.(15-17) hatte den illyrischen Namen abgelehnt, weil die Illy r ie r keine Slaven waren. 7 Vgl. Spisi II, 1, S. 202. 8 Vql. ebd. S. 6. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1. Vuk und Kopitar 16ff. 4 Vgl. KKS I, S. 120. 5 Vgl. KKS I, S. 262. 6 Vgl. KKS I, S. 262. 7 Vgl. Herder I, S. 400f. 8 Vgl. KKS I, S. 24l f f ., 252, 258. 9 In seinen Briefen an Dobrovsky, Grimm und Vuk benutzte er oft Vuks kyrillische Zeichen an Stelle der angepaßten lateinischen. 10 Vgl. Pis'ma, S. 84. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 6• Kopitars Einteilung der südslavischen Sprachen hat einen Einfluß auf Vuks Ansichten über die serbischen und kroatischen Dia- lekte und auf Grimms Verwendung des Terminus "serbisch" ausgeübt1- Kopitar hat seine seit 1810 o ft geäußerten Ansichten in der Rezen- sion von Dobrovskÿs "Institutiones" von 1822 zusammengefaßt• Er te ilte das südslavische Sprachgebiet in: "Bulgarisch, Serbisch 2 (Illyrisch), und Slowenisch" ein . Seine Einteilung, in der, entge- gen derjenigen von Schlözer und Dobrovskÿ, das Kroatische fehlte, begründete er gegenüber Dobrovsk$ so: "Croata est qui kaj habet pro što, ergo Croata = Vinidus."3 Kopitar nahm das Pronomen "kaj" als das Kriterium für seine Zuweisung des kroatischen kajkavischen Dia- lekts zur slovenischen Sprache, die er gemäß seiner "pannonischen" Theorie für die Nachfolgerin der altkirchenslavischen hielt• "Das wahre Kroatien is t nach aller Geschichte nur im Süden der Kulp, un- bestimmt wie tie f hinein in Boßnien, Dalmatien und Istrien zu su- 4 chen." Kopitars Bestimmung der Kroaten hat Vuk später in seiner Gleichsetzung des Kroatischen mit dem Cakavischen übernommen. Kopitar wollte für Kroaten und Serben, deren Sprache er als "zwey sehr nahe Varietäten einer und derselben Spezies"5 sah, den serbischen Namen an Stelle des illyrischen6 einführen. Er war sich des Problems im Zusammenhang mit dieser Benennung bewußt, da er meinte, daß man auch Kroatisch für Serbisch sagen könnte und daß man am Ende doch das "üblich gewesene Illyrisch wieder aufneh- men" müsse7. Er selbst bevorzugte und gebrauchte den Terminus "ser- bisch"®. Kopitars Ansichten über die südslavischen Sprachen sind von der Wissenschaft längst überholt worden. 5 Vgl ebd., S. 202. 6 Bereits A. Linhart, Versuch einer Geschichte von Krain, Kärnten ui Steyermark, II , 1791, Vorr. S.(15-17) hatte den illyrischen Namen abgelehnt, weil die Illy r ie r keine Slaven waren. 7 Vgl. Spisi II, 1, S. 202. 8 Vql. ebd. S. 6. 1 Vgl. F. Petrfe, Zojs, Kopitar i Karadzic, a.a.O., S. 228 2 Kopitars Vorstellung von der slavischen Einheit innerhalb öster- reichs verband ihn mit dem Kreis von Baron Zois. - Vgl. Petrê, Kopitar und Herder, a.a.O, S. 568. 3 Vgl. M. Vasmer, Kopitars Briefwechsel mit Jakob Grimm, S. XIII. 4 Vgl. A. Slodnjak, Vuk in slovensko slovstvo. In: Vukov zbornik, 1966, S. 217. 5 Vgl. ebd. S. 213; vgl. M. Popoviò, Vuk Stef. Karadziè 1787 - 1864 1964, bes. S. 71ff., 253f., 307f. 6 Vgl. V. Novak, Vuk i Hrvati, 1967, bes. S. 297ff., 393ff.; vgl. M. živančevic, Vukovi p rija te lji il i r c i . In: Vukov zbornik, 1966, S. 229 - 258. Vera Boji - 9783954793044 1. Vuk und Kopitar Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Es is t bereits bemerkt worden, daß in der damaligen Wissenschaft unklare Vorstellungen über die südslavischen Sprachen herrschten1. In Kopitars Ansichten über die Sprache wirken auch nationale und לpolitische Beweggründe. Sein Austroslavismus brachte ihn in Gegen- satz sowohl zu den čechen3,als auch zu der illyrischen Bewegung 4 bei den Kroaten , in deren A ktivität er eine Bedrohung des öster- reichischen Staates sah. Er bewertete Vuks Bemühungen um die serbi- sehe Sprache als nützlich für die slavische Einigung innerhalb Österreichs, weil sie eine Lösung von Rußland einleiteten5. Vuk ist ihm darin nicht gefolgt, indem er eine fruchtbare Zusammenarbeit mit den Illyrern entwickelte . Kopitars Bedeutung für Vuk lieg t vor allem in seinem Einfluß auf die Entwicklung von Vuks Ansichten über die Schriftsprache. Vuks Weg von der slavenoserbischen Sprache zur reinen Volkssprache war in erster Linie Kopitars Verdienst. Das is t insbesondere in der An- fangsphase deutlich sichtbar, als Vuk zwischen dem "mittleren S til" seiner serbischen Zeitgenossen und Kopitars Forderung nach der Spra- che des einfachen Volkes schwankt. Kopitar hat zur Festigung von Vuks Ansichten über die Volkssprache als Schriftsprache wesentlich beigetragen. Vuk übernahm eine Zeitlang auch Kopitars Vorstellungen von der Pflege aller Dialekte in der Literatur. In der Endphase entfernte sich Vuk von Kopitar und entwickelte eigene Ansichten über die Schriftsprache. Kopitar war außerdem der wichtigste Vermittler zwischen Vuk und dem westeuropäischen Gedankengut. Die Idee von der Volksspra- che in der Literatur hatte bekanntlich in Herder ihren bekanntesten Fürsprecher. Zweifellos hat Kopitars Berufung auf Herders Autori- tät eine große Wirkung auf Vuk ausgeübt. - 47 - 1 Vgl. F. Petrê, Kopitar und Herder, a.a.O. 2 Vuk war während der Vorbereitung des Wörterbuchs bei Mušicki. In Mušickis Briefen aus dieser Zeit sind viele Anklänge an Herders Sprachauffassung enthalten. - Vgl. Prepiska I I , S. 147ff. 3 Welche Werke Herders Vuk gelesen hat, kann nicht festgestellt wer den; in Vuks Briefwechsel und bei V. Nedic, Biblioteka Vuka Kara- dzića, in: Bibliotekar 4 (1954), 1 - 2 (1955), wird keine Schrift Herders genannt. (Uber das Schicksal von Vuks Nachlaß vgl. Nedic, ebd.). Es kann jedoch angenommen werden, daß Vuk, der von den serbischen Schriftstellern Rückkehr zur Sprache des einfachen Voi- kes verlangte, Herder gelesen hat. Vuk erwähnt ”Njemačke Pjesna- rice", bei denen es sich um Herders Liederausgaben handeln könn- te; - vgl. Sabrana dela (im weiteren Text: SD) I, 1965, S. 38. Er fordert Musicki auf, entsprechend den Vorstellungen von Her- der zu dichten; - vgl. Prepiska II, S. 57. 4 Vgl. Herder V, S. 100f. 5 Vgl Herder II S 12f 2. Vuk und Herder 2. Vuk und Herder In Vuks theoretischen Äußerungen und in seiner Konzeption der Schriftsprache sind viele Verbindungen zu Herders Gedanken vor- handen• Kopitar, der ein enthusiastischer Anhänger von Herder war1, 2 3 aber auch Musicki , haben Vuk mit Herder bekannt gemacht . Durch Herders Einfluß entstehen einerseits Gemeinsamkeiten, andererseits Unterschiede zwischen Vuks und Grimms Ansichten. Um Vuks Verbin- dung zu Herder zu veranschaulichen, werden im folgenden ihre Gedan- ken einander gegenübergestellt. Herder hat bekanntlich den Ursprung der Sprache durch die Denk- fähigkeit des Menschen erklärt: "...der Mensch... spricht, indem er denkt... Und indem er nun immer so fortdenkt", lernt er "beßer den- 4 ken, mithin auch beßer sprechen" . Er nannte die Sprache eine "Schatzkammer" der "sichtbar gewordenen Gedanken" eines Volkes, das "Werkzeug" und die "Form" der Literatur und der Wissenschaften5 Wie Herder sieht auch Vuk Sprache und Denken in engem Zusammen- hang:"Mi misiimo, da su samo misli proste, a da jezik ne može b iti prost..." (GPS I, 165; 1817). "... kad se počelo ljepše m is liti, onda su i jezici ljepši postali." (GPS II, 7; 1818). Die Sprache ist "jedino sredstvo, kojim lakše, izvjestnije i skorije к našemu obščem prosveščeniju i nravstvenom usoveršeniju doci možemo•"6 (GPS I, 93; 1816). Er wollte sein Wörterbuch von 1818 "Sokrovišče misli roda” nennen (Prepiska II, 137). 1 Vgl. F. Petrê, Kopitar und Herder, a.a.O. 2 Vuk war während der Vorbereitung des Wörterbuchs bei Mušicki. In Mušickis Briefen aus dieser Zeit sind viele Anklänge an Herders Sprachauffassung enthalten. - Vgl. Prepiska I I , S. 147ff. 3 Welche Werke Herders Vuk gelesen hat, kann nicht festgestellt wer den; in Vuks Briefwechsel und bei V. Nedic, Biblioteka Vuka Kara- dzića, in: Bibliotekar 4 (1954), 1 - 2 (1955), wird keine Schrift Herders genannt. (Uber das Schicksal von Vuks Nachlaß vgl. Nedic, ebd.). Es kann jedoch angenommen werden, daß Vuk, der von den serbischen Schriftstellern Rückkehr zur Sprache des einfachen Voi- kes verlangte, Herder gelesen hat. Vuk erwähnt ”Njemačke Pjesna- rice", bei denen es sich um Herders Liederausgaben handeln könn- te; - vgl. Sabrana dela (im weiteren Text: SD) I, 1965, S. 38. Er fordert Musicki auf, entsprechend den Vorstellungen von Her- der zu dichten; - vgl. Prepiska II, S. 57. 4 Vgl. Herder V, S. 100f. 2. Vuk und Herder 5 V l H d II S 12f g , 6 Da es hier nicht um das Problem der Genese von Vuks Orthographie Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 6 Da es hier nicht um das Problem der Genese von Vuks Vera Boji Herder hat die Sprache als Denkart eines Volkes angesehen: "Je- de Nation spricht also, nach dem sie denkt, und denkt, nach dem sie spricht. So verschieden der Gesichtspunkt war,in dem sie die Sac nahm, bezeichnete sie dieselbe."1 Auch Vuk sieht die Eigenart der Sprache durch die Denkart des Volkes geprägt: "... svi narodi ne počinju s jedne strane m is liti о stvarma; i otud je postala ona razlika izmēdu jezika, što se zove svojstvo jezika." (GPS II, 6; 1818). Herder sieht die Sprache als den Ausdruck des Volkscharakters an: "... in jede derselben ist der Verstand eines Volks und sein 2 Charakter gepräget." "Kein größerer Schade kann einer Nation zu- gefügt werden, als wenn man ihr den Nationalcharakter, die Eigen ־ heit ihres Geistes und ihrer Sprache raubt."3 Für Vuk is t die Existenz eines Volkes an seine Sprache gebunden: "Jezik je hranitelj naroda. Dokle god živi je z ik ... dotie živi i narod: moie se medu sobom razumijevati i umno sojedinjavati; ne preliva se u drugi, ne propada. ־Koliko se vecma samim djelom svi о jeziku narodnom staraino, toliko smo dalje protegnuli nravstveni život i vječnost našeg roda i ci jelog potomstva." Er nennt die Spra- che "najsvetija narodnost" (GPS I, 94; 1816). In Herders Bestimmung des Volkes steht die Sprache an erster Stelle. Die Sprache is t der Ausdruck der "Denk- und Lebensart" ei- nes Volkes; "Clima, Luft und Waßer, Speise und Trank, werden... auf die Sprache einfließen." "In diesem Gesichtspunkt wie groß 4 wird die Sprache!" Gebräuche und Religion nennt er die "heilig- ste Tradition"5. Die Sprache is t für Herder das Hauptmerkmal eines Volkes, einer Nation, die er als die natürliche Grundlage eines Staates sieht6: "der natürlichste Staat is t also auch Ein Volk, mit Einem Nationalcharakter"7. geht, werden alle Zitate seiner endgültigen Orthographie angepaßt. Seine Großschreibung und die morphologisch nicht "serbisierten" Russismen werden beibehalten. Seine Großschreibung und die morphologisch nicht "serbisierten" Russismen werden beibehalten. 1 Vgl. Herder II, S. 18, auch S. 25. - Herder trennt die Begriffe Volk und Nation nicht. 2 Vgl. Herder XIII, S. 363. 3 Vgl. Herder I, S. 1 Vgl. Herder I, S. 376. 2 Vgl. ebd. S. 378. 3 Vgl.Herder I, S. 189, 216; II, S. 10. 4 In Herders Sprach* und Poesieauffassung wirkt Rousseaus Naturbe- g riff und Hamanns Auffassung der Poesie als der Muttersprache des Menschengeschlechts nach. - Vgl. darüber B. von Wiese, Herder Grundzüge seines Weltbildes, 1939, S. 22f. 5 Vgl. Herder XXV, S. 5. 6 Vgl. Herder II, S. 13, 57; XXV, S. 12: "...singt die Gegenstände unsrer Zeit uns so natürlich... als diese Volkslieder es sangen.. 7 Vgl. Herder I, S. 302. 8 Vuk gebraucht am Anfang die Termini "Nacionalizmus" und "Nationa- litaet" neben "narodnost"; vgl. SD I, S. 361. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 2. Vuk und Herder 366. 4 Vgl. Herder V, S. 125, 136. 5 Vgl. Herder XIII, S. 387f. 6 Vgl. Herder XIII, S. 361ff. ־Vgl. dazu G. Konrad, Herders Sprach- problem im Zusammenhang der Geistesgeschichte, 1937, S. 51f. 7 Vgl. Herder XIII, S. 384. ־Vgl. dazu F. Barnard, Zwischen Auf- klärung und politischer Romantik,1964 ,׳S. 74. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vuk bezeichnete Sprache, Religion und Sitten als heiliges Ei- gentum eines Volkes, wodurch es von den anderen Völkern unterschie- den wird: "Kod svakoga su naroda najsvetije ove t r i stvari: Zakon, jezik i obicaji: tim se narodi jedan s drugim roäakaju, i jedan od drugog razlikuju. Kako naród izgubi te t r i svetinje, on izgubi i svoje ime." (GPS I, 130; 1817). Herders Gedanken spiegeln sich auch in Vuks Konzeption der Schriftsprache wider. Herder klagte in seinen Betrachtungen über die deutsche Literatur über die "mit fremden Zierrathen überlade- nie]", "ermattete Schreibart"1. Die deutsche Sprache habe durch 2 Einfluß der lateinischen ihren Nationalcharakter verloren . Die Sprachreiniger, die "ihre Denkart nach dem Latein gebildet" haben und die deutsche Sprache durch Nachahmung der griechischen, franzö- sischen und englischen verbessern wollen, nannte er "Sprachverder- ber"3. Er forderte die Rückkehr zur Volkssprache, in der er das Ursprüngliche bewahrt sah, und hob die Bedeutung der Volkspoesie 4 hervor . Die Volkslieder nannte er einen "Schatz fü r... Sprache, Dichtkunst, Sitten, Denkart..."5 "Schriftsteller der Nation" soll- ten "in die Goldgrube der Sprache herab steigen..."6 Der Geist eines Volkes sei nicht unter "Gelehrten und Leuten von vornehmer Erziehung zu suchen. Diese beiden Geschlechter gehören gleichsam keinem Lande eigen. Aber unter dem Theile der Nation liegen sie, der von fremden Sitten und Gebräuchen und Kentnissen noch nichts zur Nachahmung sich bekannt gemacht hat."7 Diese Gedanken Herders gehören zum festen Bestandteil von Vuks Ansichten über die Schriftsprache und die Poesie. Er meinte bereits 1814, daß die serbischen Gebildeten durch fremde Einflüsse D ihren "Nacionalizmus" verloren haben und daß "na tudem jeziku nauke slušati i nauőiti se... nemoSese nazvati postupkom našega 1 Vgl. Herder I, S. 376. 2 Vgl. ebd. S. 378. 3 Vgl.Herder I, S. 189, 216; II, S. 10. 4 In Herders Sprach* und Poesieauffassung wirkt Rousseaus Naturbe- g riff und Hamanns Auffassung der Poesie als der Muttersprache des Menschengeschlechts nach. - Vgl. darüber B. von Wiese, Herder Grundzüge seines Weltbildes, 1939, S. 22f. 5 Vgl. Herder XXV, S. 5. 2. Vuk und Herder 6 Vgl. Herder II, S. 13, 57; XXV, S. 12: "...singt die Gegenstände unsrer Zeit uns so natürlich... als diese Volkslieder es sangen.. 7 Vgl. Herder I, S. 302. 8 Vuk gebraucht am Anfang die Termini "Nacionalizmus" und "Nationa- litaet" neben "narodnost"; vgl. SD I, S. 361. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. Herder I, S. 376. 2 Vgl. ebd. S. 378. 3 Vgl.Herder I, S. 189, 216; II, S. 10. 4 In Herders Sprach* und Poesieauffassung wirkt Rousseaus Naturbe- g riff und Hamanns Auffassung der Poesie als der Muttersprache des Menschengeschlechts nach. - Vgl. darüber B. von Wiese, Herder Grundzüge seines Weltbildes, 1939, S. 22f. 5 Vgl. Herder XXV, S. 5. 6 Vgl. Herder II, S. 13, 57; XXV, S. 12: "...singt die Gegenstände unsrer Zeit uns so natürlich... als diese Volkslieder es sangen.. 7 Vgl. Herder I, S. 302. 8 Vuk gebraucht am Anfang die Termini "Nacionalizmus" und "Nationa- litaet" neben "narodnost"; vgl. SD I, S. 361. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 8 Vuk gebraucht am Anfang die Termini "Nacionalizmus" und "Nationa- litaet" neben "narodnost"; vgl. SD I, S. 361. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access naroda и prosveščeniju." (SD I, S. 40). Er empfahl den serbischen Schriftstellern die Volkslieder zum Vorbild, '1koje je serdce и pro- sto ti i и nevinosti bezhudožno po prirodi spevavalo"; 1 denn, die serbische Dichtung soll "po vkusu i načinu roda svoga" geschaffen werden (SD I, 42f.). Er nannte die Volkslieder die wichtigste "Na- tion alitae t", "erbo one soderžavaju и sebi narodni jezik, harakter i običaje" (SD I, 361). naroda и prosveščeniju." (SD I, S. 40). Er empfahl den serbischen Schriftstellern die Volkslieder zum Vorbild, '1koje je serdce и pro- sto ti i и nevinosti bezhudožno po prirodi spevavalo"; 1 denn, die serbische Dichtung soll "po vkusu i načinu roda svoga" geschaffen werden (SD I, 42f.). Er nannte die Volkslieder die wichtigste "Na- tion alitae t", "erbo one soderžavaju и sebi narodni jezik, harakter i običaje" (SD I, 361). Herders Einfluß auf Vuk is t besonders zu Beginn der zwanziger Jahre deutlich erkennbar. Vuk erhebt die Sprache der Bauern, die weit entfernt von den Städten und den fremden Einflüssen leben, zum Vorbild für die Schriftsprache. 1 In Vuks S til in der Anfangsphase is t der Einfluß des lite ra ri- sehen Sentimentalismus spürbar: seine Charakterisierung der Volksdichtung als der natürlichen Poesie des einfachen, unschuldi- gen Herzens steht im Einklang mit der unter Rousseaus Einfluß entstandenen Verherrlichung des Natürlichen und Kunstlosen (bez- hudožno) . Als Feier des Lebens in der Natur war u.a. auch Schäfer- dichtung beliebt. Vuk s te llt seiner Liederausgabe von 1814 eine Abbildung ("Serbska юта") voran, die eine Schäferidylle darstellt umgeben von prächtiger, unberührter Natur wirbt ein Flöte spielen- der Jüngling um das Herz des serbischen Mädchens. Die klischee- hafte Szene der Schäferliebe widerspricht eigentlich den patri- archalischen Sitten der damaligen serbischen Gesellschaft. - Uber Vuks Kenntnis der Literatur des Sentimentalismus vgl. D. Zivkovic, Odjeci sentimentalizma и srpskoj književnosti XVIII i prve poiovine XIX veka; ders., Silerovska i herderovska koncepci- ja poezije i Vukova borba za narodni jezik. In: ders., Evropski okviri srpske književnosti, 1970, S. 79-104, 119-146. - Z. sieht Vuks Auseinandersetzung mit den serbischen Schriftstelltem als Konflikt zwischen zwei Poesieauffassungen: der vereinfacuten Schillerischen bei Vuks Gegnern und der von Vuk vertretenen Herderischen. 2. Vuk und Herder Anders als bei Herder erscheinen als Sprachverderber die Schriftsteller, weil sie nicht serbisch denken: "••. ne misle Srpski, nego Njemački i l i Latinski" (GPS I I I , 154)1. (GPS II! 103, 1820)• Wie Herder sieht Vuk in den einfachen Menschen den Träger des Volkscharakters. Anders als bei Herder erscheinen als Sprachverderber die Schriftsteller, weil sie nicht serbisch denken: "••. ne misle Srpski, nego Njemački i l i Latinski" (GPS I I I , 154)1. Zitat verwendet; Vuk erklärt, daß er damit nicht etwa nur die un- gebildeten Bauern meine, sondern alle, "koji stvari upravo ne razumiju", seien sie auch vermögend und einflußreich (GPS 11,96). Er bezeichnet so oft diejenigen, deren Sprache im Vergleich mit seiner Norm"verdorben" is t. - In der hier zitierten Stelle ent- hält dieser Begriff, wie bei Herder, auch eine antiaufklärerische Komponente. Herder sprach von dem "europäischen Pöbel" der Auf- klärung und von der "gelehrten Maschine", denen er den Natur- menschen engegensetzte. - Vgl. Herder X III, S. 371f. 1 Vgl darüber S 55ff dieser Arbeit 2. Vuk und Herder Er fordert Mušicki auf, in der Volkssprache zu schreiben, so wie es " E r d e r u i ostalima onakovim" gefallen würde, "da ovu epohu utvrdimo i temelj knjižestvu da po- stavimo". (Prepiska II, 57, 1820; 243, 1819). In Vuks Auffassung, daß nur ungebildete Menschen neue Wörter schaffen können (GPS II, 26, 181), wirkt der Rousseauisch-Herderische Begriff des Natur- menschen nach. Die geistige Nähe zu Herder äußert sich auch in Vuks Distanzierung von Dositejs aufklärerischen Gedanken: "...pro- sto ti se vrlo dopao Dositej vičuci na kaludere i na namastire, na 2 post i na mošti svete, i na druge dekoje običaje crkve naSe..." 1 In Vuks S til in der Anfangsphase is t der Einfluß des lite ra ri- sehen Sentimentalismus spürbar: seine Charakterisierung der Volksdichtung als der natürlichen Poesie des einfachen, unschuldi- gen Herzens steht im Einklang mit der unter Rousseaus Einfluß entstandenen Verherrlichung des Natürlichen und Kunstlosen (bez- hudožno) . Als Feier des Lebens in der Natur war u.a. auch Schäfer- dichtung beliebt. Vuk s te llt seiner Liederausgabe von 1814 eine Abbildung ("Serbska юта") voran, die eine Schäferidylle darstellt umgeben von prächtiger, unberührter Natur wirbt ein Flöte spielen- der Jüngling um das Herz des serbischen Mädchens. Die klischee- hafte Szene der Schäferliebe widerspricht eigentlich den patri- archalischen Sitten der damaligen serbischen Gesellschaft. - Uber Vuks Kenntnis der Literatur des Sentimentalismus vgl. D. Zivkovic, Odjeci sentimentalizma и srpskoj književnosti XVIII i prve poiovine XIX veka; ders., Silerovska i herderovska koncepci- ja poezije i Vukova borba za narodni jezik. In: ders., Evropski okviri srpske književnosti, 1970, S. 79-104, 119-146. - Z. sieht Vuks Auseinandersetzung mit den serbischen Schriftstelltem als Konflikt zwischen zwei Poesieauffassungen: der vereinfacuten Schillerischen bei Vuks Gegnern und der von Vuk vertretenen Herderischen. 2 2 Der viel z itie rte Begriff p r o s t o t a is t Vuks Übersetzung des deut- sehen Wortes P ö b e l , die er zum erstenmal 1820 in einem Wieland- 2 Der viel z itie rte Begriff p r o s t o t a is t Vuks Übersetzung des deut- sehen Wortes P ö b e l , die er zum erstenmal 1820 in einem Wieland- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access (GPS II! 103, 1820)• Wie Herder sieht Vuk in den einfachen Menschen den Träger des Volkscharakters. 2 Vgl. Kopitar über "Mithridates" in: Pis'ma, S. 94ff. 3 Vgl. J. Ch. Adelung, Umständliches Lehrgebäude der deutschen Spra- che.., 1782, Bd. II, S. 618. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Zitat verwendet; Vuk erklärt, daß er damit nicht etwa nur die un- gebildeten Bauern meine, sondern alle, "koji stvari upravo ne razumiju", seien sie auch vermögend und einflußreich (GPS 11,96). Er bezeichnet so oft diejenigen, deren Sprache im Vergleich mit seiner Norm"verdorben" is t. - In der hier zitierten Stelle ent- hält dieser Begriff, wie bei Herder, auch eine antiaufklärerische Komponente. Herder sprach von dem "europäischen Pöbel" der Auf- klärung und von der "gelehrten Maschine", denen er den Natur- menschen engegensetzte. - Vgl. Herder X III, S. 371f. 1 Vgl. darüber S. 55ff. dieser Arbeit. 2 Vgl. Kopitar über "Mithridates" in: Pis'ma, S. 94ff. 3 Vgl. J. Ch. Adelung, Umständliches Lehrgebäude der deutschen Spra- che.., 1782, Bd. II, S. 618. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 3. Vuk und J. Ch. Adelung Vuk hat bereits in seiner "Pismenica" den Namen von Adelung er- wähnt (GPS I, 6) und sich auch später oft auf ihn berufen. Hinter Vuks Heranziehung von Adelung steht zweifellos Kopitar. Das zeigt sich besonders in der "Ljubomir"-Rezension von 1817, wo zur Illu - stration der Willkürlichkeit der slavenoserbischen Sprache ein "Gotho-Deutsch" anhand mehrerer Vaterunser aus Adelungs "Mithrida- 2 tes" konstruiert wird (GPS I, 158f.) In Vuks Schriften is t aber auch sonst eine Beziehung zu den Gedanken von Adelung feststellbar. Sie zeigt allerdings eine eigenartige, Vukische Interpretation von Adelung. Wir möchten zunächst Adelungs Sprachauffassung in denjeni- gen Punkten, die für Vuk von Bedeutung sind, kurz erläutern. Adelung hat in der Geschichte der deutschen Sprache eine wich- tige Rolle gespielt, vor allem in bezug auf die Festigung der schriftsprachlichen Norm. In seiner Grammatik hat Adelung u.a. sei- ne allgemeinen Überlegungen über die Sprache und die Schrift ge- äußert, die "vermittels weniger Zeichen den ganzen Reichtum der Sprache sichtbar darstellen kann". In beiden, Sprache und Schrift, sei "etwas Wunderbares und Vortreffliches, welches viele geblendet und zu dem Satze verleitet hat, daß sowohl die Sprache als Schrift das Werk eines höhern Wesens seyn müsse..." Er selbst lehnte den göttlichen Ursprung der Sprache und der Schrift ab und betrachtete 2 Vgl. Kopitar über Mithridates in: Pis ma, S. 94ff. 3 Vgl. J. Ch. Adelung, Umständliches Lehrgebäude der deutschen Spra- che.., 1782, Bd. II, S. 618. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access sie als das Mittel, die Gedanken auszudrücken- Ihre Absicht sei, "verstanden zu werden". Deshalb stellte er "das allgemeine Grundge- setz der Schrift aller Sprachen: schreib wie du sprichst..." auf1. Das bedeutete bei Adelung, daß die Orthographie "... so leicht als 2 nur möglich..." sein sollte . sie als das Mittel, die Gedanken auszudrücken- Ihre Absicht sei, "verstanden zu werden". Deshalb stellte er "das allgemeine Grundge- setz der Schrift aller Sprachen: schreib wie du sprichst..." auf1. Das bedeutete bei Adelung, daß die Orthographie "... so leicht als 2 nur möglich..." sein sollte . Adelung setzte sich auch mit der Grammatikkonzepzion nach dem griechisch-lateinischen Vorbild auseinander. Er meinte, daß die Sprachregeln "... in jeder Sprache selbst aufgesucht werden" müssen. Der Grammatiker sei kein "...Gesetzgeber der Nation, sondern nur der Sammler und Herausgeber der von ihr gemachten Gesetze... 1 Vgl. ebd., S. 64 2 2 Vgl. ebd. S. 641. 3 Vgl. ebd., Bd. I, S. 112f. 4 Vgl. Herder II, S. 40. - Herders K ritik bezog sich auf Gottsche- dianer, mit denen Adelung in diesem Punkt übereinstimmt. 5 Vgl. GKS VIII, S. 337.- Grimm führte zu Adelungs Definition von L ie b c h e n "ein nur noch in den niedrigen sprecharten übliches wort eine geliebte person zu bezeichnen, welche man auszer der ehe liebet",als Gegenargument die Verse von Hagedorn und Goethe, wo das Wort vorkommt. 6 Vgl KKS I S 314; Spisi II 1 S 20 3. Vuk und J. Ch. Adelung Er s te llt die Sprache so dar, wie sie wirklich is t, nicht wie sie seyn könnte..." In der Sprache gebe es aber Fehler, die der Grammatiker verbessern muß (z.B. e r e i g n e n , Ь е і т ѣ, richtig sei: e r ä u g n e n , Lehm) . Adelung te ilte die Sprache in eine erhabene und edle und eine niedrige und pöbelhafte; in seiner Norm hat er eine Grenze gegen- 4 über der letzteren gezogen. Herders scharfe Kritik , die J. Grimm später wiederholt und dokumentiert hat5, hat bewirkt, daß Adelungs Norm auf dem lexikalischen Gebiet durchbrochen wurde. Schon diese kurze Darstellung zeigt den wesentlichen Unterschied zwischen Adelungs und Vuks Ansichten in Bezug auf die Volkssprache. Adelungs Gedanken sind bei Vuk dennoch produktiv geworden, und zwar in seiner Grammatikkonzeption und der Orthographie. Kopitar hat in seinen Rezensionen von Vuks "Pismenica" und Wör- terbuch Adelungs Definition der Rolle des Grammatikers wiederholt6. Auch Vuk hat diesen Gedanken übernommen: "Grammatik je kakogoä istorik svoga vremena, i l i štatistik, koji opisuje stvari onako kakve su... (ne dajuci zakona nikome)."7 Aus diesem Grundsatz ent- wickelte Vuk zunächst ein methodisches Verfahren, das positive 1 Vgl. ebd., S. 64 2 2 Vgl. ebd. S. 641. 3 Vgl. ebd., Bd. I, S. 112f. 4 Vgl. Herder II, S. 40. - Herders K ritik bezog sich auf Gottsche- dianer, mit denen Adelung in diesem Punkt übereinstimmt. 5 Vgl. GKS VIII, S. 337.- Grimm führte zu Adelungs Definition von L ie b c h e n "ein nur noch in den niedrigen sprecharten übliches wort eine geliebte person zu bezeichnen, welche man auszer der ehe liebet",als Gegenargument die Verse von Hagedorn und Goethe, wo das Wort vorkommt. 6 Vgl. KKS I, S. 314; Spisi II, 1, S. 20. 7 Vgl. Vuks Rez. des "Glas narodoljubca", Archiv SANU 8552/14. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access g , ; p , , 7 Vgl. Vuks Rez. des "Glas narodoljubca", Archiv SANU 8552/14. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM i f 7 Vgl. Vuks Rez. des "Glas narodoljubca", Archiv SANU 8 Vera Boji - 97 Downloaded from PubFactory at 01/10/2019 Früchte getragen hat: die Loslösung von dem traditionellen Gramma- tiksystem, von dem seine"Pismenica" abhängig war, und die synchro- nische Beschreibung der serbischen Sprache, d.h. eines ihrer Dia- lekte. 1 Dieser Gedanke ist ein Topos seit der Renaissance. ־Vgl. A. Borst, Der Turmbau von Babel 1 - 4 , 1957 - 1963, Bd. 4, S. 1952. - Bei Herder kommt er in der Verbindung mit seiner Ansicht von der Unzulänglichkeit der Schrift, alle menschlichen Laute zu erfassen, vor. - Vgl. Herder XIII, S. 366. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. P. P. Bordevic, Vorrede, in: GPS I, S. V if. 2 Vgl. A. Belic, Vukova borba, a.a.O. S. 96. I I I . Die Entwicklung von Vuks Ansichten über die Schriftsprache I I I . Die Entwicklung von Vuks Ansichten über die Schriftsprache I I I . Die Entwicklung von Vuks Ansichten über die Schriftsprache Vuks Ansichten über die einzelnen Probleme der Schriftsprache machen im Verlaufe der Zeit eine Entwicklung durch. In der For- schung is t eine Einteilung seiner Arbeit in drei Perioden: 1814 ־ 1817, 1818 - 1835, 1836 1864 ,־üblich geworden1, die im allge- meinen auch als Entwicklungsphasen seiner Sprachkonzeption erschei- nen. Im Mittelpunkt seiner Tätigkeit stand die Schaffung der schrift- sprachlichen Norm. Das bedeutete im Hinblick auf die Sprachsitua- tion bei den Serben zunächst die Bestimmung der sprachlichen Grund- läge für die Schriftsprache, sowohl in bezug auf die Dialekte als auch auf die kirchensprachlichen Elemente in der damaligen slave- noserbischen Literatursprache, Bei Vuks Lösung dieser Probleme spielen seine Ansichten über die Funktion der Schriftsteller bei der Sprachbildung eine wichtige Rolle; sie sind für seine Sprach- auffassung aufschlußreich. 3. Vuk und J. Ch. Adelung Sein zweiter Schritt, die Schaffung der Norm für einen synthetischen Dialekt, läßt ihn dagegen als den Gesetzgeber der Sprache erscheinen. Die Schriftsprache ist aber ohne einen Kodex auch nicht denkbar. Vuk beruft sich auf Adelung auch in seinem Kampf um die Aner- kennung der Volkssprache. Seine Forderung an die Dichter, sich " ... po cistom i nepokvarenom govoru naroda Srbskoga..." zu rieh- ten, unterstrich er mit der Berufung auf Adelung, dessen Gramma- tik und Wörterbuch "...iz naroda Njemačkog skupljena pravila о njemačkom jeziku..." seien (GPS I, 82). Das ist die Vukische Inter pretation von Adelung, die auf Adelungs Ansichten und Praxis nicht z u tr ifft. Vuk hat das Adelungische Motto: "Schreibe, wie du sprichst" zum Prinzip seiner Orthographie erhoben. Die Forderung, daß die Orthographie leicht sein soll, hat er, Adelung dagegen nicht in der Praxis verwirklicht. Vermutlich hat Vuk auch folgende Ansich- ten über die Schrift von Adelung übernommen1: "Sto su god ljudi na ovome svijetu izm islili, ništa se ne može isporediti s pismom., to je nauka, koja um ljucki gotovo prevazilazi, i mogio bi se reći, da je onaj, koji je prvi nju izmislio, bio vise Bog, nego čovek. Pismo je otvorilo put umu ljuckome, da se p rib liž i к Води po mogucstvu svome." (GPS II, 281). 1 Vgl. P. P. Bordevic, Vorrede, in: GPS I, S. V if. 2 Vgl A Belic Vukova borba a a O S 96 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1. Die Rolle der Dichter und des Volkes bei der Bildung der Schriftsprache 1. Die Rolle der Dichter und des Volkes bei der Bildung der Schriftsprache A. Belic hat bereits bemerkt, daß Vuk keine Gelegenheit ver- säumt hat, die Schriftsteller anzugreifen, als wären sie seine 2 Hauptgegner . Viele von ihnen gehörten auch zu seinen Gegnern, sei es wegen der Orthographie, oder wegen seiner Konzeption der Schriftsprache. Die Tatsache steht fest, daß Vuk kein dichter!- sches Kunstwerk herausgegeben hat, obwohl auch vor ihm in der Volkssprache geschrieben wurde. Vuks Ansichten über die Rolle des Volkes und der Schriftstel- 1er bei der Bildung der Schriftsprache möchten wir im Zusammen- hang mit zwei Hauptproblemen betrachten, zu denen wir auch Jacob Grimms Auffassung gesehen haben, die aber in Vuks Tätigkeit be- sonders aktuell waren. Das sind: Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access a. Die Begründung der Schriftsprache, d.h. die Wahl des Dia- lekts und die Aufstellung der schriftsprachlichen Norm. b. Die Weiterbildung der Schriftsprache, d.h. die Schaffung von neuen Ausdrucksmitteln• b. Die Weiterbildung der Schriftsprache, d.h. die Schaffung von neuen Ausdrucksmitteln• a. Die Begründung der Schriftsprache ger sei Serbien und Montenegro; "zasto je oväe njiva za- rasla и korov, pa treba mlogo truda i vremena, dók se okrči i ocisti za pravo sjeme; a onde je njiva cista: samo treba mudri i posteni poslenika, da posiju cisto i zdravo sjeme." (GPS II, 105). ger sei Serbien und Montenegro; "zasto je oväe njiva za- rasla и korov, pa treba mlogo truda i vremena, dók se okrči i ocisti za pravo sjeme; a onde je njiva cista: samo treba mudri i posteni poslenika, da posiju cisto i zdravo sjeme." (GPS II, 105). 1821 "Ako književnici i kaku vlast и jeziku imaju, ja mislim da tu imaju, da и gramatičeskim predmetima, koji su kod na- roda neopredje lje n i, izaberu ono, što je pravilnije." Er meinte, "da je d r z u , g o v o r u , m o l u 3 r a d u (mjesto d r z e , g o v o - r e , m o l e , r a d e ) postalo od skora, kako je naród svoje kn ji- ge i književnike dobio..." (GPS II, 138). 1826 Vuk erläutert die Unterschiede zwischen der serbischen und aksl. Sprache, um die serbischen "jezikopopravitelji" und "pokondirene tikve", die die serbische Sprache "pokva- renim i svinjarskim i govedarskim" nennen, über die Tatsa- chen zu belehren (GPS I I , 261 273 .)־ 1834 Zu den Versuchen der Matica srpska, Regeln für die Schrift- spräche aufzustellen, meint Vuk: "U današnjem stanju naše literature ne mogu ni najučeniji naši spisatelji и jedan put odrediti, kako ce se pisati, jedno za to, Sto smo mi (sadašnji Srpski spisatelji) svi diletanti (da ne kažem fušeri), jer se ni jedan nije prepravio za pisanje knjiga и svom jeziku, nego je svaki učio nesto drugo; pa je и jedan put Srpski počeo pisati bez ikake u tom пайке i preprave; a drugo, što je stanje naše literature, koje и svakom početku, kője osobito kod nas zbog crkvenoga jezika i drugi kojekaki smetnji i nedoskutica, teško i preteŠko; a kamo l i da to odredi Matica..." (GPS II, 422). 1839 Vuk w irft J. a. Die Begründung der Schriftsprache "Ako književnici i kaku vlast и jeziku imaju, ja mislim da tu imaju, da и gramatičeskim predmetima, koji su kod na- roda neopredje lje n i, izaberu ono, što je pravilnije." Er meinte, "da je d r z u , g o v o r u , m o l u 3 r a d u (mjesto d r z e , g o v o - r e , m o l e , r a d e ) postalo od skora, kako je naród svoje kn ji- ge i književnike dobio..." (GPS II, 138). Vuk erläutert die Unterschiede zwischen der serbischen und aksl. Sprache, um die serbischen "jezikopopravitelji" und "pokondirene tikve", die die serbische Sprache "pokva- renim i svinjarskim i govedarskim" nennen, über die Tatsa- chen zu belehren (GPS I I , 261 273 .)־ Zu den Versuchen der Matica srpska, Regeln für die Schrift- spräche aufzustellen, meint Vuk: "U današnjem stanju naše literature ne mogu ni najučeniji naši spisatelji и jedan put odrediti, kako ce se pisati, jedno za to, Sto smo mi (sadašnji Srpski spisatelji) svi diletanti (da ne kažem fušeri), jer se ni jedan nije prepravio za pisanje knjiga и svom jeziku, nego je svaki učio nesto drugo; pa je и jedan put Srpski počeo pisati bez ikake u tom пайке i preprave; a drugo, što je stanje naše literature, koje и svakom početku, kője osobito kod nas zbog crkvenoga jezika i drugi kojekaki smetnji i nedoskutica, teško i preteŠko; a kamo l i da to odredi Matica..." (GPS II, 422). Vuk w irft J. Hadziò vor, weder deklinieren, noch konjugie- ren zu können, weil er r e c i j u neben v l a s t j u und p r o m e n u l i statt p r o m e n i l i schreibt. Wenn das letztere Wort auch im Volk "falsch" gesprochen wird, " . . .spisatelj... vaija da izbira ono, sto je pravilnije. Gdjekoji Srbi, osobito oni, koji su nesrecom jezika našega imali теви sobom više spisa- te lja i književnika govore i: p u z a t i m. p u z a t i , i m i r i s i m. a. Die Begründung der Schriftsprache In der Anfangsphase verlangte Vuk von den Schriftstellern, sich Regeln aufzustellen und sich an sie zu halten (GPS I, 82; 1815) . Das war in der Tat das Mindeste, was man von den Schrift- stellarn verlangen mußte, wenn man sich die chaotische und inkonse- quente Schreibweise von Vidakovic ansieht. Vuks zweite Forderung aus derselben Zeit war, daß sich die Schriftsteller, wenn sie Ser- bisch schrieben, nach der Sprache des Volkes richten sollten, da es noch keine vollständige Grammatik gab; in Einzelfällen verwies er sie auf seine "Pismenica". Diese Forderung hat Vuk 1817 zum obersten Prinzip, die Sprache des einfachen Volkes zum obersten Richter erklärt und den Gebrauch aller Dialekte in der Literatur zugelassen (GPS I, 150, 163; II, 138; 1821). Aber gerade in der Anwendung dieser Prinzipien hat Vuk viele Inkonsequenzen und Fehler in bezug auf die Schriftsteller begangen. Eine chronologische Übersicht ermöglicht eine bessere Beurteilung seiner Äußerungen über die Schriftsteller. 1815 Vuk hat in seinen Rezensionen Vidakovic nachgewiesen, daß 1817 er weder deklinieren, noch konjugieren kann, daß er "po pra- vilam baby Smiljani" schreibt (GPS I, 82ff., 153ff.). Vuks K ritik bezog sich vor allem auf Vidakovics willkürliche Mischung der serbischen und russ.-ksl. Sprache. Außerdem bezeichnete Vuk aber auch einige mundartliche Eigenschaften der Vojvodina als Vidakovics Fehler (po d u b r a v a л obedu usw.). 1818 Dositej habe als erster angefangen. Serbisch zu schreiben. Die Schriftsteller können aber in der serbischen Sprache nicht schreiben, weil sie vergessen haben, serbisch zu den- ken. Der Grund sei ihre Ausbildung in fremden Sprachen (GPS II, 5f.). 1820 Die Vojvodina, in deren Gymnasien von Karlovci und Novi Sad kein richtiges Serbisch gelehrt wird, könne den Fort- schritt der serbischen Literatur nicht vorantreiben, günsti- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1818 Die Vojvodina, in deren Gymnasien von Karlovci und Novi Sad kein richtiges Serbisch gelehrt wird, könne den Fort- schritt der serbischen Literatur nicht vorantreiben, günsti- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1820 000Б1004 - 56 ־ ger sei Serbien und Montenegro; "zasto je oväe njiva za- rasla и korov, pa treba mlogo truda i vremena, dók se okrči i ocisti za pravo sjeme; a onde je njiva cista: samo treba mudri i posteni poslenika, da posiju cisto i zdravo sjeme." (GPS II, 105). a. Die Begründung der Schriftsprache mole), b o l u (m. bole); ni na d o b r i m konju (m. na d o b ro m konju), и n a S im s e t u ( т. и п е в е т selu); a sad kako su se i onamo (и Srbiji) umnožill i s narodom pomijeŠali spi- satelji i književnici, ovake se pogrješke mogu čuti i и Beo- gradu i u Kragujevcu, a može b iti i u mome Trši&u. Tako: sta je u drugijeh naroda jezik ugladilo, očistilo i u nje- govij em prirodnijem pravilima utvrdilo, ono да je u nas is- kvarilo i nagrdilo (i još да kvari i grdi)i" (GPS I I I , 39). Zu dem Wort c e e t (Ehre), das er in sein Wörterbuch von 1818 als bereits zur Volkssprache gehörend aufgenommen hat- te, meint Vuk: " ... a po južnijem i jugozapadnijem krajevi- ma naroda nasega, kuda srećom jezika našega, još slabo do- piru knjige našijeh novijeh književnika, niko i ne zna za c e a t и ovom smislu nego samo za cast..." (GPS I I I , 56, Anm.). Er k ritis ie rt Musicki wegen der Nachahmung klassizisti- scher Dichtung, die dem Geist der serbischen Sprache und Volksdichtung entgegengesetzt sei (GPS I I I , 68). 1842 Uber die serbischen Schriftsteller im allgemeinen meint er: "Prije bi se mogio po pravdi reći, da narod naš sramote njegovi književnici, koji njega i jezika njegova ne poznaju i mjesto koristi eine mu štetu..." (GPS I I I , 71). 1843 "Ko je god rad, da se miješa medu književnike svoga naro- da, valjalo bi da zna, kako se и pravo govori, i kako pisa- t i valja po svojstvu narodnoga jezika; a za što onako i ot- kuda je Što postalo, to je posao drugijeh lju d i, od kojijeh je jedan и sto godina dosta za sav narod naš od sve t r i vjere." (GPS I I I , 96). Im Zusammenhang mit p r o m e n u t i und m a n ī t i bei Hadžic meint Vuk: " ... da su ljudi (ponajviše književnici), koji upravo ne znadu svoga jezika, nego ga kvare, mjesto p r o m e n i t i uze- l i p r o m e n u t i ... kao п. р. a. Die Begründung der Schriftsprache a po južnijem i jugozapadnijem krajevi- ma naroda nasega, kuda srećom jezika našega, još slabo do- piru knjige našijeh novijeh književnika, niko i ne zna za c e a t и ovom smislu nego samo za cast..." (GPS I I I , 56, Anm.) Er k ritis ie rt Musicki wegen der Nachahmung klassizisti- scher Dichtung, die dem Geist der serbischen Sprache und Volksdichtung entgegengesetzt sei (GPS I I I , 68). Uber die serbischen Schriftsteller im allgemeinen meint er: "Prije bi se mogio po pravdi reći, da narod naš sramote njegovi književnici, koji njega i jezika njegova ne poznaju i mjesto koristi eine mu štetu..." (GPS I I I , 71). "Ko je god rad, da se miješa medu književnike svoga naro- da, valjalo bi da zna, kako se и pravo govori, i kako pisa- t i valja po svojstvu narodnoga jezika; a za što onako i ot- kuda je Što postalo, to je posao drugijeh lju d i, od kojijeh je jedan и sto godina dosta za sav narod naš od sve t r i vjere." (GPS I I I , 96). Im Zusammenhang mit p r o m e n u t i und m a n ī t i bei Hadžic meint Vuk: " ... da su ljudi (ponajviše književnici), koji upravo ne znadu svoga jezika, nego ga kvare, mjesto p r o m e n i t i uze- l i p r o m e n u t i ... kao п. р. и Bačkoj što se kaže p r i k o Du- nava, i b o l e me glava... P r o m e n u t i , kao i o k a n u t i se i ostale mnoge ovakove rije č i, ja sam najprije čuo u Srijemu; a da je p r o m e n i t i pravilnije, о tome onoga, koji drukčije ne može da razumije, može u v je riti i Slavenski i Ruski je- zik. (GPS I I I , 111). ־ 57 - Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM (i gotovo sve) pogrješke и jeziku našemu postale su od na- šijeh dojakošnjijeh spisatelja i književnika. Ja dok nije- sam preiao и Srijem, n it sam čuo n. p. g o v o r u (m. govore), m o l u (m. a. Die Begründung der Schriftsprache Hadziò vor, weder deklinieren, noch konjugie- ren zu können, weil er r e c i j u neben v l a s t j u und p r o m e n u l i statt p r o m e n i l i schreibt. Wenn das letztere Wort auch im Volk "falsch" gesprochen wird, " . . .spisatelj... vaija da izbira ono, sto je pravilnije. Gdjekoji Srbi, osobito oni, koji su nesrecom jezika našega imali теви sobom više spisa- te lja i književnika govore i: p u z a t i m. p u z a t i , i m i r i s i m. m i r i s e i t . d.; a li pravi spisatelj vaija da zna, da je p u - zem p u z a t i , kao re z e m r e z a t i ; a m i r i s e m m i r i 8 a t i , k a o p i s e m p i s a t i i t.d ." In der Anmerkung dazu meint er: "Najglavnije Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 57 - (i gotovo sve) pogrješke и jeziku našemu postale su od na- šijeh dojakošnjijeh spisatelja i književnika. Ja dok nije- sam preiao и Srijem, n it sam čuo n. p. g o v o r u (m. govore), m o l u (m. mole), b o l u (m. bole); ni na d o b r i m konju (m. na d o b ro m konju), и n a S im s e t u ( т. и п е в е т selu); a sad kako su se i onamo (и Srbiji) umnožill i s narodom pomijeŠali spi- satelji i književnici, ovake se pogrješke mogu čuti i и Beo- gradu i u Kragujevcu, a može b iti i u mome Trši&u. Tako: sta je u drugijeh naroda jezik ugladilo, očistilo i u nje- govij em prirodnijem pravilima utvrdilo, ono да je u nas is- kvarilo i nagrdilo (i još да kvari i grdi)i" (GPS I I I , 39). Zu dem Wort c e e t (Ehre), das er in sein Wörterbuch von 1818 als bereits zur Volkssprache gehörend aufgenommen hat- te, meint Vuk: " ... a. Die Begründung der Schriftsprache Ovo je kva- renje jezika u carstvu Austrijskome iSLo polako, jer se na- si jem jezikom nije mnogo pisało; a li kako se u S rb iji po razliőnijem kancelarijama otvorilo i (može b iti i suviše) umnozilo pisanje, postale velike Srpske škole, postalo dru- stvo Srpske književnosti, postala državna stampari ja, и ко- joj se stampaju novine i druge različne stvari bez prestanka, a sve ove poslove rade i upravljaju ponajviše onaki lju d i, kakovi su jezik naš na ovoj strani k v a rili, tako se upravo moze reci, da kvarenje jezika ide sad mnogo brže." (GPS I I I , 154f.) Vuk lobt J. Stejic, D. Demeter und S. Vraz, die gezeigt ha- 1845 Wenn die Schriftsteller über den Mangel an Wörtern klagen, "tome nije toliko kriv jezik, koliko su k riv i oni. Sto rije - či jezika svoga ne poznaju..."; sie sollen die Volkssprache lernen, "ito kako и riječima tako i u misiima". (GPS 111,153) Zu Dositej meinte er jetzt: "... a počinje l i se zaista od onda naia literatura, о tome be se jo5 suditi i presudivati. ״ (GPS II, 98 Anm.)."Prvi Srpski spisatelj, bio on Dositije i l i Orfelin, i l i drugi koji mu drago, kako je poCeo pisati, odmah je počeo i jezik svoj kvariti, i to kvarenje tra je i upravo reći napreduje još do današnjega dana." (GPS III,154f. In der Anmerkung dazu wiederholt er seine Meinung von 1839, daß die meisten Sprachfehler von den Schriftstellern stammen und sagt: "Da ne spominjemo naštampanijeh knjiga, ne- go kad ljudi prosti čuju da kakav gospodin govori drukfcije od njih, oni misle da je ono najbolje, i odmah primaju nje- gove rije č i, da bi se barem и tome iz jednafcili s njime... Tako je narod naš u Srijemu i и Bačkoj i u Banatu bezbrojne pogrjeske u svome jeziku poprimao od svojijeh spisatelja, popova, učitelja i ostalijeh znamenitijeh lju d i. a. Die Begründung der Schriftsprache и Bačkoj što se kaže p r i k o Du- nava, i b o l e me glava... P r o m e n u t i , kao i o k a n u t i se i ostale mnoge ovakove rije č i, ja sam najprije čuo u Srijemu; a da je p r o m e n i t i pravilnije, о tome onoga, koji drukčije ne može da razumije, može u v je riti i Slavenski i Ruski je- zik. (GPS I I I , 111). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 00051004 - 58 ־ Wenn die Schriftsteller über den Mangel an Wörtern klagen, "tome nije toliko kriv jezik, koliko su k riv i oni. Sto rije - či jezika svoga ne poznaju..."; sie sollen die Volkssprache lernen, "ito kako и riječima tako i u misiima". (GPS 111,153) Zu Dositej meinte er jetzt: "... a počinje l i se zaista od onda naia literatura, о tome be se jo5 suditi i presudivati. ״ (GPS II, 98 Anm.)."Prvi Srpski spisatelj, bio on Dositije i l i Orfelin, i l i drugi koji mu drago, kako je poCeo pisati, odmah je počeo i jezik svoj kvariti, i to kvarenje tra je i upravo reći napreduje još do današnjega dana." (GPS III,154f. In der Anmerkung dazu wiederholt er seine Meinung von 1839, daß die meisten Sprachfehler von den Schriftstellern stammen und sagt: "Da ne spominjemo naštampanijeh knjiga, ne- go kad ljudi prosti čuju da kakav gospodin govori drukfcije od njih, oni misle da je ono najbolje, i odmah primaju nje- gove rije č i, da bi se barem и tome iz jednafcili s njime... Tako je narod naš u Srijemu i и Bačkoj i u Banatu bezbrojne pogrjeske u svome jeziku poprimao od svojijeh spisatelja, popova, učitelja i ostalijeh znamenitijeh lju d i. a. Die Begründung der Schriftsprache Ovo je kva- renje jezika u carstvu Austrijskome iSLo polako, jer se na- si jem jezikom nije mnogo pisało; a li kako se u S rb iji po razliőnijem kancelarijama otvorilo i (može b iti i suviše) umnozilo pisanje, postale velike Srpske škole, postalo dru- stvo Srpske književnosti, postala državna stampari ja, и ко- joj se stampaju novine i druge različne stvari bez prestanka, a sve ove poslove rade i upravljaju ponajviše onaki lju d i, kakovi su jezik naš na ovoj strani k v a rili, tako se upravo moze reci, da kvarenje jezika ide sad mnogo brže." (GPS I I I , 154f.) Vuk lobt J. Stejic, D. Demeter und S. Vraz, die gezeigt ha- ben, daß man die Volkssprache erlernen kann, und fügt hinzu, " ... da ce doci vrijeme, kad ce Srbi iz Srijema, iz Bačke i iz Srbije ic i и Zagreb da uce Srpski".(GPS I I I , 160). Man könne nicht mehr so schreiben, wie das Volk spricht, "jer su književnici nasi... iskvarili narodni jezik". (GPS I I I , 185). "...je zik nas najprije vai ja c is t it i od onijeh pogrješaka, koje su naši književnici unj u v e li..." (GPS II I , 164). Dabei müsse man unterscheiden zwischen demjenigen, "Sto su književnici naSi, ponajviSe naSega naraStaja, uveli Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vuk lobt J. Stejic, D. Demeter und S. Vraz, die gezeigt ha- ben, daß man die Volkssprache erlernen kann, und fügt hinzu, " ... da ce doci vrijeme, kad ce Srbi iz Srijema, iz Bačke i iz Srbije ic i и Zagreb da uce Srpski".(GPS I I I , 160). Man könne nicht mehr so schreiben, wie das Volk spricht, "jer su književnici nasi... iskvarili narodni jezik". (GPS I I I , 185). "...je zik nas najprije vai ja c is t it i od onijeh pogrješaka, koje su naši književnici unj u v e li..." (GPS II I , 164). Dabei müsse man unterscheiden zwischen demjenigen, "Sto su književnici naSi, ponajviSe naSega naraStaja, uveli Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM - 59 - и jedan d ije l naroda nasega, od onoga, sto je и svemu naro- du Bog zna od koliko stotina godina." (GPS I I I , 165). a. Die Begründung der Schriftsprache Auch hier führt er als Sprachfehler d v a n a j e t , p o l j a m a , p u z a t i , b o l e , kövem an und lehnt die Sprache der Vojvodina offen ab: "Sto sam do sad sve okolišio, ovdje cu izrijekom da kažem: da se и svemu narodu našemu nigdje ne govori Srpski tako ružno i pokvareno, kao u Srijemu, u Baćkoj i u Banatu. I što je god mjesto vece i ima u sebi više gospo- de i književnika, to se u njemu govori gore: tako seljaci govore bolje od varošana, a sluge od gospodara. Ja sam na- pomenuo... kako je kvarenje jezika prešlo i u Srbiju; i ako mu se kakvijem mudrijem književnijem uredbama ne stane na put, o tic i ce , kao kuga, po svemu narodu na sve strane." (GPS I I I , 181 Anm.). Zugleich betont er, daß in Bosnien und Herzegovina, aber auch in Montenegro und Dalmatien, am be- sten Serbisch gesprochen wird und schlägt den Süddialekt zur Schriftsprache vor (GPS II I , 155). 46/ Vuk lobt die Sprache der Dubrovniker Literatur und M. 47 Reljkovics, die "vece skrovište i č is t iji izvor od našijeh današnjijeh crkvenijeh knjiga" sei (GPS I I I , 260) . Aus dieser Übersicht is t zu Vuks Einschätzung der Schriftstel- 1er und ihrer Rolle bei der Begründung der Schriftsprache folgen- des ersichtlich: 1. Vuks K ritik der serbischen Schrifsteiler betraf, besonders zu Anfang, die Überfremdung ihrer Sprache durch fremdsprachige Elemente. Seine Schlußfolgerung, daß die Schriftsteller die Volks- spräche nicht kannten, war in den 20er Jahren zutreffend, in den 40er Jahren aber nicht mehr. 2. Vuk vertrat die Meinung, daß die Schriftsteller wegen ihrer fremdsprachigen Ausbildung nicht serbisch denken können. Dieser Gedanke steht im Einklang mit Herders Auffassung der Sprache als Denkart eines Volkes. Vera Boji - 9783954793044 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vuks Argumentation erinnert stellenweise stark an A. S. šiškov, so daß bei der Beurteilung seiner Meinung über die Dichter auch der Einfluß des russischen Spätklassizismus in Betracht gezogen werden muß. šiškov kämpfte gegen den Einfluß der französischen Sprache, der dadurch begünstigt werde, daß die Schriftsteller von Franzosen er- zogen werden und so "polucajut ves' obraz mysiej ich i ponjatij"1. 1 Vgl. A. S. šiškov, Sobranie sočinenij i perevodov, Bd. II, Spb. 1824, S. 6. 2 ebd., S. 23. - Vgl. Vuk über Hadžic und Simic, S. 75f. dieser Ar- b e it. 3 ebd., S. 26. - Vgl. Vuk über a o p a t v e n i k , S. 76 dieser Arbeit. 4 ebd., S. 44. - So Vuk 1845, vgl. S. 58 dieser Arbeit. 5 Vgl. dazu V. Vinogradov, Očerki po is to rii russkogo literaturnogo jazyka XVII - XIX w ., Leiden 1950, S. 195ff. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access a. Die Begründung der Schriftsprache Nur so kann seine Meinung verstanden werden, daß die serbischen Schriftsteller eine Schande für das eigene Volk sind, weil sie die Sprache verderben und mehr Schaden als Nutzen brin- gen, obwohl die Werke eines der Klassiker der serbischen Literatur, J. Sterija Popovics, bis zum Beginn der 40er Jahre bereits erschie- nen waren (Laža i paralaža 1830, Kir Janja 1Õ37, Zia žena und Pokondirena tikva 1838, Ženidba i udadba 1841). Sterija war auch einer jener Vojvodiner Schriftsteller, die für die Organisation des kulturellen Lebens in Serbien große Verdienste gehabt haben, von denen Vuk als von den Verderbern der Sprache auch südlich der Save und Donau sprach. Allerdings entsprach Sterijas Sprache den Vorstellungen von Vuk nicht. geschaffene Bild des vom Volk entfremdeten Schriftstellers zu kor- rigieren. Nur so kann seine Meinung verstanden werden, daß die serbischen Schriftsteller eine Schande für das eigene Volk sind, weil sie die Sprache verderben und mehr Schaden als Nutzen brin- gen, obwohl die Werke eines der Klassiker der serbischen Literatur, J. Sterija Popovics, bis zum Beginn der 40er Jahre bereits erschie- nen waren (Laža i paralaža 1830, Kir Janja 1Õ37, Zia žena und Pokondirena tikva 1838, Ženidba i udadba 1841). Sterija war auch einer jener Vojvodiner Schriftsteller, die für die Organisation des kulturellen Lebens in Serbien große Verdienste gehabt haben, von denen Vuk als von den Verderbern der Sprache auch südlich der Save und Donau sprach. Allerdings entsprach Sterijas Sprache den Vorstellungen von Vuk nicht. 4. Vuk lehnte die Sprache der Vojvodina als verdorben ab. D Grund war einerseits seine ungenügende Kenntnis der dortigen Mund- arten: alle Formen, die er 1839 Hadzic vorgeworfen hat, (mit Aus ־ nähme von r e S i j u und v l a s t j u , wo es um orthographische Inkonse- quenz geht), gehören auch zur Volkssprache der Vojvodina. Anderer- seits waren es die fremdsprachigen Elemente, Russismen und Germa- nismen, die in die Sprache der Vojvodina durch kulturelle Bindung an Rußland und Österreich eingegangen waren. Vuks Behandlung der Fremdwörter war widersprüchlich: er dulde- te Turzismen und lehnte Russismen und Germanismen ab: "...tako je u jednom trenutku naSe kulturne is to rije doSlo do paradoksalne situacije: proganjani su termini za odredene pojmove kulturę i civilizacije najnaprednije srpske sredine, dok, medutim, turske pozajmice - tipa Čarsija, pendžer i sl. - zatečene pretežno и za- ostalijim krajevima, nisu proganjane... 1 Vgl. M. Ivic, Vukova uloga и normiranju ekavske varijante kn ji- zevnog jezika. In: Anali FF 5, S. 96. a. Die Begründung der Schriftsprache Solche Schriftsteller "bezobrazjat jazyk svoj" durch Einführung von Fremdwörtern und durch wörtliche Übertragung fremder Gedanken in 2 die russische Sprache . So hätten sie z.B. i n f l u e n c e mit v l i j a n i e statt n a i t i e oder n a i t a t v o v a n i e übersetzt, weil "perevodčik ne znal slova naitstvovat1, izobrazajušČago tožsamoe ponjatie... Die russische Sprache sei nicht schuld daran, daß die Schriftstel- 4 1er sie nicht kennen . Vuk hat šiškov 1819 in Petersburg kennengelernt (Prepiska II, 254) und stand mit ihm im Briefwechsel von 1820 - 1838 (Prepiska I I I , 499 529 .)־Er nannte šiškov seinen Mäzen und wollte ihm die Danica für 1828 widmen (Prepiska I I I , 515). Auch in den Polemiken mit Vidakovic berief sich Vuk auf šiškov als die Autorität (GPS I, 129) . Vuks und Šiskovs Ansichten über die Schriftsprache waren grund- sätzlich verschieden, šiškov verteidigte den Gebrauch der aksl. Sprache, weil er sie für die ursprüngliche russische h ie lt5. Vuk dagegen sah die Sprache des einfachen Volkes als rein und ursprüng- lieh. Obwohl sie von verschiedenen Begriffen der ursprünglichen Sprache ausgingen, stimmten sie in der Beurteilung der Schriftstel- 1er als Sprachverderber überein. Vuk hat offensichtlich Anregungen für seine Argumente gegen die Schriftsteller von šiškov geschöpft. 3. Vuks Äußerungen über die Schriftsteller in den 40er Jah zeigen, daß er den Zeitpunkt versäumt hat, seine Haltung der ver- änderten Situation anzupassen und das durch die Spätaufklärung 1 Vgl. A. S. šiškov, Sobranie sočinenij i perevodov, Bd. II, Spb. 1824, S. 6. 2 ebd., S. 23. - Vgl. Vuk über Hadžic und Simic, S. 75f. dieser Ar- b e it. 3 ebd., S. 26. - Vgl. Vuk über a o p a t v e n i k , S. 76 dieser Arbeit. 4 ebd., S. 44. - So Vuk 1845, vgl. S. 58 dieser Arbeit. 5 Vgl. dazu V. Vinogradov, Očerki po is to rii russkogo literaturnogo jazyka XVII - XIX w ., Leiden 1950, S. 195ff. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access geschaffene Bild des vom Volk entfremdeten Schriftstellers zu kor- rigieren. 1 Vgl. dazu S.102f.dieser Arbeit. 2 Das wird durch eine große Zunahme von Personennamen aus Vidakovics Romanen bestätigt.- Vgl. M. Laüevic, Licnosti romana "Ljubomir и Jelisijumu" Milovana Vidakovica и onomastičkom fondu Vojvodine početkom 19. veka. In: Prilozi proučavanju jezika 7, S. 199-213. 3 Hinter ihren Argumenten (Wohllaut, Sprachentwicklung usw., vgl. GPS II , 124; I I I , 590) verbarg sich der Wunsch, an der ekavi- sehen Tradition und Gewohnheit festzuhalten. 4 Vgl. M. Pavlovié, Uloga i značaj vojvodanskih pisaca и razvoju Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access a. Die Begründung der Schriftsprache Dieser Widerspruch erklärt sich aus Vuks Bemühung, die Spra- che der Volkslieder, die viele Turzismen enthält, zur Schriftspra- che zu erheben. In der Vojvodina liefen aber seit Dositej die Be- mühungen um die volkssprachliche Grundlage der Schriftsprache. Vuk muß die Gefahr gefühlt haben, daß von dort eine Sprachreform gegen seine Konzeption ausgehen könnte. Seine Behandlung der Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Schriftsteller muß auch unter diesem Aspekt gesehen werden, zumal die meisten von ihnen, offen oder stillschweigend, seinen ijekavi- sehen Dialekt abgelehnt haben. Schriftsteller muß auch unter diesem Aspekt gesehen werden, zumal die meisten von ihnen, offen oder stillschweigend, seinen ijekavi- sehen Dialekt abgelehnt haben. Bei der Wahl des ijekavischen Dialekts ließ Vuk sich zu Anfang, außer der Vorliebe für den eigenen Dialekt, hauptsächlich von der romantischen Verehrung der Volksdichtung leiten1. In den 30er Jah- ren kam die Vorstellung von der sprachlichen Einigung mit den Kro- aten hinzu, die zweifellos das wichtigste Argument für das Ijeka- vische war. Vuks Hoffnung, daß die Sprache der Volkslieder eine einigende Funktion ausüben würde, war unrealistisch. Die produk- tivste Zeit dieser Poesie war bereits zu Ende. Die gebildeten Ser- ben haben zwar die Volkspoesie schätzen gelernt, nachdem sie durch Vuks Bemühungen das höchste Lob in Europa geerntet hatte. Man las aber, wie auch andernorts in Europa, offensichtlich lieber 2 die unterhaltenden Romane in Vidakovics Art . Vuks Wahl des ijekavischen Dialekts, trotz des starken Wider- stands unter den serbischen Schriftstellern3, und seine Ablehnung der Sprache und der Schriftsteller der Vojvodina zeigen deutlich seine Unterschätzung der Rolle der Schriftsteller bei der Bildung der Schriftsprache. "Vojvodina je stvorila pesnički jezik" bei 4 den Serben . Das hat dazu geführt, daß der ekavische Dialekt sich gegen Vuks Konzeption durchgesetzt hat. Diese Entwicklung begann schon zu Vuks Lebzeiten: Branko Radiceviò, der zusammen mit 0־. Danicic als Vuks beste Stütze genannt wird, und der auf die kom- mende Dichtergeneration entschieden gewirkt hat, schrieb konse- quent ekavisch. Die nachfolgenden Schriftsteller, die zunächst vorwiegend aus der Vojvodina stammten, haben nicht nur das Ekavi- sehe gefestigt, sondern auch einzelne Merkmale des Sumadiner- Vojvodiner Dialekts in die von Vuk geschaffene schriftsprachliche Norm hineingetragen. a. Die Begründung der Schriftsprache "Otuda ima pri poredenju sa Vukom pojedinih razlika i и fonetici, i u morfologiji, i и sintaksi, i u leksici - и svim jezičkim oblastima jednom rečju." Vuk hat durch seine Fehleinschätzung der Schriftsteller mit seiner Konzeption bereits den Grundstein für die spätere Spaltung der serbischen Schriftspra- che in die ekavische und ijekavische Variante gelegt. razlika i и fonetici, i u morfologiji, i и sintaksi, i u leksici - и svim jezičkim oblastima jednom rečju." Vuk hat durch seine Fehleinschätzung der Schriftsteller mit seiner Konzeption bereits den Grundstein für die spätere Spaltung der serbischen Schriftspra- che in die ekavische und ijekavische Variante gelegt. Es läßt sich zusammenfassend feststellen, daß Vuk den Schrift- stellern keine schöpferische Rolle bei der Begründung der Schrift- 2 Sprache beigemessen hat • Seine und Jacob Grimms Ansichten unter- scheiden sich stark voneinander. Während Grimm die Auffassung ver- trat, daß die Literatur, also das Schaffen der Dichter, die kultu- relie Übermacht eines Dialekts bewirkt, ging Vuk von der Sprache des einfachen Volkes aus, deren kulturelle Tradition er in der Volksdichtung sah und zu festigen bemüht war. srpskoga knjizevnoga jezika. In: Zbornik MS 1,(1954), S. 95 1 Vgl. M. Ivic, Jedno poredenje Vukovog jezika sa našim današnjim književnim jezikom. In: Zbornik FL I, S. 126. ־Eine Wissenschaft liche Untersuchung beider Varianten in diesem Sinne wäre u.E. wünschenswerter als Beteuerung der Treue zu Vuk. Erst dann wären die Grundlagen geschaffen, sich um Gemeinsamkeiten statt um Auseinandertreiben der beiden Varianten zu bemühen. 2 Die wichtigste Aufgabe, die er ihnen gestellt hat, war die Aus- wähl der "richtigeren" Formen im Rahmen der von ihm geschaffenen Norm. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 2 Die wichtigste Aufgabe, die er ihnen gestellt hat, war die Aus- wähl der "richtigeren" Formen im Rahmen der von ihm geschaffenen Norm. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM srpskoga knjizevnoga jezika. In: Zbornik MS 1,(1954), S. 95 1 Vgl. M. Ivic, Jedno poredenje Vukovog jezika sa našim današnjim književnim jezikom. In: Zbornik FL I, S. 126. ־Eine Wissenschaft- liche Untersuchung beider Varianten in diesem Sinne wäre u.E. wünschenswerter als Beteuerung der Treue zu Vuk. Erst dann wären die Grundlagen geschaffen, sich um Gemeinsamkeiten statt um Auseinandertreiben der beiden Varianten zu bemühen. b. Die Erweiterung der Ausdrucksmittel b. Die Erweiterung der Ausdrucksmittel In der Anfangsphase meinte Vuk, daß die Schriftsteller neue Wörter für die in der Volkssprache fehlenden Begriffe bilden soll- ten (SD XII, 15f.). Ab 1817 verlangte er Treue zur Volkssprache und hielt bis zum Ende an dieser Forderung fest. "Serbisch” sei das, was "naród nas и jeziku svome zaista ima i и onome istome značenju govoriH; wer etwas Anderes so bezeichnet, der "jezik naš zamršuje i kvari" (GPS II I , 173; 1845). Vuks Ansichten über die Weiterbildung der Schriftsprache können in zwei Punkte zusammengefaßt werden: 1. Zuerst soll die Volkssprache erforscht werden, bevor man neue Wörter schafft. Dieses Prinzip verrät seine Überschätzung der Ausdrucksfähigkeit der Volkssprache und ein gewisses Mißachten der Anforderungen des 19. Jhs. an die Schriftsprache. Seine Forderung hatte jedoch die positive Folge, daß der bereits vorhandene volks- srpskoga knjizevnoga jezika. In: Zbornik MS 1,(1954), S. 95 1 Vgl. M. Ivic, Jedno poredenje Vukovog jezika sa našim današnjim književnim jezikom. In: Zbornik FL I, S. 126. ־Eine Wissenschaft- liche Untersuchung beider Varianten in diesem Sinne wäre u.E. wünschenswerter als Beteuerung der Treue zu Vuk. Erst dann wären die Grundlagen geschaffen, sich um Gemeinsamkeiten statt um Auseinandertreiben der beiden Varianten zu bemühen. 2 Die wichtigste Aufgabe, die er ihnen gestellt hat, war die Aus- wähl der "richtigeren" Formen im Rahmen der von ihm geschaffenen Norm. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access sprachliche Wortschatz zur Bereicherung der Schriftsprache heran- gezogen wurde. Da er aber auf dem lexikalischen Gebiet keine Schranken in Richtung der Dialekte gesetzt hat, führte das zur Entwicklung von Wortschatzdubletten, die den wichtigsten Unter- schied zwischen der serbischen und kroatischen Variante der Schriftsprache ausmachen1. sprachliche Wortschatz zur Bereicherung der Schriftsprache heran- gezogen wurde. Da er aber auf dem lexikalischen Gebiet keine Schranken in Richtung der Dialekte gesetzt hat, führte das zur Entwicklung von Wortschatzdubletten, die den wichtigsten Unter- schied zwischen der serbischen und kroatischen Variante der Schriftsprache ausmachen1. 2. Vuk lehnte die Bildung neuer Wörter von seiten der Schr steiler ab. Er führte zwei Hauptargumente an: a. Die Gebildeten können keine Wörter schaffen, "zašto to nije za svakoga učenog čoveka (a za prostaka jest: zašto su svi jezici postali od prostaka, t . j . 1 Vgl. M. Ivic, Vukova uloga.., a.a.O., S. 96. 2 Herder I, S. 387f., meint, niemand könne sich über seinen Lebens- bereich "geläufiger und triftig e r ausdrücken als der gemeine Mann", der über "grammatische Zierlichkeiten" nichts weiß. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access b. Die Erweiterung der Ausdrucksmittel od pastira i od težaka; i prostak ni- kakvu riječ ne može drukčije načiniti, nego upravo onako, kao što su i ostale načinjene)." Diese Rousseauisch-Herderische Überhöhung der unverbildeten Natur erscheint auch in Vuks Behauptung, daß der Gebildete, der sich dessen bewußt ist, daß die Sprache ihre Regeln hat, ohne Grammatik nicht schreiben könne; der Ungebildete dagegen "...ne zna ni za kakvu gramatiku, n iti za kakav drugi jezik osim svoga, on može pisati i bez svoje gramatike; i upravo kao sto tre- ba: zašto mu ne može pasti na um da pise drukčije, nego onako kao % vsto se govori; tako je n. p. mogao Omir spjevati Ilijadu i Odise- ju ne znajući ni pisati, kao i naši starci i slijepci što su spje- vali toliku silu pjesama."2(GPS II, 26, 8; 1818). Diese Konstru- ierung des Gegensatzes zwischen dem Volk und den Gebildeten in bezug auf die Sprache steht im Widerspruch mit dem Wesen der Schrift- spräche als einer Norm, die erlernt werden muß. b. Die Gebildeten können keine Wörter im Geiste der serbischen Sprache schaffen, weil sie serbisch weder sprechen, noch denken können. Diese Meinung vertrat Vuk auch in den 40er Jahren, als die Schriftsteller bereits gelernt hatten, zwischen der serbischen und russ.-ksl. Sprache zu unterscheiden. Die junge Schriftsprache konnte auf Neubildungen für fehlende Abstrakta nicht verzichten, auch wenn sich manche von den ersten Versuchen mit der Zeit als mißlungen erweisen sollten. Grimm hat diesen, in der Literatur 00051004 ־6 5־ sich vollziehenden, sprachbildenden Prozeß als eine der Quellen gesehen, aus denen das Leben der Sprache"hervordringt, und soll ־ te sie in ihrer heftigkeit auch etwas bergschutt mitführen, er setzt sich zu boden und die reine welle flieszt darüber her." (GKS V III, 36). Vuk und Grimm haben unterschiedliche Ansichten über die Rolle der Schriftsteller bei der Entwicklung sprachlicher Ausdrucksmit ־ te l. Für Grimm sind Volk und Dichter an der Bildung der Sprache beteiligt? er sprach aber nur den Dichtern das Recht zu, die schriftsprachliche Norm auf dem lexikalischen Gebiet in Richtung der Mundarten zu überschreiten. Grimm geht von der romantischen Poesieauffassung aus1; die Poesie bildet die Sprache, sie ist ihr "geistiger duft" (GKS I, 375). Für Vuk spielen die Dichter eine in bezug auf das Volk untergeordnete Rolle. Seine Äußerungen erschei- nen als eine Verbindung von Herderischer Überhöhung des einfachen Volkes und dessen Sprache und Siskovs Meinung von den Dichtern als Sprachverderbern. b. Die Erweiterung der Ausdrucksmittel Für Vuk gab es in der Lexik keine Einschrän ־ kungen für die Dialekte und zugleich keine Vollmachten an die Dichter, die Grenzen der volkssprachlichen Lexik etwa in Richtung der aksl. Sprache zu überschreiten. 1 Vgl. dazu K. K. Polheim, Der Poesiebegriff der deutschen Roman ־ tik , 1972. 2 Hervorgehoben durch V.B. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM 1 P. Popovic, Milan Vidakovic, 1934, S. 349, nennt Vidakovics Roma ne wegen ihrer Popularität im 19. Jh. "serbische Volksbücher" (narodne knjige). 2 Vgl. J. Deretic. Roman Milovana Vidakovica. In: Književna isto- rija 17, 1972, S. 42f. 3 Archiv SANU 8552/14. 4 Vuk fragt, ob es heißen soll: jesmo, oder bicemo und sagt, es kümmere ihn nicht, wie Musicki die angestrebte Silbenzahl er- reichen wolle. 5 Vuk hat von Njegoš oft Hilfe und Unterstützung bekommen. Nje- goss Funktion als geistliches Oberhaupt wird auch einer der Grunde für Vuks Stillschweigen gewesen sein. c. Vuks philologische Methode Vuks Sprachauffassung und seine Einschätzung der serbischen Schriftsteller haben auch seine philologische Methode bestimmt, die in der Folgezeit einen großen Einfluß auf die serbische Lite ־ raturkritik ausgeübt hat. Er hat sein methodisches Verfahren in den Rezensionen von Vidakovics Romanen deutlich formuliert: "Da se svaki spisatelj mora starati koliko za stvar, о kojoj piše, 2 v toliko ( ako 8e ne a m i j e v e ò i v i s e ) za čistotu i za svojstvo onoga jezika, na kojem pise, o tom ne treba ni govoriti..." (GPS I, 82; 1815). Vuks wichtigstes Kriterium bei der Beurteilung der literarischen Werke war die "Reinheit" der Sprache, weshalb er Vidakovics Romanen jeden Wert absprach, " ... zašto je stvar na jeziku, pa ako jezik ne vaija, do stvari se ne može ni doci". Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 00051004 ־ 66 ־ (GPS I, 150; 1817). Die Tatsache aber, daß Vidakoviès Romane ge־־ rade wegen ihres unterhaltenden, aufklärerisch-sentimentalen In- halts ein breites Lesepublikum hatten1, widerlegt Vuks Argument von der üntauglichkeit seiner Sprache, die "Sache" zu vermitteln. Vom heutigen Standpunkt aus gesehen, haben Vidakovics Werke zwar keinen poetischen, aber einen literarhistorischen Wert als der Be- ginn des modernen serbischen Romans . In Vuks Blickfeld standen auch nicht die poetischen Formen. Er hat das im Zusammenhang mit Mušickis Dichtung "Glas narodoljupca" ausdrücklich betont: "A i to samo cu vám recenzirati stvar i jezik, a и poēziju necu da se mijesam." (Prepiska II, 239; 1819). In der unveröffentlicht geblie- benen Rezension3 macht er viele, zweifellos richtige, Einwände in bezug auf Mušickis Mischsprache. Wenn er aber das Fehlen der Kopu- la in solchen Versen wie: "Svoem caru verni my" k ritis ie rt4, weil das in der Volkssprache nicht üblich sei, obwohl es auch in den Volksliedern vorkomme, so zeigt dies, daß er die Reinheit der Spra- che mit der gesprochenen Volkssprache gleichsetzt. Vuk betrachtet die Sprache vor allem in ihrer Funktion als Kommunikationsmittel und übersieht ihre Eigenheiten als Medium der Kunst. Seine Auffas- sung der Reinheit der Sprache war u.E. der Grund, weshalb er keine Dichtung von Njegoš rezensiert hat. NjegoŠs "Górski vijenac" ist zwar inhaltlich der Volksdichtung verpflichtet, seine syntheti- sehe Sprache mit vielen Russismen und Kirchenslavismen is t aber durchaus nicht im Sinne von Vuks Sprachkonzeption. Man könnte sich Vuks Beurteilung dieses Werkes leicht vorstellen5. 1 Vgl. J. Skerlic, Istorija srpske književnosti, 1967, S. 437. 2 Eine ähnliche Auffassung hat auch M. Vidakovic in seiner Vor ־ rede zu "Ljubomir и Jelisiumu" von 1814 vertreten. (GPS I, S. 107. c. Vuks philologische Methode Vuks methodisches Verfahren in seinen Rezensionen is t einsei- tig . Obwohl er bei der Auswahl der Volkslieder für seine Sammlun- gen ein Gefühl für das Poetische gezeigt hat, hat er es bei der Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Beurteilung der Kunstdichtung vernachlässigt und die dichterische Sprache nur mit dem Maßstab der reinen Volkssprache gemessen• Die serbische Philologie hat aus diesem Ansatz bei Vuk einen Dogmatis ־ mus entwickelt, der lange Zeit die Dichtung nur nach der Reinheit der Sprache geprüft und die ästhetischen Werte vernachlässigt hat1. 2 . Die Beziehung der serbischen Schriftsprache zur kirchen- slavischen Sprache 2 . Die Beziehung der serbischen Schriftsprache zur kirchen- slavischen Sprache 2 . Die Beziehung der serbischen Schriftsprache zur kirchen- slavischen Sprache Als Vuk seine Reform mit dem Ziel der Einführung der Volks ־ spräche in die Literatur begann, stand er vor der Aufgabe, die Be ־ Ziehung der künftigen Schriftsprache zur kirchenslavischen (russi- scher Redaktion) zu regeln. Die Lösung dieses Problems war von größter Bedeutung und hatte für die serbische Schriftsprache weit- reichende Folgen. Vuks Ansichten darüber wechseln im Laufe der Zeit und wir möchten ihre Entwicklung und deren Gründe verfolgen. Zu Beginn hat sich auch Vuk die serbische Schriftsprache als eine Art von "stilus medius" vorgestellt. In seiner Rezension der "Novine Serbske" von 1814 meint er, daß "oni, koji pišu, moraju paziti na govor prostoga Naroda; i neotstupajuci ot onoga treba 2 svoj jezik da uglaQavaju i da popravljaju." Das beste Mittel da- für seien die Zeitungen, da sie von den Gebildeten gelesen wer- den, die "maio ugladeniji jezik"verstehen; die Bücher sollte man aber so schreiben, daß sie auch von den einfachen Bauern verstan ־ den werden. Diese "Verfeinerung" der Sprache beschreibt Vuk so: "A akoli koje rije č i Serbske пета,., to onda može se i tuda upotre- b iti, i l i onome označeniju prilična nova skovati... Mi istina то- zemo rije č i, koje ņemamo и obštemu govoru, uzimati iz Slavenskoga jezika; a li takove rije č i treba po Serbljavati sto se vise može..." (SD XII, 15f.). Bereits hier aber zeigt sich in Vuks Auffassung des Verhält ־ nisses zwischen der serbischen und ksl. Sprache ein Unterschied gegenüber seinen Zeitgenossen: die ksl. Elemente sollten "serbi־ siert" werden. Damit meinte er eine Anpassung an die phonetischen und morphologischen Eigenarten der serbischen Sprache, wie es die von ihm angeführten Beispiele zeigen {meüu statt m e z d u , p r i j a t e l j e statt p r i v a t e l e usw.) , In bezug auf die Lexik neigt er aber schon hier zum Purismus, den er später vertreten hat. Zu k a p i j a - d v e v i - -ovata meinte er nämlich: ״Sto je god Turska kapija, to su Slaven- ske dveri, a štasu Slavenske dveri, to su Serbska vrata! Kad nebi smo imali naše rije č i, to bi drugo bilo." (SD XII, 18) . 1 Vgl. A. Leskien, Grammatik der serbo-kroatischen Sprache 1. T., 1914, S. 362. 2 Er selbst hat Sreznevskij angegeben, Mrazovics Grammatik als Muster benutzt zu haben. ־Vgl. О Vuku Karadzicu, 1968, S. 26. 3 Vgl. dazu Th. Butler, The War for a Serbian Language, a.a.O. S.1 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 2 . Die Beziehung der serbischen Schriftsprache zur kirchen- slavischen Sprache Indem er die ersten beiden Wörter aus der serbischen Sprache vertreiben wollte, mißachtete er den semantischen Unterschied, der hier be- reits zu seiner Zeit bestand, und handelte sich den berechtigten Vorwurf ein, die Verarmung der Sprache voranzutreiben. In der Rezension des "Usamljeni junoša" von 1815 erläuterte er seine Auf- fassung: "A i s p e e c v e n premda je Slavenska rijeČ, no mi bi je mogli űzeti, kad ne bi imali Srbsku riječ, koja to isto znači, a li kad imamo na što na? Zašto bi č in ili od jasna dana pomrčinu..." (GPS I, 83). Wortschatzdubletten würden nach seiner Meinung zur Begriffs Unklarheit führen. Er hat die semantische Differenzierung und stilistische Schichtung, die die Literatur schaffen und ausnützen könnte, offensichtlich nicht bedacht. Vuks erste grammatische Arbeit, die "Pismenica" von 1814 gibt uns weitere Auskunft über seine derzeitigen Ansichten. In der Darstellung der Deklination führt er mehrmals doppelte, serbische und ksl. Formen an, z.B. im Dat. Sg. der Adjektive: " Pitom om e i l i p i t o m o m u ; pervo (na me)jest padež prostoga naroda; a onaj su drugi (na mu) književnici izvadili iz cerkovni knjiga: sad ko koje vo li." Gen. PI. p i t o m i j e und p i t o m i , "pervo po govo- renju prostoga naroda... A onaj drugi padež rod. množ. nácinjen je od Slavenskoga samo što mu je otsečeno h; no može podnijeti i jedno i drugo." (GPS I, 26f.). Er selbst gebraucht beides. Zu der Deklination der Pronomina bemerkt Vuk: "U vinitelnom je- dinstvenom, и ženskom rodu ima trojako: nju, je, ju, ovo poslednje (ju) izvadili su Srbski Spisatelji iz cerkovni knjiga, a li medu prostim Serbljima nigdi se cuti nemože, nego svuda ono drugo (je) na mjesto njega. .. Ovo istina vidise da je nepravilno skraceno (od nju) a li je kod naroda и običaju.( ״GPS I, 34) . Er nimmt auch Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. In der Darstellung der Konjugation führt er u.a. die ksl. Parti- zipia a u s t i (zu jesam) und b u d u e t i (zu b iti) an, (GPS I, 43), als auch das Part. Präs. Pass, auf -m { k a r a e m i ) , zu dem er meint: "Ovo pričastije sadašnjeg vremena kod naroda nečujese, nego je uze- to iz Slavenskoga, i vec je ušlo и običaj." (GPS I, 54). Er selbst gebraucht diese Partizipia. Aus diesen Beispielen is t folgendes ersichtlich: Beim Vorhandensein doppelter, volks- und kirchensprachlicher Formen s te llt Vuk es den Schriftstellern zur freien Wahl, welche sie gebrauchen werden ("ко koje voli"). In Einzelfällen mißt er die Richtigkeit der volkssprachlichen Formen an den altkirchenslavischen als dem Maßstab (je,das der alte Gen. Sg. is t aksl. sei "nepravilno skraòeno"). Ein ähnliches Verfahren hat auch Dobrovsk^ angewandt und viele "Fehler" in der serbischen Sprache gefunden. Er läßt auch solche ksl. Formen in der Schriftsprache zu, die beim Volk nicht gebräuchlich sind. Das steht im Widerspruch mit seiner Behauptung, die Deklination und Konjugation der Bauernspra- che entnommen zu haben (GPS I, 7). Er hat vielmehr auch die Spra- che der Gebildeten, an der er teilhatte, in seine Grammatik ein- bezogen. Diese Tatsache erklärt sich aus folgenden Gründen: Vuks "Pismenica" is t von ihrem Vorbild, der Grammatik von 2 Mrazovic stark beeinflußt. Er hat zwar die Flexionsparadigmen der Volkssprache entnommen, seine Grammatik is t aber stellenweise, besonders in den theoretischen Ausführungen und in der Darstellung der Konjugation stark von Mrazovic abhängig^. Außerdem hat Vuk selbst die serbische Volkssprache zu dieser Zeit nicht gut be- herrscht, wie seine Sprache in der Vorrede deutlich zeigt. Er konnte beim Schreiben ohne den slavenoserbischen Vorrat an Aus- drucksmitteln nicht auskommen. Seine Auffassung der Beziehung zwischen der ksl. und der ser- bischen Schriftsprache hat Vuk am ausführlichsten in der Ankündi­ via free access gung des Wörterbuchs von 1816 vorgetragen• HSlavenski jezik ostaje istočnik obogaščenija Serbskom jeziku, kao i Rossijs k o m .Zu- nächst sei aber ein Wörterbuch der serbischen Volkssprache nötig, "•.- и kojem ce se soderzavati samo one rije č i, koje narod Serbski upotrebljava.•. da poznamo cijelo polje našeg jezika... da smotre pisatelji i obrazovatelj i jezika, kako narod proizvodi rije č i, ka- ko ih slaže... 1 Ähnliche Gedanken finden wir auch bei šiškov: "Drevnij Slavenskij jazyk, otec mnogich narečij^ est' koren' i načalo Rossijskago jazyka... istočnik krasnorecija našego..." Vgl. šiškov, a.a.O. S. I f . , 4. 2 So Belic, Vukova borba.., S• 97. - Außerdem vertrat Mušicki die Auffassung von einer reinen ksl. Sprache neben der Volkssprache. Vgl. ebd., S. 45ff. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Ähnliche Gedanken finden wir auch bei šiškov: "Drevnij Slavenskij jazyk, otec mnogich narečij^ est' koren' i načalo Rossijskago jazyka... istočnik krasnorecija našego..." Vgl. šiškov, a.a.O. S. I f . , 4. 2 So Belic, Vukova borba.., S• 97. - Außerdem vertrat Mušicki die Auffassung von einer reinen ksl. Sprache neben der Volkssprache. 1 Ähnliche Gedanken finden wir auch bei šiškov: "Drevnij Slavenskij jazyk, otec mnogich narečij^ est' koren' i načalo Rossijskago jazyka... istočnik krasnorecija našego..." Vgl. šiškov, a.a.O. S. I f . , 4. das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. da iz svega toga Filolog pravila za sebe izvede••• nakonac da vidimo koliko nam oskudjeva za izobraženije, usoverše- nije i obogaščenije jezika..•" Diesen Dienst soll sein Wörterbuch leisten, das außer dem Wortschatz der sog. "illyrischen" Wörter- biicher, noch ein D rittel "pravih Serbskih rije č i, koje se ni и jednome, ot rečenih Rječnika, ne nalaze" enthalten soll. "Za Sla- venske Riječi, koje blagoobražena i učena cast naroda vec i usmeno i pismeno upotrebljava, к tomu i za druge, koje nam trebaju za p rija tije bogatstva i krasote čuzeg knjižestva, nuždan nam je dru- gi Rječnik, kao pribavljenie Srbskome... Medu njima ce b iti tije - san, lak i prijatan sojuz... Óba 6e se Rječnika, s vremenom, и jedno s lit i. . . " (GPS I, 92ff.). Diese Gedanken von Vuk stehen im Einklang mit denjenigen aus der Rezension der "Novine Serbske" von 1814. Die Schriftsprache wird nicht mit der Dorfsprache gleichgesetzt, sie sollte vielmehr aus der kroatischen Schrifttradition und der aksl. Sprache schöp- 2 fen• Musickis Einfluß auf Vuk liegt u.E. hier nicht vor • Vuk erscheint vielmehr als Angehöriger derjenigen geistigen Strömung bei den Serben seit Dositej, die für die Einführung der Volksspra- che in die Literatur, aber auch für ihre Bereicherung aus der ksl. Quelle war• Zu ihr gehörten auch spätere Gegner־ von Vuk, u.a. auch J. Hadžic. In Anbetracht der Tatsache aber, daß Vuk bereits in dem darauf- folgenden Jahr eine völlig entgegengesetzte Meinung vertrat, muß man bei der Beurteilung dieser Gedanken die Möglichkeit berück- sichtigen, daß er in dieser Ankündigung einige Äußerungen aus 1 Ähnliche Gedanken finden wir auch bei šiškov: "Drevnij Slavenskij jazyk, otec mnogich narečij^ est' koren' i načalo Rossijskago jazyka... istočnik krasnorecija našego..." Vgl. šiškov, a.a.O. S. I f . , 4. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access taktischen Gründen gemacht hat1. Man kann aber mit Sicherheit so viel behaupten, daß er noch keine feste Position zwischen Kopitars Forderung nach der Sprache des einfachen Volkes und den Ansichten der übrigen gebildeten Serben eingenommen hat. Seine ursprüngli ־ che Vorstellung von der Ausbildung der serbischen Schriftsprache auf volkssprachlicher Grundlage mit gleichzeitiger "Serbisierung" der aksl. Elemente hätte, wäre sie ohne puristische Einschränkungen verwirklicht, der serbischen Sprache die Möglichkeit geschaffen, bereits zu Beginn einen reichen Wortschatz zu entwickeln. In Vuks Ansichten über die Beziehung der serbischen Schrift- spräche zur aksl. 1 Er hat z.B. aus taktischen Gründen die neue Orthographie vor dem Erscheinen des Wörterbuchs verheimlicht. ־Vgl- P. Ivic, О Vukovom Rjecniku, a.a.O., S. 41f. das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. t r i t t 1817 eine Wende ein, die er zum erstenmal in der Rezension des "Ljubomir и Jelisiumu" deutlich formuliert. Er verlangt eine klare Trennung zwischen den beiden Sprachberei- chen, lehnt das Slavenoserbische als künstlich, willkürlich und inkonsequent völlig ab und fordert zum erstenmal entschieden sei- ne Ablösung durch die serbische Dorfspräche (GPS I, 159). Diese Forderung unterstreicht er mit der Behauptung, daß bei allen VÖ1- kern "književni jezici narječija zivi naroda" sind; "n iti je коте ikad palo na um da pise jezikom, kojim n iti ко govori, n iti je kad govorio!" (GPS I, 157). Zugleich betont er die Eignung der Dorf ־ spräche zu literarischen Zwecken: ״Mi ne znamo bilise mogio kaza ־ t i ikakvim srednjim slogom što ljepše i blagorodnije, nego Srbskim seljačkim jezikom? Mi misiimo... n.p. što je god prosto u Srbskom jeziku, ono mora b iti prosto i u Jelinskom i u Latinskom; a što je visoko u Omirovoj Ilija d i, ono ne može b iti prosto ni u Srbskom jeziku." (GPS I, 165). Die Notwendigkeit der Entlehnungen aus dem Ksl. in den Fällen, wo es der Dorfspräche an Begriffen fehlt, wird zunächst nicht mehr diskutiert. Diese Wende in Vuks Haltung gegenüber der ksl. Sprache is t durch verschiedene Gründe hervorgerufen. Zweifellos hat Kopitar viel da ־ zu beigetragen. Zwei weitere Gründe haben u.E. eine wichtige Rol ־ le gespielt. Vidakovics Widerstand gegen Vuks Kritik war stärker, als Vuk es vermutlich erwartet hatte. Vidakovic g riff Vuks Vorschlag der Vidakovics Widerstand gegen Vuks Kritik war stärker, als Vuk es vermutlich erwartet hatte. Vidakovic g riff Vuks Vorschlag der 1 Er hat z.B. aus taktischen Gründen die neue Orthographie vor dem Erscheinen des Wörterbuchs verheimlicht. ־Vgl- P. Ivic, О Vukovom Rjecniku, a.a.O., S. 41f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Orthographiereform in der "Pisinenica" scharf an, worin er viele Anhänger gegen Vuk hatte, und lehnte es ab, in der "Küchensprache" zu schreiben (GPS I, 113ff.). Seine Forderungen bedeuteten prak- tisch die Beibehaltung des regellosen Zustandes in der Sprache, und Vuk mußte eine Alternative bieten. Vidakoviòs fehlerhafter und inkonsequenter Sprachgebrauch hat Vuk die Schwierigkeiten einer Verbindung der beiden Sprachen verdeutlicht und ihn zur Forderung nach völliger Trennung der beiden Sprachbereiche geführt. Der sicherste und unmittelbarste Weg, diese Trennung zu erreichen, war die lebendige und erlernbare Dorfspräche, die frei von ksl. Einflüssen war. 1 Vgl. P. Ivic, 0 Vukovom Rjecniku.., a.a.O. S. 163f Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. Es kommt noch eine wichtige Tatsache hinzu. Die Dorfspräche hatte zu dieser Zeit bereits ihre erste Affirmation in der Litera- tur erhalten. Vuk hatte schon zwei Bände mit Volksliedern herausge- geben, deren ästhetischer Wert von Jacob Grimm und Kopitar hervor- gehoben wurde. Das hat Vuk die Sicherheit gegeben, die Dorfsprache, ohne die altkirchenslavische als Stütze und Quelle, für literatur- fähig zu erklären. Das auf Herder zurückgehende Interesse der deut- sehen Romantik für Volkspoesie hat zweifellos einen Einfluß auf Vuks Sprachkonzeption ausgeübt. Die Grenze zwischen der serbischen und ksl. Sprache, sowohl in bezug auf die Phonetik, Morphologie und Syntax als auch die Lexik, soll nach Vuks Auffassung der Sprachgebrauch des einfachen Volkes bestimmen. Dieses Prinzip hat er in seinem Wörterbuch von 1818 angewandt. Er hat darin eine verhältnismäßig kleine Anzahl von ksl. Wörtern aufgenommen, die er in der Volkssprache festge- s te llt hat. Es sind Wörter, deren Lautgestalt auf ihre Abstammung entweder aus der serb.-ksl. ( e t , v a - , - a n e t v o ), oder russ.-ksl. ( e , о * ь, ъ; а < ç ; - j u 80 i ) Redaktion des Altkirchenslavischen hinweisen, oder solche, die aus den beiden stammen könnten (z d , - i j e ) . Zugleich hat er den Gebrauch ähnlicher, in der Volksspra- che nicht vorkommender Wörter entschieden bekämpft (GPS I, 81f., 148ff ., 170ff.). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Wie schwer dieses Prinzip zu verwirklichen war, hat Vuk selbst am besten demonstriert: seine Vorrede zum Wörterbuch und seine ser- bische Grammatik wären allein mit Hilfe des im Wörterbuch aufgenom- menen Wortschatzes nicht zu verstehen . Auch seine Diskussion mit Musicki im Zusammenhang mit der Benennung des Wörterbuchs is t für seine Arbeitsweise bezeichnend. "Kakvo cerno ime naöesti Srbskom Rječniku? Rjecnik, Slovar i Leksikon, to je sve maio. Sokroviice misli roda, najboljebi bilo, a li evo sijaseta: što sokrovišča пета и тот Rječniku!" (Prepiska II , 137). Die heutige Form e k r o v i š t e , die er in seiner Sprache hatte, (GPS I I I , 207, 260), gab es eben- fa lls in seinem Wörterbuch nicht, und das nächstliegende r i z n i c a kannte er nur aus den Volksliedern und als Benennung der Schatzkam- 2 mer in den Klöstern . 1 Vgl. P. Iviè, 0 Vukovom Rjecniku.., S. 53 2 Das endgültig gewählte r j e c n i k , das sicher besser war, als die Nachahmung des Herderschen Begriffes "Schatzkammer", gab es in seinem Wörterbuch auch nicht. Vera Boji 9783954793044 1 vgl. M. Kicovic, Jovan Hadzifc (Milos Svetic),1930, S. 242f. 2 Die angeführten Beispiele stellen eine Auswahl dar. Sie sind in de heutigen Orthogrphie und im Nominativ angegeben, da es Vuk nur um das lexikalische Problem geht. Die Abkürzungen AR und MR meinen die Wörterbücher der JAZU und das der Matica srpska und Matica hrvatska (ab Bd. 3 Matica srpska). R bezeichnet Vuks Wörterbuch von 1852. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. Das is t eines der Beispiele von puristischer Anwendung des Prinzips von dem volkssprachlichen Charakter der Schriftsprache, die bei Vuk zahlreich sind. So wollte er z.B. das Fremdwort І 8 t o r i j a i keineswegs durch das ksl. p o v e e t b , sondern durch p r i p o v i j e t k a ersetzt sehen, statt epoha sollte p o e t a t geschrieben werden (GPS II, 110; 1820). Die spätere Sprachentwicklung hat diesen Purismus überwunden und alle diese Wörter unter semantischer Diffe- renzierung übernommen. Vuk hat in seiner Ergänzung des St. Petersburger vergleichenden Wörterbuchs festgestellt, daß der serbischen Volkssprache Wörter für Ь г а к ъ , v o z d u c h b , ѵ к и в ъ fehlten (GPS II, 183ff.; 1822). Dennoch hat er sowohl in den 20er, als auch 40er Jahren alle Versuche der Beseitigung solcher Lücken durch Entlehungen aus der ksl. Sprache bekämpft. Seine Ansichten über die Beziehung zwischen der kirchenslavischen und der künftigen Schriftsprache enthalten folgende Komponenten: 1. Er überschätzte den Reichtum der Volkssprache, da er ungen gende Kenntnis ihrer Worte immer wieder als das Hauptproblem bezeich- nete (GPS I I I , 152; 1845). Auch wenn er zugeben mußte, (daß man ohne neue Wörter nicht auskommen wird, meinte er, daß man sie nicht ohne äußerste Not einführen soll. (GPS I I I , 153, 163, 1845). Das aber is t ein sehr subjektives Kriterium, bezogen auf verschiedene Verwendungs- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access bereiche der Sprache. 2. Vuk vertrat die Meinung, daß die Sprache sich aus eigenen Mitteln, ohne fremde Einflüsse, bilden kann. "A zar se jezik sam po sebi ne može izobraziti i uljepšati bez tuöi jezika?" (GPS I I , 137; 1821). In Bezug auf die Lexik is t diese Vorstellung unreali- sierbar. Keine der neueren Sprachen hat auf Entlehungen und Lehn- bildungen verzichten können, noch weniger konnte das die erst im 19. Jh. sich bildende serbische Schriftsprache. 2. Vuk vertrat die Meinung, daß die Sprache sich aus eigenen Mitteln, ohne fremde Einflüsse, bilden kann. "A zar se jezik sam po sebi ne može izobraziti i uljepšati bez tuöi jezika?" (GPS I I , 137; 1821). In Bezug auf die Lexik is t diese Vorstellung unreali- sierbar. das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. z e n i d b a an, die verschiedene Begriffe sind. Wahrscheinlich e. Russismus (Naš jezik 3,255); zu Vuks Zeit in der ma. Form d o z v o l e t i gebräuch- lieh (MR). Russismus, den Vuk in d. Grammatik 1818, XLIX, gebraucht- (AR). Russismus, den Vuk in d. Grammatik 1818, XLIX, gebraucht- (AR). In alten serb. Schriften nachgewiesen. (AR). In alten serb. Schriften nachgewiesen. (AR). Russismus, bei Dositej u. in Nov.Srbske 1834, 114, festgestellt- (AR). Serbisiert aus d. russ. o t s u t s t v i e (AR). Serbisiert aus d. russ. o t s u t s t v i e (AR). griech., Vuk: NPj 1,128: p a r o k ; R^: p a r o k , p a r o k i j a . Dositej: p a r o h i j a l a n . Serbisiert aus d. russ. o t s u t s t v i e (AR). griech., Vuk: NPj 1,128: p a r o k ; R^: p a r o k , p a r o k i j a . Dositej: p a r o h i j a l a n . (AR, MR). griech., Vuk: NPj 1,128: p a r o k ; R^: Russismus; v. Vuk 1814 gebraucht (GPS I, S.7). (AR)- Russismus; v. Vuk 1814 gebraucht (GPS I, S.7). (AR)- Vom Stamm in p r e - p e t i ; in d.Volkssprache von Lika verzeichnet; v. Njegoš in Gorski vijenac, Widmung, gebraucht. (AR). Vom Stamm in p r e - p e t i ; in d.Volkssprache von Lika verzeichnet; v. Njegoš in Gorski vijenac, Widmung, gebraucht. (AR). r a z n i , allg.slav., von Vuk selbst gebraucht; p o l (sexus) v. Danicic u.a. b. Domentijan festgestellt- (AR). r a z n i , allg.slav., von Vuk selbst gebraucht; p o l (sexus) v. Danicic u.a. b. Domentijan festgestellt- (AR). v. Rajic gebraucht; v. Vuk Vrčevic in der Volkssprache verzeichnet. (AR). 2 v. Rajic gebraucht; v. Vuk Vrčevic in der Volkssprache verzeichnet. (AR). 2 2 Regelmäßige Ableitung zu 8 p o r , das im R aufgenommen is t. (AR)- Drei Jahre vorher hat Vuk, ebenfalls bei Hadžic, folgende Wörter beanstandet (GPS I I I , 115f.): Es sei nach d. russ. das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. griech., Vuk: NPj 1,128: p a r o k ; R^: p a r o k , p a r o k i j a . Dositej: p a r o h i j a l a n . (AR, MR). zu: p r e n e b r e c i is t russ. p r e n e b r e c ' v. Rajic u- Dositej gebraucht. (AR). Russismus; v. Vuk 1814 gebraucht (GPS I, S.7). (AR)- Vom Stamm in p r e - p e t i ; in d.Volkssprache von Lika verzeichnet; v. Njegoš in Gorski vijenac, Widmung, gebraucht. (AR). r a z n i , allg.slav., von Vuk selbst gebraucht; p o l (sexus) v. Danicic u.a. b. Domentijan festgestellt- (AR). v. Rajic gebraucht; v. Vuk Vrčevic in der Volkssprache verzeichnet. (AR). 2 Regelmäßige Ableitung zu 8 p o r , das im R aufgenommen is t. (AR)- vom aksl. e ç p r ç g b , s ç p r ç g a . Vuk gebraucht s u p r u g a für die Fürstin Milica (Milos 82) (AR). s u p r u z n i k (socius) in d. Volkssprache v. Montenegro verzeichnet. (AR). b r a k / b r a o n i • b r a k o l o m e t v o d o z v o l i t i n e i z v ( j ) e s t a n n e v i n o p r e d ( i j ) e l i t i o t s u s t v o p a r o k i j a p a r o h i j a l a n p r e n e b r e g n u t i p r i m ( i j ) e t i t i p r e p o n a r a z n i p o l ehodno 8 p o r a n e u p r u g , s u p r u g a , e u p r u z n i k Das Wort kommt in alten serb. Denkmälern vor, z.B. bei Sv.Sava, auch in den sog. illyrischen Wörterbüchern. (AR)• Vuk gibt stattdessen u d a j a u. das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. Keine der neueren Sprachen hat auf Entlehungen und Lehn- bildungen verzichten können, noch weniger konnte das die erst im 19. Jh. sich bildende serbische Schriftsprache. 3. Er unterschätzte den Wert der Wortschatzdubletten, (Typ: v r a t a - d v e r i , oder g l a s - v i j e s t ) . Deshalb forderte er, daß nichts ohne Not entlehnt wird. 4ê Seine Ablehnung der Lehnbildungen beruht auf seiner Auffassun der Rolle der Schriftsteller und des Volkes bei der Sprachbildung. 4ê Seine Ablehnung der Lehnbildungen beruht auf seiner Auffassun der Rolle der Schriftsteller und des Volkes bei der Sprachbildung. Es is t zur Beurteilung dieser Ansichten von Vuk aufschlußreich zu sehen, welche von den Wörtern, die er in seiner K ritik der Spra- che von J. Hadziò als "unserbisch" und "unnötig" abgelehnt hat, zum festen Bestandteil der heutigen Schriftsprache gehören. Jovan Hadžic gehörte zu denjenigen Schriftstellern, die von An- fang an für die Volkssprache in der Literatur waren; er forderte die klassizistischen Dichter auf, im Geiste der Volkspoesie zu Schreiber selbst schrieb er im Deseterac . Sein Streit mit Vuk betraf vor al- lem die Orthographie, (Hadžic war einer der konservativsten Verfech- ter der sog. historischen Orthographie), und seine unglückliche Ein- mischung in die linguistischen Probleme, von denen er nicht viel Ahnung hatte. Als Verfasser der serbischen Gesetze hatte er mit dem Problem der fehlenden Abstrakta in der Sprache zu kämpfen. Vuk schrieb 184S gegen Hadziès Sprache: "Da je Srpski zakonopise upravo poznavao naród, za koji pisę zakone, i da je znao, kako naroc misli i govori o onijem stvarima,koje on pise...", hätte er die Ge- 2 setze ohne folgende Worte geschrieben (GPS I I I , 154) : 00051004 - 75 - Das Wort kommt in alten serb. Denkmälern vor, z.B. bei Sv.Sava, auch in den sog. illyrischen Wörterbüchern. (AR)• Vuk gibt stattdessen u d a j a u. z e n i d b a an, die verschiedene Begriffe sind. Wahrscheinlich e. Russismus (Naš jezik 3,255); zu Vuks Zeit in der ma. Form d o z v o l e t i gebräuch- lieh (MR). Russismus, den Vuk in d. Grammatik 1818, XLIX, gebraucht- (AR). In alten serb. Schriften nachgewiesen. (AR). Russismus, bei Dositej u. in Nov.Srbske 1834, 114, festgestellt- (AR). Serbisiert aus d. russ. o t s u t s t v i e (AR). das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. Das Wort is t aber eine regelrechte Ableitung zum aksl. e o p s t v o über 80p 8 t v e n , das fast alle "illyrischen" Wörterbücher verzeichnen. (AR). Vuk meint: " ... kad mjesto h a b e r imamo g l a s > sta ce nam v i j e s t ? " (GPS I I I , 172f.) Die Übernahme dieser Wörter in die serbische Schriftsprache hat sich gegen Vuk durchgesetzt. Eine ähnliche Haltung gegenüber den vermeintlichen Kirchensla- venismen zeigt Vuk auch in seiner K ritik von V. Laziò^. Man brauche v ( j ) e r o z a k o n und v ( j ) e r o i a p o v d j ) e d nicht, da das Volk v j e r a und z a k o n habe; 8p 080ban und s o p s t v e n i k seien unserbisch, das letztere sei eine Lehnübersetzung des dt. Eigentümer, das Volk habe dafür s a i b i j a . Das Wort is t aber eine regelrechte Ableitung zum aksl. e o p s t v o über 80p 8 t v e n , das fast alle "illyrischen" Wörterbücher verzeichnen. (AR). Vuk meint: " ... kad mjesto h a b e r imamo g l a s > sta ce nam v i j e s t ? " (GPS I I I , 172f.) Die Übernahme dieser Wörter in die serbische Schriftsprache hat sich gegen Vuk durchgesetzt. Die meisten der betreffenden Wörter kommen also auch in den alten serbischen Schriften vor. Diese Schriften sind zwar in der serb.-ksl. Sprache geschrieben, sie gehören aber zur serbischen Schrifttradidtion. Es is t zweifellos richtig, daß viele von die- sen Wörtern erst durch den Einfluß der russ.-ksl. Sprache bei den Serben wieder gebräuchlich geworden sind. Die meisten der betreffenden Wörter kommen also auch in den alten serbischen Schriften vor. Diese Schriften sind zwar in der serb.-ksl. Sprache geschrieben, sie gehören aber zur serbischen Schrifttradidtion. Es is t zweifellos richtig, daß viele von die- sen Wörtern erst durch den Einfluß der russ.-ksl. Sprache bei den Serben wieder gebräuchlich geworden sind. Bekanntlich hat sowohl die russ. als auch die serb. Sprache, neben der besonderen Weiterentwicklung, einen Teil des ksl.Erbes unverändert bewahrt. das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. t o r z i e c e gebildet, das Volk habe dafür v a 8 a r , p a n a f t u r , p a z a r , s a j a m d e r n e k . Das Wort ist e. regelrechte Ableitung zu Ьгъдъ , das Danicic in d. alten serb. Schriften festge- s te llt hat. (AR). In d. heutigen Sprache sind р а п а З и г und d e r n e k ungebräuchlich, die übrigen sind alle unter semantischer Differenzierung übernommen worden. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vuk: " u narodu ja do danas n iti sam čuo ove r i- ječi, n iti korijena... (p o z o r , p o z r e t i i l i barem z r e t i ) ." p o z o r i s t e p o z o r i s t e ) Das Wort is t e. Bildung zu p o - z r e t i , das Danicić b. Domentijan festgestellt hat. (AR). p o n o b p r e v a l i l a sei falsch, richtig sei: p o r t o t i p r e v a l i l o . Die heutige Schriftsprache zieht Hadzics Ausdrucks- weise vor. (MR). Wiewohl Vuk in den sprachlichen Angelegenheiten Hadzi& bei wei- tem überlegen war, war Hadziòs Einwand richtig, daß es eine Sache für sich sei, wenn im Volk seit Jahrhunderten über Heirat gesprochen wird, und eine andere, diese Begriffe gemäß den Anforderungen der Zeit präzis mit Worten fassen zu müssen (GPS I I I , 252). Eine ähnliche Haltung gegenüber den vermeintlichen Kirchensla- venismen zeigt Vuk auch in seiner K ritik von V. Laziò^. Man brauche v ( j ) e r o z a k o n und v ( j ) e r o i a p o v d j ) e d nicht, da das Volk v j e r a und z a k o n habe; 8p 080ban und s o p s t v e n i k seien unserbisch, das letztere sei eine Lehnübersetzung des dt. Eigentümer, das Volk habe dafür s a i b i j a . 1 Vuks K ritik der extrem puristischen Sprachreinigungsversuche von Lazifc war sonst richtig, wenn sie nicht die Kirchenslavismen betraf. (GPS I I I , 162ff.). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. Während die slavenoserbischen Schriftsteller die einzelsprachlichen Besonderheiten irrtümlicherweise als zwei Stilarten betrachteten und nebeneinander verwendeten, ging Vuk extrem in die entgegengesetzte Richtung, indem er den gemeinsamen, in der serbischen Volkssprache nicht nachgewiesenen Wortschatz 1 Vuks K ritik der extrem puristischen Sprachreinigungsversuche von Lazifc war sonst richtig, wenn sie nicht die Kirchenslavismen betraf. (GPS I I I , 162ff.). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access (brak, suprug) ablehnte. Der Versuch, solchen Wörtern den Eingang in die serbische Schriftsprache zu versperren, war sowohl falsch als auch unhaltbar. Die serbische Sprache konnte auch auf die Ent- lehnungen aus dem Russischen nicht verzichten. Vuks Prinzip, bei der Übernahme aus den Fremdsprachen auf die phonetisch - morpholo- gischen Eigenarten des Serbischen zu achten, hat sich dabei im We- sentliehen durchgesetzt. Vuks Haltung gegenüber den Kirchenslavismen in den 20er Jahren mit seiner Forderung nach einer strengen Grenzziehung zwischen den beiden Sprachen hat sich als sinnvoll erwiesen, weil sie dazu bei ־ getragen hat, daß die volkssprachliche Grundlage der Schriftsprache gefestigt wurde. In den 40er Jahren, (alle angeführten Beispiele stammen aus dieser Zeit), erscheint sie als puristisch. Das Problem lag in den 40er Jahren nicht mehr in der Gefahr einer Überfremdung durch die russ.־ksl. Sprache, es ging vielmehr darum, die volks ־ sprachliche Grundlage auf dem schnellsten Wege in eine elastische und wendige Schriftsprache auszubauen, die den Anforderungen der Zeit genügen könnte. Einen bedeutenden Beitrag dazu hat Vuk selbst mit seiner Bibelübersetzung geleistet, indem er von seinem Prinzip des Festhaltens an der reinen Volkssprache, vor allem in Bezug auf Lexik, abgewichen is t. Vuks Übersetzung des Neuen Testaments wird mit Recht als ein Kunstwerk und eine Tat von großer philologischer Bedeutung gewür- digt1. Vuk hat darin nicht nur seinen synthetischen Dialekt in der schriftstellerischen Praxis gezeigt, sondern auch ein Beispiel dafür gegeben, wie die Schriftsprache aus der ksl. Quelle und durch Neubil- dungen bereichert werden kann. In seiner Übersetzung hat Vuk, wie er in der Vorrede selbst an ־ gegeben hat, neben den in der Volkssprache vorkommenden dreißig Tur ־ zismen auch "49 rije č i slavenskijeh koje se и našemu narodu ne govore, a li se lasno mogu razumjeti i s narodnijem rijecima pomiješļati" und "47 rije č i koje su od Slavenskijeh posrbljene" aufgenommen. 1 vgl. A. Belic, Vukova borba... S.240; vgl. I Grickat, Vukov prevod Novog zaveta kao spomenik velikog filoloskog nastojanja. In: JF 26, 1963/64, S. 219-245. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. P. Вогбіс, О Vukovu Novom zavjetu. Sonderdr. aus: Bogoslovlje IX, 2 , 1934 ; vql. M. Popovic, Vuk Stef. Karadzic, 1787 1864 ־־, Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. Außerdem "ima i rije č i 84 kojijeh nijesam čuo u narodu da se govore, nego sam ih ja načinio." (GPS I I I , 266f.) Schon J. Stejić hat darauf hingewie ־ sen (GPS I I I , 582; 1849), und die spätere Forschung hat es bestätigt# 1 vgl. A. Belic, Vukova borba... S.240; vgl. I Grickat, Vukov prevod Novog zaveta kao spomenik velikog filoloskog nastojanja. In: JF 26, 1963/64, S. 219-245. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 78 ־ daß darin mehr ksl• Wörter als von Vuk angegeben, wie auch viele Gräzismen,enthalten sind1. Belic hat diese drei von Vuk genannten Wortgruppen untersucht und dabei folgendes festgestellt: 1• Bei der Übernahme aus dem Kirchenslavischen hat Vuk darauf geachtet, daß die betreffenden Wörter in ihrer phonetischen und morphologischen Struktur der serbischen Sprache entsprechen (also: ž r t v a und nicht z e r t v a ; die der ksl. und serbischen Sprache gemein- samen Suffixe - t e l j , - n i k , - o s t , - 8 t v o , - n i ) . Neun von ihnen hat er in die zweite Auflage seines Wörterbuchs aufgenommen. Weniger als die Hälfte dieser Kirchenslavismen is t zum allgemeinen Gut der ser- bischen Schriftsprache geworden. 2. Bei der Serbisierung hat Vuk die betreffenden Wörter den pho- netisch-morphologischen Eigenarten der serbischen Sprache angepaßt: er hat also an Stelle der russ.-ksl. die serbischen Laute gesetzt. Viele von diesen Wörtern waren wahrscheinlich in dem damaligen Sprachgebrauch, allerdings in der russ.-ksl. Form, vorhanden. Dar- auf deutet die Tatsache hin, daß Vuk viele von ihnen (11) in sein Wörterbuch aufgenommen hat. Fast alle sind sie in die serbische Schriftsprache eingegangen. 2. Bei der Serbisierung hat Vuk die betreffenden Wörter den pho- netisch-morphologischen Eigenarten der serbischen Sprache angepaßt: er hat also an Stelle der russ.-ksl. die serbischen Laute gesetzt. Viele von diesen Wörtern waren wahrscheinlich in dem damaligen Sprachgebrauch, allerdings in der russ.-ksl. Form, vorhanden. Dar- auf deutet die Tatsache hin, daß Vuk viele von ihnen (11) in sein Wörterbuch aufgenommen hat. Fast alle sind sie in die serbische Schriftsprache eingegangen. 3. das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. Bei der Bildung von neuen Wörtern hat Vuk sich nach den in der Volkssprache produktivsten Bildungskategorien gerichtet: die meisten der 84 Wörter haben folgende Suffixe: - а с , - a c , - i c a , - a r , ־־i k , - I j i v , - n j e , -o a t , - 8 t v o , —8 1vo . Vuk hat dabei eine ausge- zeichnete Kenntnis der serbischen Sprache gezeigt; sein Verfahren hätte den damaligen Schriftstellern als Vorbild dienen können. Die meisten dieser Wörter sind in die Schriftsprache eingegangen, ob- wohl in manchen Fällen Neubildungen mit anderen Morphemen entstan- den sind (z. B. p o u ö a n zu Vuks p o u o l j i v , das eine andere Bedeutung erhalten hat)- Vuk hat 29 von diesen Wörtern in sein Wörterbuch 2 von 1852 aufgenommen . 3. Bei der Bildung von neuen Wörtern hat Vuk sich nach den in der Volkssprache produktivsten Bildungskategorien gerichtet: die meisten der 84 Wörter haben folgende Suffixe: - а с , - a c , - i c a , - a r , ־־i k , - I j i v , - n j e , -o a t , - 8 t v o , —8 1vo . Vuk hat dabei eine ausge- zeichnete Kenntnis der serbischen Sprache gezeigt; sein Verfahren hätte den damaligen Schriftstellern als Vorbild dienen können. Die meisten dieser Wörter sind in die Schriftsprache eingegangen, ob- wohl in manchen Fällen Neubildungen mit anderen Morphemen entstan- den sind (z. B. p o u ö a n zu Vuks p o u o l j i v , das eine andere Bedeutung erhalten hat)- Vuk hat 29 von diesen Wörtern in sein Wörterbuch 2 von 1852 aufgenommen . Vuks Sprache in der Übersetzung des Neuen Testaments zeigt nicht nur im lexikalischen Bereich Neuerungen, sondern sie is t auch durch ihre syntaktischen Eigenschaften mit der Volkssprache nicht identisch: mit den archaisierenden syntaktischen Wendungen nähert sie sich der ksl. Sprache- In seiner Übersetzung hat Vuk den Gedanken von Jacob Grimm, daß ein "dichter ,.״über Stoff, geist und regel der spräche gewisz ganz anders zu gebieten weisz, als grammatiker und Wörterbuchmacher zusammengenommen", und daß die Schriftsprache aus dem Zusammenwir- ken von Volk und Dichtern entsteht (GKS V III, 30, 36), in der Praxis verwirklicht. Belgrad 1964, bes. S. 370. 2 Vgl. A. Belic, Vukova borba.., S. 244ff. 1 Vgl. Skerlic, a.a.O., S. 437. 2 Vgl. A. Belic, Vukova borba.., S. 255f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. Es is t interessant zu sehen, welche von die sen Wörtern Vuk in sein Wörterbuch von 1852 aufgenommen hat und wie- viele von ihnen zum Bestandteil der heutigen serbokroatischen Schrif spräche gehören. Es handelt sich dabei nicht um Steji&s Neubildungen sondern um die Wörter, die im damaligen serbischen Schrifttum ge- bräuchlich waren^. daß auch den anderen Schriftstellern das erlaubt sein muß, was Vuk praktiziert hat. Er hat eine beträchtliche Anzahl von Wörtern ange- führt, die nach seiner Meinung in die serbische Schriftsprache auf- genommen werden sollten. Es is t interessant zu sehen, welche von die sen Wörtern Vuk in sein Wörterbuch von 1852 aufgenommen hat und wie- viele von ihnen zum Bestandteil der heutigen serbokroatischen Schrif spräche gehören. Es handelt sich dabei nicht um Steji&s Neubildungen sondern um die Wörter, die im damaligen serbischen Schrifttum ge- bräuchlich waren^. Stejic Vuk Heutige Stejic Vuk Heutige R2 Schrift- R Schrift Sprache Sprache sposobnost1 — + priznatelnost 1 + strogost' - + uljudnostי+ + sobstvenost1 — + ugodnost* - + gordost' - + ljutost* — + dražest' — + udobnost 1 - + živost 1 с + gladkost' - + cestnost 1 - — rapavost' -־+ blagost' + + mekost* - + opasnost 1 - + krutost 1 - + jarost' + + oblost 1 - + podlost ״ —+ okruglost י+ + naustraiimost' - kratkost' + + povinost' - — plitkost 1 - + nevinost 1 — + radinost 1 — + skromnost' — + nejakost' - + uspešnost' - + jakost' - + ravnost ״ —+ krepost' + + raznost 1 - ungew. odvažnost' - + izobraženost' - + covecnostי- + obrazovanost' - + umnost 1 - + stalnost' — + zakonost 1 — arch. samostalnost' - + zakonitost 1 - + prevratnost1 - ustavnost' - + nedoraslost1 - + državnost״ —+ + - — - - -־ - - - + + - — - - + - - - — - - — 1 Stejics Beispiele mit seiner Orthographie werden in der für das Russ. üblichen Transliteration angeführt. Zur Überprüfung ihres Eingangs in die serbokroat. Schriftsprache wurde überwiegend das Wörterbuch der beiden Matica und z.T. das Wörterbuch der JAZU herangezogen. Die Bezeichnungen: hist(orisch), veralt(et), ar ־ ch(aisch) und ungew(öhnlich) , (das letztere meint die nur in ver- schiedenen Lexika nachgewiesenen Wörter), wurden aus dem Matica- Wörterbuch übernommen. Die undeutlichen Beispiele von Stejic wur- den in meiner Lesart [ . das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. Der Grammatiker Vuk hat aber seine schriftstel- lerischen Erfahrungen in seine Theorie nicht explizite aufgenommen. Theoretisch gefaßt bedeutete sein Verfahren, vor allem in bezug auf die Lexik, die Verwirklichung derjenigen Prinzipien, die wir in der Anfangsphase seiner Arbeit festgestellt haben. Die ksl. Spra- che erscheint als eine Quelle der Bereicherung der serbischen Schriftsprache unter dem obersten Grundsatz, daß die phonetisch- morphologische Struktur des Serbischen beachtet wird. Vuks Spra- che in der Bibelübersetzung entspricht der Ansicht von Jacob Grimm, daß die Volkssprache als Schriftsprache gleich zu Beginn eine not- wendige Distanz zur gesprochenen Sprache gewinnt. Es is t als ein Versäumnis von Vuk anzusehen, daß er sein Ver- fahren bei der Bibelübersetzung nicht theoretisch begründet hat. Das hätte der jungen Dichtergeneration als Leitsatz in der Praxis helfen können und dem späteren philologischen Dogmatismus, der die Dichtung nur nach der Reinheit der Volkssprache bewertete1, den Weg versperrt. Vielleicht hätte es auch dazu beigetragen, daß die Bedeutung dieses Werkes für die serbische Schriftsprache von Vuks Zeitgenossen erkannt worden wäre; statt dessen entzündete sich der Streit um seine angeblich schädlichen Folgen für die orthodoxe Kirch 2 Daničics hohe Einschätzung dieses Werkes von Vuk is t bekannt . Wir möchten uns mit J. Stejićs sprachlichen Bemerkungen zu Vuks Übersetzung (GPS I I I , 471 - 593) befassen, die in verschiedener Hinsicht bedeutend sind. Stejic war einer der beachtenswertesten Kritiker von Vuk, der ihm wohlgesonnen war, obwohl er zu einzelnen Fragen (u. a. zur Orthographie) eine andere Meinung vertrat. Er setzte sich vor allem für mehr Freiheit in bezug auf die Entlehnun- gen aus der ksl. Sprache ein, als Vuk sie zulassen wollte. Nach Erscheinen von Vuks Übersetzung stellte er die Forderung auf, Belgrad 1964, bes. S. 370. 2 Vgl. A. Belic, Vukova borba.., S. 244ff. 1 Vgl. Skerlic, a.a.O., S. 437. 2 Vgl. A. Belic, Vukova borba.., S. 255f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Belgrad 1964, bes. S. 370. 2 Vgl. A. Belic, Vukova borba.., S. 244ff. 1 Vgl. Skerlic, a.a.O., S. 437. 2 Vgl. A. Belic, Vukova borba.., S. 255f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access daß auch den anderen Schriftstellern das erlaubt sein muß, was Vuk praktiziert hat. Er hat eine beträchtliche Anzahl von Wörtern ange- führt, die nach seiner Meinung in die serbische Schriftsprache auf- genommen werden sollten. - ־ - - - - - - - - - - - ־ - - - - - bezzazornost' — + bezzakonst1 — arch. bezkonačnost' + bezmernost' + + bezopasnost1 - + bezplotnost' - - bezgrësnost1 + + bezpoleznost' - arch . bezbëdnost' — + bezosnovanost1 - — bezmyslenost' — + bezporočnost' - -־ bezcislenost1 - - bez čuvstvenost1 — — bogopodobnost' — - tu zite l1 + slušatel' — + upravitel' + + popecitel' - h is t. osmotritel1 - arch. nadziratel' - + predsedatel1 - + zasedatel' — — ravnitel1 — + iskupitel1 — + delovoditel1 — — perovoditel ' - — skupitel' — — obitatel• — — z ite l’ - arch. staratel' — + laskatel' - arch. razvratitel' - — prositelי —arch. molitel1 — + uvreditel1 - — spasitel1 - + stvoritel1 + + meritel1 — pokazatel1 — + donositel1 - ungew izvestitel״ —+ isçytatel1 — arch. racunovoditel' + izsleditel' — — izvršite l1 — + gubitel'־+ narušitel' — + pravitel1stvo _ h is t. upravitel1stvo - + popeCitel1stvo - h is t. das ksl. o t o neben dein serb. 8 t o auf (GPS I, 36).. . . ] nachgeprüft. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 00051004 - ו8 - - ־ - - - - - - - - - - - ־ - - - - - nadziratel1stvo - + predsedatel' stvo — - zasedatel1stvo — - + ־+ - + - + - + - + - + - + - + - + - + - + - + ־+ - + - + - + - + - + - + - + - + ־+ - + - + - + - + - + - + - + [+] [+] - + - + - + - + - + - + - + - + + + - + - + - + - + - + - + Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM družstvenostי - narodnost' ־ sklonost' - naklonost' - blagonaklonostי - pristojnost' - blagopristojnost' dobrovol'nost' - blagovol' nost' zlovo^nost* - zavisimost' nezavisimostי bytnost' - sustnost* odkupnost' - pristupnost' - osnovnost* - sadasnjost' - proslost' ־ buducnost' - postepenost1 - smotrenost' - nesmotrenost1 - tocnost' - netocnost' - ispravnost' - neispravnost' - slicnost' ־ neslicnost' - izmyslenost' - istin ito st' - razvratnost' - raskosnost' - razuzdanost’ - zanimatel' nost' nadulost' - 'olost' [oholost] [+] lazlivost' - bojazl1ivost' - pozorl1ivost' podkupi״ivost' - podmitl1ivost1 - rastocl'ivost' pohotl*ivost' - svojstvenost' - podozritelnost' izvestnost* - neizvestnostי - pravednost* + nepravednost1 - pristrastnost' - nezakonost1 vaznost' - nistetnost' - okolnost' - nadleznost* - - - - - ־ - - - - - - - - ־ - - - - - - - [+] - - - - - - - [+] - - - - - - - - + - - - - - - zanimatel' nost' okolnost' 00051004 00051004 ־ 82 ־ staratel'stvo - + razpis + nacalstvo *־ —popis - nacalnicestvo - — prepiska čovečestvo — veralt. grimetka cest ' otečestvo - veralt. + božestvo w - « počest1 - sustestvo — — oglas - jedinstvo + + vkus — spokojstvo - + sluh — nespokojstvo - + duh + malolëtstvo - + vozduh -» punoletstvo - + (povetarce?) — jedinozenstvo - + uspeh - mlogoženstvo — + os nov — bezbožstvo - + savez bezvërstvo — + obzor nevërstvo - + zadatak — ustrojstvo - + odskok + svojstvo — + naskok - rodstvo - + odboj — blagoustrojstvo - + iz liv — samoubijstvo laskatel1stvo — + uliv — - + odbor + obstoja te l' stvo — + izraz — slëdstvo - arch. - ־ - - - - - - - - - - - ־ - - - - - cel' — sredstvo - + molba — veličestvo - - prosba — vysoãestvo - - tražba — obštestvo - arch. žalba + rukovodstvo - + utok + pro i z vod s tvo - + natisk — vërolomstvo - + potisk — odsustvo — + podlog — prisustvo — + izlog - blagorodstvo - + predlog — dokazatelstvo — + zavitak + ę svemogucstvo — + popravka - ratarstvo — + uslove - rudarstvo - + povod - postrojstvo — — spëv־ voinstvo — veralt. plovitva - v è poslëdica - vest1 w — + raznoglasica — cin + + izvoz + po jav - + uvoz - dokaz - + unos - ob jav — — iznos + krug + + vi'd [vid] [ + ] pozyv — + uzrast + prizyv — + nagon + padezנ+ pobëda by'ce [bice] + razkaz — — [ + ] navod — + prestupak + uvod — + otačbina + izvod - + brak - opis - + ukaz - - + razpis + *־ —popis - - — prepiska — veralt. grimetka cest ' - veralt. + - « počest1 - — — oglas - + + vkus — - + sluh — - + duh + - + vozduh -» - + (povetarce?) — - + uspeh - — + os nov — - + savez — + obzor - + zadatak — - + odskok + — + naskok - - + odboj — - + iz liv — — + uliv — - + odbor + — + izraz — - arch. cel' — - + molba — - - prosba — - - tražba — - arch. žalba + - + utok + - + natisk — - + potisk — — + podlog — — + izlog - - + predlog — — + zavitak + — + popravka - — + uslove - - + povod - — — spëv־ — veralt. - ־ - - - - - - - - - - - ־ - - - - - plovitva - poslëdica - — + raznoglasica — + + izvoz + - + uvoz - - + unos - — — iznos + + + vi'd [vid] [ + ] — + uzrast + — + nagon + נ+ pobëda by'ce [bice] + — — [ + ] — + prestupak + — + otačbina + - + brak - - + ukaz - + - + - - — — + -» — - — — + - — — — + — — — — — + 00051004 00051004 - 83 ־ - + meteznik + - + oblastnik _ veralt — — posadnik — — + obsadnik + + + odvažnik - + odrodnik _ - + učastnik veralt - — umnik — + prijemnik — + + + punomocnik — + — + punovlastnik — + - + bezdëlnik — veralt — + vlastnik + + + + državnik _ + - + umivalnik _ - + naselënik я. + + istočnik _ + - + izobražen + — — obrazovan — + - + stalan + + - - samostalan + + - + sposoban — + — + strog — + - + točan + + + ispravan — + — — čestan + — — opasan — + — razdražen — + — + sobstven - + - + svojstven — + — + podal - + - + bezopasan — + + + bezkonačan — + + + bezgrešan + + - + bezčislen — — - + bezčuvstven — ־־+ bezurnan + + — - bezsmylen — + [+1 M bezporocan — + A - povin - — # + + nevin — + veralt. porocan — + + 1jubavan + + — + 1jubezan - + - + savëstan — + bezsavëstan — + — + okruzen + ־+ spokojan — + - veralt. pristupan — + - + izvanredan — + + črezvičajnyj — - + + podoban — + - — uspēšan — + — + slëdstven — + + _ vera — + _ vera — + — + — + — vera + + _ + _ я. + _ + + — + + + + + — + — + + — + + — + — + - + — + - + — + — + + + — — — + + — + — + - — — + — + + + - + — + — + + — + — + — + — - — + — + — + — + — - 9783954793044 — — + — — + — — - — — + - — — — — — — — — — — — 978395 2019 05 uvoznik izvoznik 00051004 00051004 - 84 - zakonno odsudno pobuditi uzbuditi izvëstiti kazniti izslëditi is tra ž iti ispytati zanimati odobriti u s tro jiti ustanoviti opunomociti oblagodariti obožavati predstaviti predložiti pokolebati narušiti vospitati proizvoditi prisustvovati odsustvovati iz ja v iti izjasniti zlosloviti preduprediti opredeliti sačiniti izobraziti prosvëtiti nadležati uvažiti rēšiti zaklju č iti dëlati razsuditi razsmotriti nastavljāti umstvovati čestvovati žertvovati nadzirati obnarodovati dējstvovati rukovoditi zapostaviti pobudenē uzbudenē odobrenē predstavlënë predloženē skroman - + blagovoljan - - naklonēn - + blagonaklonēn — + bogopodoban — + izvēstan — + neizvēstan - + važan - + malovažan — + ništetan - + priznatelan - - blagodaran - + udoban - + uman + + bezuman + + nesmotren - + razvratan - + raskošan — + buran - + kaznēn - + pobuden - + uzbuden — + sluzben - + zvaničan - + dužnostan — podozritelan - - počastan pravitel* stven - + - — božestven - - otečestven - vera upotrebitelan — — zanimatelan — — primënl' iv pozorl1iv — + — + trudoljubiv — + čovekoljubiv — + slavoljubiv - + častoljubiv - + bogoljubiv - + strastoljubiv - + pristrastan - + neustraŠim - — zavisim - - nezavisim - - nevredim - - neosporym — — nepokolebim - — vidim - — uslovno - + bezuslovno - + burno — + prenosno — + odnositelno - - ispravno + + zakonno odsudno pobuditi uzbuditi izvëstiti kazniti izslëditi is tra ž iti ispytati zanimati odobriti u s tro jiti ustanoviti opunomociti oblagodariti obožavati predstaviti predložiti pokolebati narušiti vospitati proizvoditi prisustvovati odsustvovati iz ja v iti izjasniti zlosloviti preduprediti opredeliti sačiniti izobraziti prosvëtiti nadležati uvažiti rēšiti zaklju č iti dëlati razsuditi razsmotriti nastavljāti umstvovati čestvovati žertvovati nadzirati obnarodovati dējstvovati rukovoditi zapostaviti pobudenē uzbudenē odobrenē predstavlënë predloženē izslēdene iz javlënë izjasnënë - + - - - + — + — + — + - + - + — + - + - - - + - + + + + + - + - + — + - + - + - + — + - + - + — - - - + - — - - - vera — — — — — + — + — + — + - + - + - + - + - + - — - - - - - - — — - — - — - + - + — + — + - - + + — + — + - + Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM 00051004 00051004 - 85 - (izjasnenije) - — obštavanē — — kolebanë - + nastavljanë + + vospitavanë - - (nastavlënije) - - (vospitanije) - — umstvovanë - + oprëdëlënë - + ispravljanë + + (opredëlënije) - - čestvovanē — — sačinjavanë — + Žertvovanē - - (sačinēnije) - - obnarodovanë — + izobraženē — + (obnarodovan i j e) — — uobraženē — + dëjstvovanë — + uvaženē - + rukovodenë - + rēšavane - + ustrojavanë - + (rēšenije) - — (ustrojenije) — — zaključenē - + razsmatranë + + dëlanë + + razsmotrenë — - odnoŠenē + + ustanovlënë — + sporazumlënë - ungew. nadziravanë + + povërëne - + (nadziranije) - - Schon auf den ersten Blick f ä llt es auf, daß Vuk verhältnismäßig weniqe der angeführten Wörter (64 von 518) in sein Wörterbuch aufge- nommen hat, während die meisten von ihnen (412) in die serbokroa- tische Schriftsprache eingegangen sind. Vuks Leitsatz bei der Ab- fassung des Wörterbuchs war der Nachweis in der lebendigen Volksspra- che, z.T. auch der städtischen (vgl. z.B. j a r o e t ); das, was nach sei- ner Meinung Schriftserbisch war, hat er nicht aufgenommen. Daraus er- klärt sich die Tatsache, daß sein Wörterbuch so wenige Abstrakta auf - o e t verzeichnet, obwohl es die meisten Adjektive, von denen diese Abstrakta gebildet sind, enthält. Die meisten der 106 übriggebliebe- nen Wörter von Stejic sind in etwas veränderter Form in die Schriftsprache übernommen worden. Bei den wenigen übrigen handelt es sich um Russismen, die der phonetisch-morphologischen Struktur der serbischen Sprache nicht entsprechen, z.B. die Fälle mit dem e an Stelle der reduzierten Vokale { z e r t v o v a t i usw.), oder die Ad- jektive auf -гт (russ. - i m y j ) und die Substantive mit dem Suffix - n i j e , (der bekanntlich ein Kirchenslavismus in der russ. Sprache is t), an Stelle von - n j e ; diese Fälle wurden später duch andere B il- dungen ersetzt. So wurde auch die Produktivität des Suffixes - t e l j später durch - l a c eingeschränkt. In bezug auf die morphologische Struktur dieser Wörter is t es auffällig, daß die meisten von ihnen mit den produktivsten Suffixen der serbischen Sprache gebildet sind, denjenigen nämlich, die auch In bezug auf die morphologische Struktur dieser Wörter is t es auffällig, daß die meisten von ihnen mit den produktivsten Suffixen der serbischen Sprache gebildet sind, denjenigen nämlich, die auch Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM in Vuks Kirchenslavismen, Serbisierungen und Neubildungen in der Bibelübersetzung dominieren: - 0 8 t , - t e l j , - 8 t v o , - i k , - e n , - a n , - I j i v , - n j e . Das is t ein Zeichen dafür, daß bei den damaligen Schriftstellern das Gefühl für die Natur der serbischen Sprache be- reits entwickelt war; auch die Simplizia sind dafür besonders be- zeichnend• Im Hinblick auf die semantische Seite f ä llt auf, daß es sich überwiegend um abstrakte Zivilisationsbegriffe handelt, an denen in der Volkssprache Mangel bestand. Die meisten dieser Wörter sind auch für das heutige Leben unentbehrlich (vgl. 1 Matica srpska hat die Ausarbeitung eines Wörterbuchs der slaveno- serbischen Literatursprache geplant. - Vgl. I. Grickat, U сети je značaj i kakve su specificnosti slavenosrpskog perioda, a.a.O. S. 63ff. 2 Vgl. ebd., S. 64 . Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Das g ilt offensichtlich auch für nichtslavische Fremdwörter. Ein großer Teil der von V. Mihajlovic, Grafla za rečnik stranih reci и predvukovskom periodu I- II, 1972/74, verzeichneten Fremdwörter eu- ropäischer Herkunft gehört zum festen Bestandteil der heutigen Schriftsprache. 2 Vuks Ansichten über die Dialekte sind eines der besterforschten Probleme im Zusammenhang mit Vuks Tätigkeit. Die grundlegenden Stu־ dien von A. Belic wurden in der neueren Zeit besonders durch die Arbeiten von P. Ivic sowohl mit neuen Erkenntnissen als auch me- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access etwa u a t a v n o s t , d r z a v n o a t , p r 0 8 v e t a , a a d a a n j o a t , p r o s l o a t , b u d u b n o a t ). Was ihre Herkunft be- t r i f f t , so is t es nur für die Sprachgeschichte von Bedeutung, ob sie durch den Einfluß der russ.-ksl. Sprache in die serbische Spra- che gekommen sind; vom sprachstrukturellen Standpunkt aus sind we- nige als Russismen zu bezeichnen, von denen wiederum die meisten zu der veralteten und archaischen Sprachschicht gehören. Dieser kleine Exurs in die sprachliche Entwicklung der 40er Jah- re und ihr Vergleich mit Vuks Sprachkonzeption und der späteren Sprachentwicklung weist darauf hin, daß die heutige Schriftsprache, als deren Begründer Vuk g ilt, eine Synthese von Vuks Prinzipien und der Entwicklung, die vor ihm begonnen hatte, darstellt. Der Umfang und die Bedeutung des Beitrags, den die slavenoserbi- sehen Schriftsteller für die heutige Schriftsprache geleistet haben, soll erst untersucht werden1, nachdem sie lange im Zuge der romanti- sehen Glorifizierung von Vuk vernachlässigt wurden. I. Grickat führt eine Reihe der heute gängigen und unentbehrlichen Wörter an, wie: b e z u a l o v n o , d v o a m i a l e n o , o b 0 8 t r a n , p r e t p l a t a usw., die das Društvo srpske slovesnosti vorgeschlagen hat und "...ko jih nije bi- 2 lo и širokoj upotrebi i l i ih nikako nije bilo ra n ije ..." Vuk hat bekanntlich den von dem Društvo vorgeschlagenen Wortschatz abgelehnt Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vermutlich hat sich der von slavenoserbischen Schriftstellern ge- schaffene Vorrat an Ausdrucksmitteln im lexikalischen Bereich in demselben Maße durchgesetzt, wie die von Vuk geschaffene phone- tisch-morphologische Norm1. a. Vuks Weg zum synthetischen Dialekt Seine "Pismenica*1 von 1814 schrieb Vuk nach eigenen Worten mit dem Ziel, den in Fremdsprachen gebildeten Serben "neznajuóim svoga maternjeg jezika", die Erlernung der serbischen Sprache zu erleich- tern. Die Deklination und Konjugation habe er der Sprache derjenigen Serben entnommen, die "po selima daleko od gradova” leben (GPSI, 5ff. In den Dörfern sei die Volkssprache überall gleich, den größten und wichtigsten Unterschied bilde die Aussprache des alten e. Die Ver- tretung des è nimmt Vuk zum Kriterium für seine e r s t e Dialektein ־ teilung in Hercegovačko, Sremsko i Slavonsko narjecije, die der heu- tigen Einteilung in Ijekavisch, Ekavisch und Ikavisch im wesentli- לchen entspricht . Außer diesem Unterschied gebe es nur kleine, un- bedeutende Abweichungen im Akzent, ("Hercegovci sve rije c i izgovara- ju berze i ostrije, nego Sremci, a Slavonci opet raztežu vise"), und im Wortschatz (z.B. d j e v o j k a - c u r a , l o z i c a - k a s i k a ) (GPS 1,79). Zu Vuks Ansichten über die Dialekte in der Anfangsphase is t folgendes festzustellen: Zu Vuks Ansichten über die Dialekte in der Anfangsphase is t folgendes festzustellen: 1. In seinem Blickfeld steht nur der štokavische Dialekt mit sei- nen drei e־Ausprachen. Er nennt diesen Dialekt ”serbisch", und unter den "Serbij i " versteht er alle Stokaver. 2. Mit der serbischen Volkssprache meint Vuk hier die Sprache des Dorfes. 1 Das g ilt offensichtlich auch für nichtslavische Fremdwörter. Ein großer Teil der von V. Mihajlovic, Grafla za rečnik stranih reci и predvukovskom periodu I- II, 1972/74, verzeichneten Fremdwörter eu- ropäischer Herkunft gehört zum festen Bestandteil der heutigen Schriftsprache. 2 Vuks Ansichten über die Dialekte sind eines der besterforschten Probleme im Zusammenhang mit Vuks Tätigkeit. Die grundlegenden Stu־ dien von A. Belic wurden in der neueren Zeit besonders durch die Arbeiten von P. Ivic sowohl mit neuen Erkenntnissen als auch me- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 3. Die Reduzierung der Dialektunterschiede auf die drei ë-Aus- sprachen verrät seine ungenügende Kenntnis der Dialektunterschiede, weshalb er 3. Die Reduzierung der Dialektunterschiede auf die drei ë-Aus- sprachen verrät seine ungenügende Kenntnis der Dialektunterschiede, weshalb er 4. die Dorfspräche, abgesehen von dem ё-Unterschied, als über- a ll gleich bezeichnet. 4. die Dorfspräche, abgesehen von dem ё-Unterschied, als über- a ll gleich bezeichnet. thodisch ergänzt. In unserer Fragestellung, in der die Vuk-Grimm- Beziehung im Vordergrund steht, wird auf diese Ergebnisse verwiese 1 Vgl. A. Belic, Vukova borba, a.a.O., S. 88, vgl. auch S. 100- 2 Vgl. P. Ivic, О Vukovom Rjecniku, a.a.O. S. 79. a. Vuks Weg zum synthetischen Dialekt Vuks Ansicht von der Einheitlichkeit der Dorfsprache deutet darauf hin, daß ihm, im Gegensatz zu Kopitar, bereits zu Beginn eine einheitliche Schriftsprache, eben auf der Überall gleichen Volks- spräche, vorgeschwebt hat- Seine Arbeiten bis in die 20er Jahre zei- gen, daß er die serbische Volkssprache mit seinem eigenen Dialekt, dem Neuštokavischen vom Typ des Ostherzegovinischen, gleichgesetzt hat- Das beweist sein Verfahren, alle ihm unbekannten mundartlichen Formen, besonders diejenigen aus der Vojvodina, dem verderblichen Einfluß der Schriftsteller zuzuschreiben ( z.B. o ö e d u , r a d u , na k o l i , po 8 e l i usw.; GPS I, 46, 83). *1Svoj jezik on je izjednačio sa narodnim jezikom; i kako je, sem jednog glasa, narodni jezik svugde is ti, gramatika njegova u isto je vreme zajednička za ceo narod. Ko greSi protiv nje, greši protiv narodnoga jezika."1 Auch sein Wörterbuch von 1818 is t, im Ganzen gesehen, "u osnovi rečnik govo- ra j e d n o g čoveka"; es enthält den Wortschatz einer organisch ent- 2 wickelten Rede innerhalb eines einheitlichen Sprachsystems . In der Rezension von Vidakovics "Ljubomir и Jelisiumu" von 1817 hat Vuk die ersten Angaben über den sog. Ressaver Dialekt gemacht. Er hat zum erstenmal zugegeben, daß die Volkssprache Abweichungen auch in der Deklination zeigt, wie z.B. Dat. Sg. der Feminina ("Dao devojke jabuku"), oder Akk. Pi. Mask. ("Dočekao Turee") (GPS. I, 151). Trotzdem unterstrich er nachdrücklich: "Sklonenija, soprja- Ženija i Sintaksis (jednom rije č i svu gramatiku) moramo uzeti od pastira i od orača--." (GPS I, 159). Diese Forderung erscheint zunächst widersprüchlich: im Hinblick auf das neue Dialektmaterial wäre die Frage berechtigt, nach welcher Hirten- und Pflügersprache man sich in bezug auf die Flexion richten thodisch ergänzt. In unserer Fragestellung, in der die Vuk-Grimm- Beziehung im Vordergrund steht, wird auf diese Ergebnisse verwiese 1 Vgl. A. Belic, Vukova borba, a.a.O., S. 88, vgl. auch S. 100- 2 Vgl. P. Ivic, О Vukovom Rjecniku, a.a.O. S. 79. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access sollte. 1 Vgl. A. Belic, Vukova borba..., S.80 1 Vgl. A. Belic, Vukova borba.., S. 109ff Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access a. Vuks Weg zum synthetischen Dialekt Eine Antwort darauf hat Vuk selbst mit seinem Aufruf an die Schriftsteller gegeben: " To je najpreci i jedini nacin, kojim se może podignuti naia literatura: da počne pisati svaki Spisatelj onako, kao sto se govori и опот predjelu, gd je se on rodio i uzrastao..." (GPS I, 150) . Vuks Zulassung aller Dialekte in der Literatur beruht weiterhin in erster Linie auf der ungenügenden Kenntnis ihrer Unterschiede. Trotz der festgestellten Abweichungen im Ressaver Dialekt meinte er, auf diese Weise würde man sehen, "da je izmeöu sviju tije predjela najveca razlika, ito Ercegovci, Boinjaci, Crnogorci, Dalmatinci, Rvati, Slavonci (Grčkog zakona) i Srbijanci ozgo do Loznice, do Va- ljeva i do Karanovca, govore 6evojka...i t.d. a ovi svi ostali de- vojka..." (GPS I, 151). Er vertrat weiterhin die Meinung, daß die Volkssprache bis auf kleinere Abweichungen und den e-Unterschied überall gleich is t. "On je mislio da jedinstvo književnog jezika proističe sasvim prirodno iz osobina naših dijalekata. . . " 1. Das war offensichtlich der entscheidende Grund für eine solche Behandlung der Beziehung zwischen der zu begründenden Schriftsprache und den Dialekten. Zum Glück wurde seine Empfehlung an die Schriftsteller nicht befolgt;das wäre sicher nicht der "kürzeste" Weg zu einer ein- heitlichen serbischen Schriftsprache gewesen. Das wird durch die jahrhundertelang andauernde Pflege mehrerer Literatursprachen bei den Kroaten bestätigt. Es kommt u. E. noch ein wichtiger Grund hinzu. 1817 spitzte sich Vuks Polemik mit Milovan Vidakovic als dem wichtigsten Vertre- ter und Verfechter des "mittleren Stils" zu. Nachdem Vuk zu der Auf- fassung gekommen war, daß zwischen der serbischen und der Kirchenspra che zunächst eine klare Grenze zu ziehen sei, konnte er zur Ablösung der slavenoserbischen Sprache nur die ungenormte Volkssprache, den Sprachgebrauch des einfachen Volkes nennen? die einzige Norm, auf die er verweisen konnte und es auch tat, war seine Pismenica von 1814, die er selbst für unvollkommen hielt (GPS I, 6, 163). Im eigenen Hei- matdialekt zu schreiben, war die einzige Forderung, die er zu diesem Zeitpunkt stellen konnte. "Piši kao što narod govori, pa ako ne uzval ne t e s t i b iti kriv, nego narod, kao i и svima ostalim (kako starim. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access tako i novim) jezicima." (GPS I, 163). Dieser Grundsatz bedeutete aber, objektiv gesehen, das Gegenteil einer einheitlichen Schriftsprache. tako i novim) jezicima." (GPS I, 163). 1 Vgl. A. Belic, Vukova borba.., S. 109ff Vera Boji Downloaded from PubFactory at 01/10/2 a. Vuks Weg zum synthetischen Dialekt Dieser Grundsatz bedeutete aber, objektiv gesehen, das Gegenteil einer einheitlichen Schriftsprache. Es is t aber feststellbar, daß Vuks Zulassung aller Dialekte von Anfang an nur ein theoretisches Postulat war, das mit seiner empi- rischen Arbeit im Widerspruch stand, indem er nämlich die mundart ־ liehen Formen als Sprachfehler behandelte, v e il er von seinem Hei- matdialekt als der Norm ausging. Hinter Vuks Zulassung aller Dia- lekte stand u.E. auch der Wunsch, seinen herzegovinischen Dialekt in die Literatur einzuführen. Das vordringliche Problem, die Ablösung der slavenoserbischen Sprache hat u.E. zu dieser Zeit auch Vuks Interesse für die Dia- lekte beeinflußt. In der "Ljubomir"־Rezension und in seiner "Ser- bischen Grammatik" von 1818 hat er den "slavonischen" (ikavischen) Dialekt völlig ausgelassen. Belic sah den Grund dafür in Vuks un- genügender Kenntnis des Ikavischen: die "illyrischen" Lexika, die Vuk bei der Verfassung seines Wörterbuchs von 1818 benutzt hat, könnten ihn zu der Ansicht gebracht haben, daß es sich beim Ikavi- sehen nur um eine sporadische Spracherscheinung handele1. Vuks obige Angabe aber, daß von den Slavoniern nur die Orthodoxen zum Ijekavischen gehören, deutet auf sein Festhalten an der Bestimmung des ikavischen Dialekts von 1814 hin, wonach zu ihm "Serblji Rimo- katoliceskog Vjeroispovijedanja, koji žive po Slavoniji, po Horvat- skoj, i po Dalmaciji" gehören (GPS I, 79). Es scheint vielmehr, daß Vuk sich zu dieser Zeit auf die Einführung der Volkssprache, ihre Kodifizierung und Absicherung gegenüber der Kirchensprache und auf die Orthographiereform konzentriert hatte. Diese Aufgaben hatte er mit seiner Grammatik und dem Wörterbuch bereits in An- g r iff genommen; sie betrafen bekanntlich nur die Serben, also den ekavischen und ijekavischen Sprachbereich. Vuks Einschränkung auf die serbische Problematik bestätigt auch sein Brief an J. Grimm vom 14. Nov. 1823: "Wie wohl meine Grammatik is t nur für die Ser- ben der griechischen Religion geschrieben..." (Prepiska I I , 13f.) P. Iviè hat bemerkt, daß unter den Abonnenten auf Vuks Wörterbuch keine Katholiken vertreten sind und daß für die Auslassung des Ikavischen auch taktische Gründe eine Rolle gespielt haben könn- ten1. Vuk mußte sich u.a. wegen der Einführung des j gegen An- schuldigungen wehren, daß er mit seiner Tätigkeit der orthodoxen Kirche schade. ten1. Vuk mußte sich u.a. wegen der Einführung des j gegen An- schuldigungen wehren, daß er mit seiner Tätigkeit der orthodoxen Kirche schade. 1 Vgl. P. Ivic, О Vukovom Rječniku.., S. 51. 2 Eine Analyse des dialektologischen Materials in Vuks Grammatik und Wörterbuch geben A. Belic, Vukova borba.., S. l l l f f . und P. Ivic, О Vukovom Rjecniku.., S. 79ff. 3 Vgl. P. Ivic, О Vukovom Rječniku.., S. 50. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. A. Belic, Vukova borba.., S. 189. 2 Vgl. ebd., S. 175f. 3 über J. Grimms Einfluß auf diese Dialekteinteilung von Vuk vgl. unten S.152. 4 Vgl. den unveröffentlichten Brief Vuks an J. Stejic, Archiv SANU 8552/30(2). a. Vuks Weg zum synthetischen Dialekt Der Einengung des Dialektgebietes in Vuks z w e i t e r Dialektein- te ilung, die er in der Vorrede zum Wörterbuch vorgetragen hat, steht eine ausführlichere Beschreibung der angeführten Dialekte und die Unterteilung des Ekavischen in Resavsko und Sremsko narječije gegenüber. Das Hauptkriterium is t weiterhin die e-Vertretung, die er mit vielen richtigen Beobachtungen ergänzt (z.B. daß der Ressa- ver Dialekt am konsequentesten in der Entwicklung des alten e ist) 2 (GPS II, 22) . Vuks Dialekteinteilung is t, im ganzen gesehen, rieh- tig, aber unvollständig; seine Beschreibung der Dialekte dagegen is t ziemlich unvollkommen: es fehlt u.a. das neben der e-Vertretung wichtigste Kriterium der modernen Dialektologie, die Akzentuation3. Die ständige Konfrontierung mit einer immer größeren Anzahl von mundartlichen Unterschieden in der Volkssprache hat Vuk die Widesprüchlichkeit seiner theoretischen Begründung der Schriftspra- che sehr früh bewußt gemacht. Bereits 1821 kam er zu der Auffassung, daß bei der Schaffung einer einheitlichen Schriftsprache die Schrift steiler durch Auswahl "richtigerer” Formen Hilfe leisten könnten (GPS II, 138). An Stelle der Auffassung von der spontanen Entwick- lung der Schriftsprache t r i t t die Ansicht, durch das eklektische Verfahren eine einheitliche Schriftsprache zu schaffen. Er hat den Schriftstellern keine Prinzipien für dieses Verfahren gegeben; aus seiner Diskussion des Falles k u r j a c i - k u r j a k o v a geht her- vor, daß dasjenige als richtiger g ilt, was "mlogo obienije (tako i u narodu Ijepše i pravilnije) ,״also allgemein verbreitet is t (GPS II, 138). Auch diese Forderung Vuks mußte in der gegebenen Situation des Fehlens einer allgemein anerkannten Norm zu Wider- Sprüchen führen, die sich im Streit um die Volksmäßigkeit ver- schiedener Formen und in deren Vertreibung äußerten. Vuk hatte Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. P. Ivic, Die Entwicklung von Vuk Karadzics Ansichten über die serbische Schriftsprache. Erscheint im Sammelband der Vor- träge bei der wiss. Tagung in Jena 1973 anläßlich des 150. Ju- biläums von Vuks Promotion zum Dr. phil. an der Jenaer Univer- s itä t• Mühe, dieser Entwicklung Einhalt zu gebieten1. Mühe, dieser Entwicklung Einhalt zu gebieten1. Mitte der 30er Jahre hat Vuk seine Theorie korrigiert: der Begriff der Volkssprache gewinnt bei ihm eine neue Bedeutung. H0n je dijalekatske oblike i cela narečja počeo tada smatrati kao matērijai iz kojega treba da se obrazuje jedan opšti književni 2 jezik.” Diese Auffassung spiegelt sich auch in seiner d r i t t e n und letzten Dialekteinteilung in istocno, juSno i zapadno narje- cije wider3(GPS I I I , 36; 1839); die ё-Vertretung bildet weiterhin das ausschließliche Kriterium, obwohl er während seiner Reisen durch westliche Gegenden die bis dahin größte Anzahl von mundart- liehen Unterschieden festgestellt und in seiner Vorrede zu "Poslo- vice" von 1836 veröffentlicht hat. In der Schriftsprache wollte Vuk nur den Unterschied in der ё-Aussprache zulassen; alle übri- gen mundartlichen Eigenschaften sollten ausgeglichen werden, um eine allgemeine Regelmäßigkeit ("opeena pravilnost") zu erreichen (GPS I I I , 186; 1845). Vuk hat seinen Begriff der "opeena pravil- nost” nicht definiert; aus seinen Polemiken und seiner schrift- stellerischen Praxis is t ersichtlich, daß er damit neben einer konstanten ë-Vertretung die von ihm aufgestellte Norm für seinen Heimatdialekt mit den Innovationen von 1836 und 1839 meinte. Die- se Innovationen betreffen die Einführung des h und die Beseiti- gung der sog. "neuesten Jotierung" des t und d { d j e v o j k a , t j e r a t i statt d e v o j k a , b e r a t i ) . Bei der Bestimmung der "opeena pravilnost" t r i t t an Stelle des vagen Prinzips des im Volk üblicheren die grammatische Regelmäßig- keit als Hauptkriterium:"Kad se u narodu jedne rije č i dvojako govore, onda je, mislim, spisatelj dužan iz b ira ti ono, što je pravilnije, ne gledajuci, i l i да govori naroda manje, i l i više. Tako i medu Bokezima ako se nade sto ljepŠe i pravilnije, nego sto se govori amo po nasim krajevima, mi smo dužni űzeti ono и opšti srpski jezik, ne gledajuci, što je njih samo 30.000 duša." Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Mit dem Begriff der grammatischen Regelmäßigkeit schränkt Vuk den Begriff der Volkssprache ein. Mühe, dieser Entwicklung Einhalt zu gebieten1. Er meint, daß auch das Volk ״,ver- dorben" sprechen könne, macht zwar die Schriftsteller für die mei- sten Fehler verantwortlich, nennt aber dabei auch montenegrinische mundartliche Eigenarten, die er den Schriftstellern nicht zu- schreiben konnte. "Dokle narod cisto i nepokvareno govori, knji- ževni se jezik ne mora razlikovati od narodnoga... " (GPS II, 184; 1845). Die ״prostota" mag sagen z.B.: m i r i e i m , m i r i e i t i (statt: miriŠem, m irisa ti), b o l e , b o l u (statt: boli, bolв ) , д о ѵ о т и , g o v o - r i d u (statt: govore), o b e j u , obedu (statt: hoce), o z e n i t , e l o m i t (statt: oženjen, slomljen), z e t e o c (statt: žetelac), и n a s im se- l u (statt: и našem selu), b r a à a m a , decama (statt: braci, deci), znam, к ѵ з п о i m e , b a c i t i , I j u H i (statt: znam, krsno ime, baciti, ljudi) , p v i k o (statt: preko) , sie mag p r e und ргг, Akkusativ und Lokativ verwechseln, "spisatelj nas mora da se trudi raspoznati c is ti narodni jezik od onoga sto se govori pokvareno: koji tako uzradi onaj ce i po ž e lji mojoj pisati narodnijem jezikom, i po že lji našijeh jezikopopravitelja i jezikograditelja ne ce pisati kao što prostota pokvareno govori, nego ce imati književni jezik." "Kako stanemo svi ovako pisati, odmah cemo imati književni jezik, koji ce se razlikovati od jezika pokvarene prostotę, a opet ne ce b iti ništa drugo do c is ti narodni Srpski jezik." (GPS III , 185f.) In Vuks Sprachkonzeption bedeutet der Begriff der Volksspra- che hier so viel, daß die Schriftsprache aus den volkssprachlichen Elementen zusammengesetzt werden soll. P. Ivic hat treffend be- merkt: "Vuku je ovde promaklo da njegova vizija književnog jezika kao selekcije erta iz raznih dijalekata isključuje slobodu pisa- nja bilo kojim konkretnim dijalektom, a pogotovu svakim dijalek- tom•"1 Seine Schriftsprache is t ein synthetischer Dialekt, für dessen morphologische und syntaktische Struktur sein Süddialekt bestimmend war, während die phonetische Struktur Korrekturen nach der süd-westlichen Aussprache erhalten hat. Belic sah Vuks Verfah- 1 Vgl. P. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. A. Belic, Vukova borba.., S. 180. 2 Vuk hat das h eingeführt, nachdem er es in der Volkssprache gehört hat. Die Regel für die Schriftsprache hat er aber aus der dia- chronischen Sicht gestellt: das Zeichen soll geschrieben werden, "gdje mu je po etimologij i mjesto." (GPS I I I , 300). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Mühe, dieser Entwicklung Einhalt zu gebieten1. Ivic, Die Entwicklung von Vuk Karadzics Ansichten über die serbische Schriftsprache. Erscheint im Sammelband der Vor- träge bei der wiss. Tagung in Jena 1973 anläßlich des 150. Ju- biläums von Vuks Promotion zum Dr. phil. an der Jenaer Univer- s itä t• Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ren gerechtfertigt "na osnovu toga sto su se t i oblici nahodili i и jednom delu istočnog govora, i to и onome koji se nalazio и Srbiji и b liz in i Vukova maternjeg govora, a, možda, i и jednom delu zapadnog (onde gde se on dodiruje sa istocnim i južnim)."1 ren gerechtfertigt "na osnovu toga sto su se t i oblici nahodili i и jednom delu istočnog govora, i to и onome koji se nalazio и Srbiji и b liz in i Vukova maternjeg govora, a, možda, i и jednom delu zapadnog (onde gde se on dodiruje sa istocnim i južnim)."1 Zusammenfassend kann festgestellt werden, daß Vuks Ansichten über die Dialekte und die Schriftsprache sich erst in der Endpha- se denjenigen von Jacob Grimm nähern. Vuks Konzeption der Schrift- spräche als eines synthetischen Dialekts erscheint verwandt mit Grimms Ansichten von der Entwicklung der Schriftsprache auf der Grundlage einer "stärkeren", kulturtragenden Mundart, die "durch ihre wachsende ausdehnung unvermerkt eigenheiten der ändern Stämme an sich ziehen... musz" (GKS V III, 12). In Vuks Verfahren bei der Schaffung eines synthetischen Dia- lekts sind für uns zwei Komponenten von besonderem Interesse, an denen die Veränderung in seiner Sprachauffassung deutlich erscheint. Das sind einerseits die Heranziehung der Sprachgeschichte und Sprachvergleichung bei der Bestimmung der grammatischen Regelmäßig- keit und andererseits die Korrektur der Auffassung von der Dorf- spräche als dem Vorbild für die Schriftsprache. 1 Vgl. A. Belic, Vukova borba.., S. 180. b. Die Diachronie im Dienste der Synchronie bei Vuk b. Die Diachronie im Dienste der Synchronie bei Vuk Vuk hat in seiner Ausgabe der Sprichwörter von 1836 die Schrei- bung des h wieder eingeführt "...fle mu je goä pravo mjesto (po etimologij i ), pa č ita te lji пека да izgovaraju, kako je коте obič- n ije ..." (GPS I I I , 9). Er habe die Aussprache dieses Lautes in Dubrovnik, Boka Kotorska, Ста Gora und in den Städten von Bosnien gehört. Er selbst gebraucht dieses Zeichen konsequent nach dem etymologischen Prinzip, es gehört zu seinem Begriff der grammati- 2 sehen Regelmäßigkeit . Das is t der erste Fall, in dem Vuk bei der Bestimmung der schriftsprachlichen Norm die Sprachgeschichte aus- drücklich heranzieht. Das ursprüngliche Prinzip "schreibe, wie du Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access sprichst" wurde damit für den überwiegenden Teil der NeqStokaver, (für die Angehörigen des Ostdialekts insgesamt), aufgehoben, da zum richtigen Schreiben die Kenntnis der Etymologie erforderlich war1. sprichst" wurde damit für den überwiegenden Teil der NeqStokaver, (für die Angehörigen des Ostdialekts insgesamt), aufgehoben, da zum richtigen Schreiben die Kenntnis der Etymologie erforderlich war1. Vuks Abweichung von seinem ursprünglichen Prinzip war aber von größtem Vorteil für die Schriftsprache. P. Ivic hat Vuks Einfüh- rung des h und die Beseitigung der sog. neuesten Jotierung unter » dem strukturellen, dialektologischen, diachronischen und kompara- tiven Aspekt betrachtet und festgestellt, daß dadurch eine größe- re Regelmäßigkeit des sprachlichen Systems mit gleichzeitiger Ar- chaisierung und Annäherung an die übrigen slavischen Sprachen er- reicht worden is t; zugleich bedeuteten aber Vuks Innovationen die Abschaffung des einheitlichen Dialektsystems, indem er in seinen Dialekt Elemente anderer aufgenommen hat ; dieser über den Dialek- ten stehende Charakter seiner Schriftsprache hat die Voraussetzun- gen für die sprachliche Annäherung der Serben und Kroaten geschaf- 2 fen . In unserer Fragestellung is t Vuks Weg von der reinen synchro- nischen Sprachbetrachtung zur Heranziehung des diachronischen Aspekts von besonderem Interesse, weil dabei u.E. Grimms Einfluß auf Vuk eine Rolle gespielt hat. Jacob Grimm hat das Erscheinen von Vuks Grammatik sowohl in serbischer (1Ô18) als auch in deutscher Sprache (1824) mit einer Rezension bzw. Anzeige begleitet. Er hat u.a. 1 Durch den Einfluß der Schriftsprache auf die Aussprache wurde diese orthographische Schwierigkeit später aufgehoben. 2 Vgl. P. Iviè, Tendencije и konačnoj fazi Vukove jezičke reforme. In: Vukov zbornik, 1966, S. 65-71; ders., Dva aspekta Vukovog dela. In Anali FF 5, 1965, S. 99 - 107. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access b. Die Diachronie im Dienste der Synchronie bei Vuk die Bedeutung von Vuks Arbeit für die Sprachgeschichte und Sprachvergleichung her- vorgehoben (GKS IV, lO lff.) und entsprechend seiner historisch- vergleichenden Methode Vuk darauf hingewiesen, daß keine der sia- vischen Sprachen durch sich allein ganz erklärt werden könne, "dazu müssen sie theils alle untereinander verglichen, theils auf das altslavische zurückbezogen werden, dann wird sich das verworre- ne zu ordnen anfangen." (GKS V, 225). Er k ritis ie rte auch Vuks Auslassung des h (GKS V, 226) und meinte, eine serbische Grammatik nach dem Vorbild von Dobrovskjs böhmischer Grammatik und "Insti- tutiones linguae slavicae" wäre sehr wünschenswert (GKS V, 187;1823) - 96 ־ Grimms Forderung nach Einbeziehung der Sprachvergleichung und Sprachgeschichte hat einen Einfluß auf Vuk ausgeübt• Vuk veröffent- lichte in der Danica für 1826 und 1828 zwei Schriften: "Glavne razlike izmēdu današnjega Slavenskoga i Srpskoga jezika" und "Glav- na svrsivanja susteštvitelni i prilagatelni imena и Srpskom jezi- ku" (GPS II, 261-272, 292-370); in der Einleitung zu der letzteren sagt er: "Ja sam još prije nekoliko godina počeo prepravljati gra- du za Srpsku gramatiku po načinu G. Dobrovskoga česke i Slavenske gramatike", wozu die vorliegende Schrift gehöre, die er auf Anre- gung von Jacob Grimm angefertigt habe (GPS II, 292)1. In diesen beiden Schriften is t in methodischer Hinsicht eine Neuerung feststellbar; entsprechend den Forderungen von Grimm ver- sucht Vuk, die historisch-vergleichende Methode anzuwenden: er erläutert u.a. die Entstehung des sog. beweglichen a aus den redu- zierten Vokalen und seine Entsprechung in verschiedenen slavischen Sprachen. Er greift die von Grimm verschiedenenorts behandelten oder hervorgehobenen Probleme auf, wie z.B.: den Übergang des aus- lautenden I zu o , die einzelsprachliche Entwicklung der reduzier- ten Vokale, die Entnasalisierung, die 1. Pers. Sg. Präs. auf -m gegenüber -u im Altkirchenslavischen und Russischen, das Fehlen des 2 Aorists im Russischen . Auch Grimms K ritik der Auslassung des h findet ihren Nieder- schlag: Vuk meint, daß man es zwischen zwei Vokalen und am Ende des Wortes schreiben könnte, vor Konsonanten jedoch nicht, "jer ne samo što bi na takijem mjestima ružilo narodni izgovor, nego ni od stotine jedan ne bi znao svuda de да valja pisati." (GPS 11,268) Den Verlust des h erkannte Vuk bereits hier als eine sprachliche Neuentwicklung (najmlade svojstvo). Dafür daß er es nicht zu die- ser Zeit wieder eingeführt hat, waren u.E. die damit verbundenen orthographischen Schwierigkeiten ausschlaggebender als die prinzi- pielle Treue zur Volkssprache. 1 Vgl. auch Grimms Brief an Vuk v. Silvester 1824 (Prepiska 11,49). 2 Vgl. GKS IV, 10f., 189, und Grimms Vorrede zur deutschen Fassung von Vuks Grammatik. g ( p 2 Vgl. GKS IV, 10f., 189, und Grimms Vorrede zur deutsc von Vuks Grammatik. sprachlichen Norm, die als solche erlernt werden muß • Diesen diachronisch-komparativen Ansatz hat Vuk mit der Zeit ausgebaut und er is t bei ihm besonders in der Endphase bei der Bestim mung der grammatischen Regelmäßigkeit produktiv geworden. Außer der bereits genannten Archaisierung (h , d j e , t j e ) wendet Vuk die- ses Verfahren in seinen Polemiken bei der Bestimmung des Richtige- ren im Einzelfall an. Er sagt ausdrücklich, man könne "pravila jednoga jezika uporediti s pravilima drugoga, osobito da se čo- vjek na gdjekojim mjestima uvjeri. Sta je pravilnije." (GPS 11,94)* So meint er z.B. in seiner Argumentation gegen Hadžic im Zusammen- hang mit p r o m e n u t i und p r o m e n i t i :" ... da je p r o m e n i t i pravilnije, о tome ... može u v je riti i Slavenski i Ruski jezik." (GPS I I I , 111 1843); zu k o r e t i , koremx es sei "pravilnije (prema Slavenskom i Ruskom jeziku) k o r i t i , k o r i m " . (GPS I I I , 167; 1845)."Od glagola u m e t i , umem и treòem licu množ. broja sadašnjega vremena ponajvi- Še pišu umu , a ja mislim da bi pravilnije bilo u m e j u , i sve mi se Čini da sam u narodu tako slušao: staroslovenski je o u m e j ç t b , Ruski urne j u t , a Srpski po južnome narječiju u m i j u i tako mislim da bi i po sjeveroistočnome morālo b iti u m e j u . . . n (GPS I I I , 167; 2 1845) . Auf den Einwand, daß der Einschub des a im Part. Perf. b. Die Diachronie im Dienste der Synchronie bei Vuk Diese Schwierigkeiten waren objek- tiv nicht anders 1836, aber Vuks Sprachkonzeption enthielt bereits die Vorstellung von der grammatischen Regelmäßigkeit der schrift- 1 Vgl. auch Grimms Brief an Vuk v. Silvester 1824 (Prepiska 11,49). 2 Vgl. GKS IV, 10f., 189, und Grimms Vorrede zur deutschen Fassung von Vuks Grammatik. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 97 ־ sprachlichen Norm, die als solche erlernt werden muß • 1 Vgl. M. Ivic, Vukova uloga и normiranju ekavske varijante knji- ževnog jezika. In: Anali FF 5, 95-98, S. 98. 2 Nach seiner Meinung werden im Aksl» und Russ. nur 1 und n vor e als l j und nj ausgesprochen, während in Herzegovina, Crna Gora und Boka Kotorska auch d und t zu Ű und с geworden sind (GPS I I I , Bekanntnlich hat er nur die beiden letzten konsequent abgeschafft Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM sprachlichen Norm, die als solche erlernt werden muß • mask, { m o g a o , v u k a o statt mogo, v u k o ) die Sprache mit unnötigen Ausnahmeregeln belaste, argumentiert Vuk ebenfalls mit der Sprach- geschichte: er erläutert nochmals die Entwicklung der reduzierten Vokale und sagt: "Kakogod što je и riječima Staroslavenskog jezi- ka: (n.p.) р г і ё ъ і ъ , о г ъ і ъ , и današnjem Slavenskom jeziku ( po Ruskome narječiju) e а и Srpskome a , tako je u nas blagoglasija radi umetnuto a pred Z i u riječima: г е к і ъ , р е к і ъ , т о д і ъ . . usw. (GPS I I I , 95; 1843). 1 Die Verbindung zu den angeführten Worten aus Grimms Rezension is t offensichtlich. Auch Grimms Meinung aus der Vorrede zu Vuks Grammatik, daß die Erforschung der nomina propria zur Erhellung der Geschichte des ehemaligen Illyriens von Bedeutung sei, fin- den wir bei Vuk: "Ovakijeh ce se imena i vise naci, koja mogu b iti od koristi ne samo za današnji naš jezik, nego i za sta- ru is to riju naroda našega•" (GPS I I I , 178). 2 Vuk gebraucht hin und wieder Grimms Bezeichnung: nordöstlicher Dialekt. - Vgl. dazu S.152 dieser Arbeit. Bayerische Staatsbibliothek München Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Bayerische Staatsbibliothek München Do In unserer Betrachtung der Gemeinsamkeiten und Unterschiede zwischen VukundGrimm is t zusammenfassend festzustellen, daß Vuks Einbeziehung der Diachronie in seine sonst überwiegend synchroni- sehe Behandlung der Sprache auf Grimms Anregung zurückgeht und eine Annäherung an Grimms diachronisch-vergleichende Methode be- deutet. Die Konsequenzen, die sich aus Vuks Korrektur der Auffassung von der Vorbildlichkeit der Dorfsprache ergeben, bringen ihn eben- fa lls in die gedankliche Nähe zu Jacob Grimm. c. Von der Dorfspräche zur Stadtsprache c. Von der Dorfspräche zur Stadtsprache Seine Antwort auf Hadžics "Sitnice jezikoslovne" schrieb Vuk 1839 nach eigenen Worten in der Dubrovniker Mundart des Süddialekts Außer dem 1836 bereits eingeführten h gebrauchte er hier zum er- stenmal d j e , t j e an Stelle von Ű e , be mit der Angabe: " ... za ovo mogio bi se reci, da je varoški - gospodski - govor južnoga narje- čija ." (GPS I I I , 37). Diese Eigenart behält er konsequent in seiner eigenen Sprache. Damit korrigiert Vuk offen seine ursprüngliche Auffassung von der Dorfsprache als dem Vorbild für die Schriftspra- che. Einen ähnlichen Schritt hatte er bereits 1828 stillschwei- gend getan, mit seiner in der städtischen Aussprache des Ostdia- lekts verfaßten Schrift "Milos Obrenovic", worüber er 1839 berich- tet: " ... nijesam smjeo da kažem nésam, jer sam čuo, gdje se и Bio- gradu podsmijevaju onima, koji tako govore? a li svatko, ko posao ovaj razumije, mora priznati, da je i u ovome narječiju pravilnije i p rilič n ije nesam nego nisam, kakogod i gde nego gdi..." (GPS I I I , 38). Die spätere Entwicklung der ekavischen Variante, der er eigent lieh die Norm gegeben hat1, hat die Richtigkeit seines Schrittes bestätigt. 1839 handelt Vuk bewußter, seine Heranziehung des Altkirchen- slavischen und Russischen deutet darauf hin, daß er auch in diesem 2 Falle nach dem Richtigeren gesucht hat . "Tzv. najnovije jotova- via free access via free access nje t i d je ... komplikovalo gramatički sistem uvoflenjem alternaci- ja t : ô odn. d i d u kategorije u kojima se te alternacije inače ne bi pojavljivale, l e t i m : l e b e t i , d i j e t e : d e t e t a , s a d i j e v a t i : a a â e e t i . Još ve&u kompleksnost unosiła je činjenica da pravilo d j > Ű, t j > b u Vukovom jeziku nikad nije delovalo dosledno."1 Durch die Beseitigung der neuesten Jotierung wurde eine größere Regelmäßigkeit des sprachlichen Systems erreicht. Die städtische Sprache bot sich als Vorbild an. Vuk hatte in der Zwischenzeit das Wesen der Schriftsprache besser begriffen, um sich ausschließlich nach der Dorfspräche richten zu können. Für Vuks Zuwendung zur Dubrovniker Sprache waren folgende Grün- de von entscheidender Bedeutung. 1 Vgl. P. Ivic, Tendencije и konačnoj fa z i.., S. 67 c. Von der Dorfspräche zur Stadtsprache Er schrieb an Kopitar kurz nach seinem Aufenthalt in Dubrovnik: ״DanaSnji je Dubrovački jezik pra- vi jezik Ercegovački; ovo su najvece izmeflu n ji razlike: 1) Sto se и Dubrovniku izgovara h ( i to ne kao h, nego upravo kao ch) ; 2) što ne kažu n.p. Л е са , nego d j e c a i t.d .; a 3) Sto imaju mnogo i premnogo rije č i Talijanski. . . . i po Dubrovniku pjevaju uz gusle, kao BoSnjaci po Bosni, i Ercegovci po Ercegovini." (Prepiska I, 4 52). Vuk betont die Verwandtschaft zwischen der Dubrovniker und seiner herzegovinischen Sprache und die Existenz der epischen Lie- der ("uz gusle"). Es kommt noch eine wichtige Tatsache hinzu, die er 1839 anklin- gen läßt (GPS III, 38) und die er 1845 als eines der Argumente für seine Schriftsprache anführt: die Dubrovniker Literatur (GPS I I I , 156). Während des ganzen Kampfes um die Anerkennung der Volksspra- che in der Literatur haben sowohl Vuk als auch Grimm die Volks- dichtung als das Muster "edler Schreibart" hervorgehoben, und Vuk hat in unermüdlicher Sammeltätigkeit seiner Sprache eine "zeitge- nössische" Tradition in der Dichtung geschaffen. Jetzt konnte er sich für seine modifizierte Schriftsprache auch auf die reiche l i - terarische Tradition der Dubrovniker Literatur berufen: " ... ono je narječije i и Dubrovačkijeh spisatelja..." (GPS I I I , 155). Er nennt die Dubrovniker Literatur immer wieder als Vorbild und schlägt ihre Herausgabe auch in kyrillischer Schrift vor (GPS I I I , c, Tendencije и konačnoj fa z i.., S. 67 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 261). Mit dieser Einbeziehung der literarischen Tradition in seine Konzeption der Schriftsprache nähert sich Vuk in der Endphase der Auffassung von Jacob Grimm, daß die Schriftsprache auf der Grundlage eines kulturtragenden Dialekts mit literarischer Tradi- tion entsteht. Grimm hat diese Ansichten auch in seinen slavisti- sehen Rezensionen geäußert (GKS IV, 186f., 200); sie könnten eine Klärung bei Vuk eingeleitet haben, die zu seinem Schritt von der Folklore zur literarischen Tradition geführt hat. d. Die einheitliche Schriftsprache v 1 Vuk hat irrtümlicherweise die Kroaten mit den Cakavern gleichge- setzt (vgl. S.!104ff. dieser Arbeit) Richtig dagegen war seine Betonung der Verwandtschaft der beiden Dialekte. 2 Vuks Festhalten an dem serbischen Namen wurde sowohl durch das na tional-historische Bewußtsein bei den Serben als auch durch Kopi- tars, Dobrovskys und Grimms Ansichten unterstützt (vgl. S.'150 dieser Arbeit). 3 Vgl. Vuks Brief an den Ban Jelačic vom 3. Dez. 1861; z it. nach V. Novak, a.a.O., S. 321. 4 Vgl. dazu Barnard, a.a.O., S. 77, 203ff. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access d. Die einheitliche Schriftsprache d. Die einheitliche Schriftsprache Am Ende der Entwicklung von Vuks Sprachkonzeption steht sein Vorschlag, seinen synthetischen Dialekt zur einheitlichen Schrift- spräche der Serben und Kroaten zu nehmen. Er nennt fünf Argumente: 1. Die räumliche Ausdehnung vom Westen bis nach Srem, "do Vukovara i uz Dunavo gotovo do Budima, a od juga и S rbiji gotovo do Bijo- grada"; 2. " ... da su gotovo sve naie narodne pjesme и njemu posta- le"; 3. der Süddialekt unterscheide am besten die Wörter verschie- dener Bedeutung wie a j e d i m und s i j e d i m ś, 4. er sei dem Altkirchen- slavischen am nächsten; 5. er sei auch der Dialekt der Dubrovniker Literatur, "... i tako se samo erez njega то zemo ujediniti s naŠom braćom Rimskoga zakona, koja s radošeu nama ruke pruŽaju: mi svi valja da se trudimo, dotie da dotjeramo, da nam jezik и knjigama bude tako jednak, da se svaka knjiga može od slova do slova pre- stampati od Latinskijeh slova Slavenskima a od Slavenskijeh Latin- skima, pak cemo onda (i samo onda) b iti jedan narod i imati jednu književnost, kao n.p. Nijemci zakona Rimskoga i Lutoranskoga (a kad nam se po vremenu pridruže i ona po rodu i jeziku braca naŠa zako- na Turskoga... onda cemo b iti kao Nijemci zakona Rimskoga, Lutoran- skoga i Kalvinskoga..." (GPS I I I , 155f.; 1845). Das erste, dritte und vierte Argument bestehen eine kritische Prüfung nicht: Vuks synthetischer Dialekt wurde in seiner Ganzheit nirgendwo gesprochen; der Ostdialekt z.B. macht auch durch die Akzentuation einen Unterschied in den genannten Fällen (allerdings nicht nach Vuks Orthographienorm, die die Akzente nicht berücksich- tig t). Die prosodischen Elemente (Akzent und Quantität) spielen Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access aber auch sonst o ft eine distinktive Funktion { g r å d : g r a d ű, Gen. Sg. r u k ē : Nom./Akk. PI. r u k e ) . Vuks Dialekt steht dem Altkirchensla- vischen nicht am nächsten. aber auch sonst o ft eine distinktive Funktion { g r å d : g r a d ű, Gen. Sg. r u k ē : Nom./Akk. PI. v 1 Vuk hat irrtümlicherweise die Kroaten mit den Cakavern gleichge- setzt (vgl. S.!104ff. dieser Arbeit) Richtig dagegen war seine Betonung der Verwandtschaft der beiden Dialekte. g 2 Vuks Festhalten an dem serbischen Namen wurde sowohl durch das na- tional-historische Bewußtsein bei den Serben als auch durch Kopi- tars, Dobrovskys und Grimms Ansichten unterstützt (vgl. S.'150 dieser Arbeit). , , 4 Vgl. dazu Barnard, a.a.O., S. 77, 203ff. Downloaded from PubFactory at 01/10/2019 v d. Die einheitliche Schriftsprache r u k e ) . Vuks Dialekt steht dem Altkirchensla- vischen nicht am nächsten. Zwei der genannten Argumente waren ausschlaggebend, nämlich: 1. die Idee von einer einheitlichen Schriftsprache der Serben und Kroaten, die 2. auf der Grundlage des - nach dem Vorbild der Dubrov- niker Sprache modifizierten - Dialekts der Lieder geschaffen wer- den sollte. 1. In Vuks Vorstellung von der einheitlichen Schriftsprache der Gedanke enthalten, daß die Serben und die Kroaten e i n Volk mit zwei Namen sind. Vuk geht in seiner Bestimmung des Volkes von der Sprache als dem Hauptkriterium aus: die sprachlichen Unterschiede seien, "kad se govori о dva razlicna jezika i naroda, vrlo male- ne"*(GPS I I I , 292). Die Gemeinsamkeiten betrachtet er als ursprüng- lieh, die Unterschiede als das Resultat der späteren Differenzie- rung. Die gemeinsame Schriftsprache sieht er, ähnlich wie Grimm, als das Mittel der Einigung der Serben und Kroaten an. Er beruft sich ausdrücklich auf das Vorbild der Deutschen, die trotz der un- terschiedlichen Glaubensbekenntnisse e i n Volk sind. Wie Herder und Grimm sieht Vuk offenbar das Volk als eine geschichtliche Leistung der Sprache an. Er meint nämlich auch nach seiner Ablehnung des 2 von den Illyrern vorgeschlagenen gemeinsamen illyrischen Namens : ...״ja mislim da se sad ništa korisnije ne može č in iti nego da se trudimo da bismo književni jezik sa svijem izj ednačili. .. - pa kad dovde dotjeramo, onda možemo b iti da cemo se nekako slo žiti i и imenu, a bez toga slabo bi nam pomagaio i kad bismo i jedno ime imali." 3 Der Herderische Gedanke von der Sprache als dem Hauptmerkmal des Volkes war zur damaligen Zeit, insbesondere unter den Slaven 4 der Donaumonarchie , allgemein verbreitet. Er wurde auch von den Illyrern vertreten, die eine Einigung der Südslaven durch die Spra- v 1 Vuk hat irrtümlicherweise die Kroaten mit den Cakavern gleichge- setzt (vgl. S.!104ff. dieser Arbeit) Richtig dagegen war seine Betonung der Verwandtschaft der beiden Dialekte. ) 3 Vgl. Vuks Brief an den Ban Jelačic vom 3. Dez. 1861; z it. nach V. Novak, a.a.O., S. 321. 4 Vgl. dazu Barnard, a.a.O., S. 77, 203ff. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 102 ־ che zu erreichen hofften. Die Formulierung in der Wiener Sprachver- einbarung zwischen Vuk, Danicic und den Vertretern der illyrischen Bewegung: ” ... d. Die einheitliche Schriftsprache da j e d a n narod treba j e d n u književnost da ima..." steht im Einklang mit dieser Idee (GPS I I I , 299). In Vuks Vorstellun- gen von Sprache und Volk spielt offenbar das deutsche Vorbild eine wichtige Rolle. Bei den Deutschen is t zwar der Vereinheitlichung der Schriftsprache auch die nationale Einigung gefolgt; die Tatsache aber, daß sie nicht den gesamten deutschsprachigen Raum erfaßt hat, zeigt deutlich, daß dabei die sprachlichen Voraussetzungen nicht entscheidend waren. Die Bestimmung eines Volkes durch Sprache ist vom heutigen Stand punkt aus nicht vertretbar; denn die Sprache is t nur eines der Merk- male des Volkes. Die Erfahrungen aus der Geschichte beweisen, daß mehrere Völker dieselbe Sprache sprechen können; das Beispiel des jüdischen Volkes u.a. zeigt wiederum, daß das Zusammengehörigkeits- gefühl auch ohne eine gemeinsame Sprache entwickelt sein kann. Die Gefahren, die die Gleichsetzung von Sprache und Volk in sich birgt, haben sich deutlich in der Diskussion zwischen Vuk und den Illyrern über den štokavischen, čakavischen und kajkavischen Dia lekt gezeigt. Die Vermischung des Sprachlichen mit dem Nationalen, die dabei stattfand, trug zur Klärung der linguistischen Probleme nicht bei. 2. Vuks synthetischer Dialekt is t, wie bereits festgestellt w de, sein nach dem Vorbild der Dubrovniker Sprache modifizierter her- zegovinischer Dialekt. Bei diesen Modifikationen waren für Vuk zwei Momente von Bedeutung: erstens, die von ihm hervorgehobene Zugehörig- keit der Dubrovniker Sprache zum herzegovinischen Dialekt und die Existenz derselben epischen Lieder auf dem ganzen Gebiet; zweitens, die Bemühungen der Illy re r, den stokavisch-ijekavischen Dialekt zur kroatischen Schriftsprache zu erheben. I. Mazuranic und S. Vraz hatten die Bedeutung der Dubrovniker Literatur und der Liederausgabei Vuks als Vorbild für die kroatische Schriftsprache hervorgehoben . In Vuks Sprachkonzeption is t von Anfang an seine Entscheidung für den herzegovinischen Dialekt feststellbar. Dabei hat, außer der Vorliebe für den eigenen Dialekt, offenbar auch die Tatsache eine wichtige Rolle gespielt, daß die meisten von ihm gesammelten Lieder, darunter alle epischen, in diesem Dialekt entstanden sind. Grimms Bevorzugung der Heldenlieder (GKS IV, 221) kann auch einen Einfluß Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 103 - auf Vuk ausgeübt haben. Vuk beruft sich immer wieder auf die Volks- lieder: "Sve su gotovo junačke pjesne, kője se po narodu Serbskom pjevaju, и Hercegovačkom dijalektu spjevane." (SD I, 141? 1815). 1 Vgl. P. Ivifc, О Vukovom Rječniku.. , S. 87. 2 Vgl. Vuks Brief an Grimm vom 14. Nov. 1823: "Das is t einzig für unsere Sprache was Sie... über pöbelhafte und edle Sprache etc. gesagt haben." (Prepiska I I , 12). 1 Vgl. P. Ivifc, О Vukovom Rječniku.. , S. 87. 2 Vgl. Vuks Brief an Grimm vom 14. Nov. 1823: "Das is t einzig für unsere Sprache was Sie... über pöbelhafte und edle Sprache etc. gesagt haben." (Prepiska I I , 12). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access d. Die einheitliche Schriftsprache In der Polemik mit den Rezensenten seines Wörterbuchs von 1818 gibt er eine Begründung für seine Wahl des herzegovinischen Dialekts, in der schon die wesentlichen Komponenten seiner Argumentation von 184 5 enthalten sind: "Ja mislim... da Srbalja пета manje, koji govore Ercegovačkim nerječijem, nego onije, koji govore Sremačkim i Resav- skim, ü narodnim našim pjesmama, kője su jedino i vjecno blago i ukrašenije literature naše, pjeva se i današnji dan usred Bačke i Srijema, kao i usred Braničeva i Resave, bijelo, lijepo, dijete, evi jet i t.d." (GPS II , 133f; 1821). Mit dieser Äußerung steht auch die folgende von 1842 im Einklang: "U našim je narodnim pjesmama za nas najdragocjenija a i s t o t a i a l a d o s t našega jezika: kad bi književni- ci naši to poznavali, onda ne bi kva rili i g rd ili najveèu dragocje- nost svoga naroda, svoj narodni jezik, nego bi se tru d ili naučiti да iz narodnijeh pjesama." (GPS I I I , 70). Die Volkspoesie als Vorbild und Schatz für die Sprache is t ein Herderischer Gedanke. Außerdem v e rtritt Vuk offensichtlich die Auf- fassung, daß die Verbreitung der Lieder über das ganze Gebiet zur Durchsetzung ihrer Sprache als Schriftsprache beitragen würde. In Vuks Grammatik und Wörterbuch von 1818 zeigt sich seine Be- strebung, die Spache der Lieder zu kodifizieren. Zur Zeit als die Treue zur Volkssprache sein oberstes Prinzip war, hat er eine Ka- tegorie von Wörtern aufgenommen, die in der lebendigen Volksspra- che nicht gebräuchlich waren. Es sind die sog. "stajace reci", die zum Vorrat "izražajnih sredstava folklora koji ima naddijalekatski karakter"* gehören. Vuk hat zur gleichen Zeit durch die Herausgabe der Volkslieder seinem Dialekt eine Tradition von poetischer Verwendung geschaffen. Jacob Grimms hohe Einschätzung des ästhetischen Wertes dieser Poe- sie und sein Lob ihrer "edlen" Sprache haben Vuk in seiner Uber- 2 zeugung zweifellos bestärkt . Vuks Konzeption der Schriftsprache auf der Grundlage der Volks- lieder und der Dubrovniker Literatur erscheint in der Endphase als eine Synthese von Herders Ansichten über die Volkssprache und Volks- poesie und Grimms Auffassung von der Bedeutung der schriftlichen Tra dition für die Entwicklung der Schriftsprache. 4. Vuks Ansichten über den čakavischen und kajkavischen Dialekt In Vuks Blickfeld stand zu Beginn seiner Arbeit nur der štokavi- sehe Dialekt, den er irrtümlicherweise mit der serbischen Sprache gleichsetzte. 1 Vgl. A. Belic, Jezičko ģedinstvo Srba, Hrvata i Slovenaca kod Bure Danioica, In: Danicicev zbornik, 1925, S. 33ff. 2 Er hat sie zum erstenmal in der zusammen mit Miklosich angefer- tigten Rezension von Auers ,,Sprachenhalle” vertreten. (GPS I I I , 285 - 289) . Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access d. Die einheitliche Schriftsprache Seine erste Bekanntschaft mit dem kroatischen čakavischen Dia- lekt stammt aus der Zeit seiner Reisen durch die südwestlichen Ge- genden und Dalmatien. In der Vorrede zu "Poslovice" von 1836 macht er die ersten Angaben über die čakavismen in der Sprache von Korču- la, ohne sie als solche zu bezeichnen. Erst 1843 gibt er eine Defi- nition des Čakavischen ( c a , r e k a l : GPS I I I , 95). 1845/46 geht er ausführlicher auf die Problematik des Čakavischen und Kajkavischen ein (GPS I I I , 181f., 195f.) Dabei führt er überwiegend das dialekto- logische Material aus dem Čakavischen an, während er zum Kajkavi- sehen die Form h o c e j u nennt, die er auch in Vojvodina verzeichnet hatte, und meint, daß auch die Vojvodiner Lokativformen na k o l i usw. Kajkavismen sein könnten. Zu Vuks Ansichten über die beiden kroatischen Dialekte hat be- reits A. Belic festgestellt, daß Vuk am Anfang das čakavische in einer näheren Gemeinschaft mit dem Stokavischen, das Kajkavische dagegen als Kroatisch angesehen hat1. Eine solche Theorie hat zu jener Zeit auch P. J. Safarik vertreten. Ihr gegenüber stand Kopi- tar-Miklosichs Theorie, die das čakavische dem Rest der bei Por- phyrogenetos erwähnten Kroaten zuschrieb und das Kajkavische dem Slovenischen zuordnete. Dieser Theorie hat sich Vuk seit ca. 1846 2 angeschlossen, wahrscheinlich unter Miklosichs Einfluß . Seine Ansichten trug er in dem Aufsatz "Srbi svi i svuda" von 1849 vor. in dem er die Štokaver mit den Serben, die Čakaver mit den Kroaten und die Kajkaver mit den Slovenen gleichsetzte1• in dem er die Štokaver mit den Serben, die Čakaver mit den Kroaten und die Kajkaver mit den Slovenen gleichsetzte1• In Vuks Verfahren is t der Herderische Gedanke über die Identität von Volk und Sprache deutlich erkennbar; auch die Gefahren, die ei- ne praktische Anwendung dieser Idee in sich birgt, sind deutlich zum Ausdruck gekonmen. Die Bestrebung, das Volk durch Sprache zu be- stimmen , ließ ihn einerseits die Existenz der Štokaver Kroaten übersehen, andererseits wurden die Dialekte derselben Sprache zu "Merkmalen" verschiedener Völker erhoben. Solche Fehler wurden auch durch die damalige Unklarheit über die linguistischen Tatsachen, d.h. über das Verhältnis der betreffenden Dialekte untereinander, begün- s tig t. Die Denkkategorie: Sprache - Volk war zur damaligen Zeit allge- mein verbreitet. Sie erscheint auch bei B. 1 Diese Ansichten Vuks konnten von den Illyrern selbstverständlich nicht geteilt werden. Dennoch haben die Meinungsunterschiede die begonnene Zusammenarbeit, aus der die Wiener Sprachvereinbarung hervorging, nicht gefährdet. - Über die pankroatischen Aktivitä- ten von A. StarČevifc, die nicht nur gegen Vuk, sondern auch gegen die Ideale der illyrischen Bewegung gerichtet waren, vgl. V. No- vak, a.a.O., S. 337ff. , , 2 Bekanntlich nannte Vuk am Ende an Stelle der Sprache die Religion, die er, wie auch Herder, zu den heiligen Eigentümern des Volkes rechnete (GPS I I I , 467; 1861). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 3 Vgl. B. Sulek, Srbi i Hrvati, 1856; In: GPS I I I , S. 461. 4 Uber verschiedene Theorien zu dieser Frage und die Literatur dazu vgl. die Aufsätze unter: Jezik srpskohrvatski, in: Enciklopēdija Jugoslavije IV. 1 Vgl. darüber die Arbeiten von A. Belic: 0 Vukovim pogledima na srpske dijalekte i književni jezik. Glas SKA LXXXII (1910); Vuko- va borba.., a.a.O.; Jezičko jedinstvo Srba Hrvata i Slovenaca.., a.a.O.; Vgl. auch V. Novak, a.a.O., S. 360ff. 3 Die Unterstreichungen stammen von Vuk. Meine Ergänzungen sind durch f ] gekennzeichnet. d. Die einheitliche Schriftsprache Šulek, der in die Diskus- sion über das serbische und kroatische Sprachproblem vermittelnd einzugreifen versuchte. Ausgehend von dem Grundsatz: "narodi se upra- vo jezikom razlikuju", bemühte sich šulek zu beweisen, daß Serben und Kroaten als "jedan narod, jednoga roda i koliena" Štokaver sind und betrachtete dabei die Čakaver als den Rest der slavischen Bai- kaneinwohner vor der Einwanderung der Kroaten und Serben3. Die Klärung der Beziehung zwischen dem Štokavischen, čakavi- sehen und kajkavischen Dialekt gehörte zweifellos zu den schwierigen linguistischen Fragen, über die auch in der nachfolgenden Zeit un- 4 terschiedliche Auffassungen vertreten wurden . Das Verfahren von g , , ; , 4 Uber verschiedene Theorien zu dieser Frage und die Literatur dazu vgl. die Aufsätze unter: Jezik srpskohrvatski, in: Enciklopēdija Jugoslavije IV. g 4 Uber verschiedene Theorien zu dieser Frage und die Literatur dazu vgl. die Aufsätze unter: Jezik srpskohrvatski, in: Enciklopēdija Jugoslavije IV. Vuk und seinen Zeitgenossen hat jedoch deutlich gezeigt, daß lin - guistische und nationale Probleme auseinandergehalten werden müssen. Vuk und seinen Zeitgenossen hat jedoch deutlich gezeigt, daß lin - guistische und nationale Probleme auseinandergehalten werden müssen. Zu Vuks Beschäftigung mit den Dialekten ist festgestellt worden, daß er außer dem štokavischen auch dem čakavischen Dialekt große Aufmerksamkeit gewidmet hat1. In seinen veröffentlichten Schriften sind sehr wenige Angaben über das Kajkvaische enthalten; außer den bereits genannten Formen erwähnt er lediglich, das Wort k o t a c für t o a a k in Kroatien gehört zu haben (GPS I I I , 197) und erläutert in seinem Wörterbuch von 1852 unter K e kava cz der Kroate (Windische), der k a j (statt 8 t a ) spricht". 2 In Vuks unveröffentlichtem Nachlaß befindet sich ein Blatt mit seinen Aufzeichnungen der Merkmale des kroatischen kajkavischen Dia- lekts, die wir hier mitteilen möchten, weil sie bisher unbekannt ge- blieben sind und weil sie uns eine Auskunft über den Umfang seiner Kenntnisse dieses Dialekts geben3. mene se ne rači (nemam volje) 1) e mjesto a, n.p. šiрек (ruža) 6 â v v ko se k, otec, ves; zešiti (sasiti) zestàti se (sastati se) 2) 1 (pisai, kotel etc) 3) v m[jesto] u, n.p. vmra i vmrl (итгъо), vpirati (upirati), ѵъ hižu; vubiti, navučitelj , vdîlj (udilj) v č in iti 4) u rod. mn. jabuk, žen, hrušek, volov, gradôv; roditeljev, krajev, konj, penjéz, 5) u skaz. jed. e (kao u Resav[skom] [durchgestrichen] ) (mfjesto] u) n.p. a.a.O.; Vgl. auch V. Novak, a.a.O., S. 360ff. 2 Vgl. Archiv SANU 8552/30(27). d. Die einheitliche Schriftsprache vremena na kraju kao g a [durchgestrichen] e (kao maio kroz nos); a и tr[ecem] lic[u ] ju n.p. hoceju, piseju, govoriju, loviju i t.d. (veliju i velē) 9) и prvtom] licfu] m[n].br[oja] sad. vremena na kraju kao g a [durchgestrichen] e (kao maio kroz nos); a и tr[ecem] lic[u ] ju n.p. hoceju, piseju, govoriju, loviju i t.d. (veliju i velē) 10) U Zagorju u skaz. [durchgestrichen] vin. jed. kao o m[jesto] и n.p. vodo, ženo i t.d. 11) ë jedni govore kao i Herc[egovci], a jedni (ponajviše) kao e ponek[i] i (osobit[o] и Zagorju) 12) о svuda kao Ö, e ae, a kao a°, и kao ÿ (ü) 13) osobite rije č i: gorica, vinograd, zdje (i ozdje) ovde; vezdej, vezdaj, sad, kotač (kolo) m na kraju svagda kao n (kao и Pastrovièima) 9) и prvtom] licfu] m[n].br[oja] sad. vremena na kraju kao g a [durchgestrichen] e (kao maio kroz nos); a и tr[ecem] lic[u ] ju n.p. hoceju, piseju, govoriju, loviju i t.d. (veliju i velē) 9) (veliju i velē) 10) U Zagorju u skaz. [durchgestrichen] vin. jed. kao o m[jesto] и n.p. vodo, ženo i t.d. 11) ë jedni govore kao i Herc[egovci], a jedni (ponajviše) kao e ponek[i] i (osobit[o] и Zagorju) 12) о svuda kao Ö, e ae, a kao a°, и kao ÿ (ü) 13) osobite rije č i: gorica, vinograd, zdje (i ozdje) ovde; vezdej, vezdaj, sad, kotač (kolo) m na kraju svagda kao n (kao и Pastrovièima) 10) U Zagorju u skaz. [durchgestrichen] vin. jed. kao o m[jesto] и n.p. vodo, ženo i t.d. 11) ë jedni govore kao i Herc[egovci], a jedni (ponajviše) kao e ponek[i] i (osobit[o] и Zagorju) 10) U Zagorju u skaz. [durchgestrichen] vin. jed. kao o m[jesto] и n.p. vodo, ženo i t.d. Es is t deutlich zu sehen, daß Vuks Aufzeichnungen als Notizen zum eigenen Gebrauch gedacht waren. Die erste, nicht numerierte Zeile is t offensichtlich aus Platzmangel an die erste Stelle ge- raten; sie gehört zu den unter 13) verzeichneten lexikalischen Eigentümlichkeiten. In diesem Zusammenhang erinnern wir uns an ei- nen Brief von Jacob Grimm, in dem er an Vuk schrieb: "Sollten sich überhaupt nicht mehr einzelne Wörter zur bezeichnung der dialecti- sehen Spielarten ausmitteln lassen?" (Prepiska II, 22f.; 1823, un- d a tiert). Grimms Anregung hat hier ihren Niederschlag gefunden. 1 Vgl. Lj. Stojanovic, a.a.O., S. 757f. 2 Das wird auch durch seinen Brief an Kopitar während der ersten Reise durch die westlichen Gegenden bestätigt, in dem er auch über Kajkaver in Lika und Istrien schreibt (Prepiska I, 453). 1 Vgl. Lj. Stojanovic, a.a.O., S. 757f. 2 Das wird auch durch seinen Brief an Kopitar während der ersten Reise durch die westlichen Gegenden bestätigt, in dem er auch über Kajkaver in Lika und Istrien schreibt (Prepiska I, 453). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access d. Die einheitliche Schriftsprache na stole, na kvartire, pri boge, na brege 6) ženski u dat. i skaz. jed. де i l i e, n.p. gospoje m[jesto] gospe; kobile (podaj kobile sena) 7) compar[ativ]: zeleneji (zeleneja, zeleneje); [1 Wort unleserl.] zelenejši; mudreji, mudrejsi 8) h se izgovara svuda mene se ne rači (nemam volje) 1) e mjesto a, n.p. šiрек (ruža) 6 â v v ko se k, otec, ves; zešiti (sasiti) zestàti se (sastati se) 2) 1 (pisai, kotel etc) 3) v m[jesto] u, n.p. vmra i vmrl (итгъо), vpirati (upirati), ѵъ hižu; vubiti, navučitelj , vdîlj (udilj) v č in iti 4) u rod. mn. jabuk, žen, hrušek, volov, gradôv; roditeljev, krajev, konj, penjéz, 5) u skaz. jed. e (kao u Resav[skom] [durchgestrichen] ) (mfjesto] u) n.p. na stole, na kvartire, pri boge, na brege 6) ženski u dat. i skaz. jed. де i l i e, n.p. gospoje m[jesto] gospe; kobile (podaj kobile sena) 7) compar[ativ]: zeleneji (zeleneja, zeleneje); [1 Wort unleserl.] zelenejši; mudreji, mudrejsi 8) h se izgovara svuda ) u skaz. jed. e (kao u Resav[skom] [durchgestrichen] ) (mfjesto] u) n.p. na stole, na kvartire, pri boge, na brege ) ženski u dat. i skaz. jed. де i l i e, n.p. gospoje m[jesto] gospe; kobile (podaj kobile sena) 8) 1 Vgl. darüber die Arbeiten von A. Belic: 0 Vukovim pogledima na srpske dijalekte i književni jezik. Glas SKA LXXXII (1910); Vuko- va borba.., a.a.O.; Jezičko jedinstvo Srba Hrvata i Slovenaca.., a.a.O.; Vgl. auch V. Novak, a.a.O., S. 360ff. 2 Vgl Archiv SANU 8552/30(27) 3 Die Unterstreichungen stammen von Vuk. Meine Ergänzungen sind durch f ] gekennzeichnet. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 107 ־ 9) и prvtom] licfu] m[n].br[oja] sad. vremena na kraju kao g a [durchgestrichen] e (kao maio kroz nos); a и tr[ecem] lic[u ] ju n.p. hoceju, piseju, govoriju, loviju i t.d. (veliju i velē) 10) U Zagorju u skaz. [durchgestrichen] vin. jed. kao o m[jesto] и n.p. vodo, ženo i t.d. 11) ë jedni govore kao i Herc[egovci], a jedni (ponajviše) kao e ponek[i] i (osobit[o] и Zagorju) 12) о svuda kao Ö, e ae, a kao a°, и kao ÿ (ü) 13) osobite rije č i: gorica, vinograd, zdje (i ozdje) ovde; vezdej, vezdaj, sad, kotač (kolo) m na kraju svagda kao n (kao и Pastrovièima) 9) и prvtom] licfu] m[n].br[oja] sad. 1 Vgl. Lj. Stojanovic, a.a.O., S. 757f. d. Die einheitliche Schriftsprache Die letzte, durch das Fehlen der Numerierung abgesetzte Zeile enthält eine cakavische Eigenart mit dem Verweis auf Paštrovici, der in Bezug auf die Datierung uns den Hinweis gibt, daß diese Aufzeichungen von Vuk nach seinen Reisen durch Montenegro in den Jahren 1834 - 1839 entstanden sind. Auch das h in der Sprache von Vuk (vgl. unter 11): Hercegovci), deutet auf die Zeit nach 1836 hi Vuk war in den 30er Jahren mehrmals in Kroatien (1835, 1838, 1839) so daß die Vermutung naheliegt, daß seine intensivere Beschäfti- 2 gung mit dem kajkavischen Dialekt zu dieser Zeit begann . Vuks Aufzeichnungen enthalten phonetische und morphologische Eigenarten des kajkavischen Dialekts. Es is t auffallend, daß un- ter Vuks Anmerkungen keine der kajkavischen Akzentuation gewidmet is t. Er gibt zwar bei sechs Wörtern den Akzent an, verwendet da- bei aber seine drei Akzentzeichen für das Neuštokavische (A 4 *), die für das kajkavische Akzentsystem, das auch den Neoakut kennt1, unzutreffend sind. den Besonderheiten des kajkavischen Lautsystems verzeich Von den Besonderheiten des kajkavischen Lautsystems verzeich- net Vuk Von den Besonderheiten des kajkavischen Lautsystems verzeich- Von den Besonderheiten des kajkavischen Lautsystems verzeich- net Vuk a. zum Konsonantismus: 2 - die Aussprache des h (8) ; - die Bewahrung des auslautenden I (2); - er führt Beispiele zur Entwicklung des anlautenden u- und der Gruppe ѵъ- an (3) . Unter dem Akzent is t die Entwicklung von u - und ѵ ъ - in v u - zusammengefallen; außerhalb der Akzentstelle ent- wickelt sich in beiden Fällen ein v - , das verschiedene mundart- liehe Reflexe hat (h , g \ h m r l a , g m r l a ) ; b. zum Vokalismus: - die Entwicklung der Halbvokale zu e (1). Das g ilt für die meisten kajkavischen Mundarten; es gibt jedoch auch solche, in denen der 4 Halbvokal erhalten geblieben is t ; - die Vertretung des è durch einen 0-Laut in den meisten Mundarten. Es is t ein, wie im Falle des e < ъ, ь, halbgeschlossenes e (also: ъ = ь = e > e ) , das Vuk mit é bezeichnet (11). Seine Angaben über die ijekavische e-Vertretung kann sich auf die Diphthongierung des langen e beziehen5, die ikavische dagegen auf den Zusammen- fa ll des kurzen, nicht akzentuierten e mit г . 1 S.Ivšic hat als erster die kajkavischen Akzente untersucht und die Mundarten in zwei Gruppen (mit jeweils zwei Untergruppen) ein geteilt: die *,konservative" Gruppe, die das alte Akzentsystem von drei Akzenten bewahrt, und die "revolutionäre” Gruppe, die eine neue Akzentuation entwickelt hat. - Vgl. S. Ivsic, Jezik Hrvata kajkavaca, Ljetopis JAZU 48, jetzt in: Gesammelte Schriften zum slavischen Akzent, 1971, S. 673ff. , , 2 Vgl. dazu M. Hraste, Jezik Srpskohrvatski. Kajkavski dijalekt, In Enciklopēdija Jugoslavije IV. 5 Vgl. P. Ivic, Procesi rasterecenja vokalskog sistema.., S. 58ff. 6 Vgl. ebd., S. 6 2. 1 Vgl. S. Ivšic, a.a.O., S. 660. 2 Vgl. P. Ivic, Procesi rasterecenja vokalskog sistema.., S. 58ff. 3 Vgl. P. Ivic, Die serbokroatischen Dialekte I, 1958, S. 29. 4 Es is t jedoch unklar, was mit dem unter 1) angeführten ko se к meint. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access d. Die einheitliche Schriftsprache 1 S.Ivšic hat als erster die kajkavischen Akzente untersucht und die Mundarten in zwei Gruppen (mit jeweils zwei Untergruppen) ein geteilt: die *,konservative" Gruppe, die das alte Akzentsystem von drei Akzenten bewahrt, und die "revolutionäre” Gruppe, die eine neue Akzentuation entwickelt hat. - Vgl. S. Ivsic, Jezik Hrvata kajkavaca, Ljetopis JAZU 48, jetzt in: Gesammelte Schriften zum slavischen Akzent, 1971, S. 673ff. 2 Vgl. dazu M. Hraste, Jezik Srpskohrvatski. Kajkavski dijalekt, In Enciklopēdija Jugoslavije IV. 3 Vgl. P. Ivic, Prilozi poznavanju dijalekatske slike zapadnę Hrvat ske, in: Godišnjak FFNS VI, 1961, 193.; ders. Izveštaj o teren- skom dijalektološkom radu и severnoj Hrvatskoj i južnoj Dalmacij i ebd. II, 1957, 403. 4 Vgl. ders.. Procesi rasterecenja vokalskog sistema и kajkavskim govorima. In: Zbornik FL XI, 1968, 58. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Bereits Ivšic hat festgestellt, daß in der Entwicklung des kaj- kavischen Vokalismus der Akzent, insbesondere aber die Quantität eine wichtige Rolle spielen . Die kurzen Vokale, vor allem die un- betonten, werden offener gesprochen oder reduziert; die langen Vo- kale dagegen erhalten eine geschlossenere Aussprache; 3, 5, г wer- den oft diphthongiert. Vuk verzeichnet einige Besonderheiten der kajkavischen Vokale (12), nämlich: die allgemein kajkavische Öffnung des e ("e ae"),die sehr verbreitete Verschiebung der Artikulation des a nach hinten ("a kao a°"), des и und des о zur vorderen Vokalreihe ( ü , ö ) . Die Verschiebung des nichtaukzentuierten о reicht bis zu seinem Zusam- 2 menfall mit e hin . Vuks Anmerkung zum "e (kao maio kroz nos)" in der 1. PI. Präs. (9) bezieht sich auf diese Entwicklung. Aus dem Bereich der Morphologie führt Vuk wenige Angaben an: a. zur Deklination der Substantive die älteren Kasusformen: Dat. Sg. Fern, und Lok. Sg. Fern./Mask, auf e < ë (5. 6.); Akk. Sg. Fern, auf - о < ç (10); Gen. PI. ohne Endung, oder bei den Mask, auf - o v / - e v (4); a. zur Deklination der Substantive die älteren Kasusformen: Dat. Sg. Fern, und Lok. Sg. Fern./Mask, auf e < ë (5. 6.); Akk. Sg. Fern, auf - о < ç (10); Gen. PI. ohne Endung, oder bei den Mask, auf - o v / - e v (4); b. d. Die einheitliche Schriftsprache zum Adjektiv führt er außer der charakteristischen Komparativ- bildung mit - e ( j ) 8 i und - 5 ־auch das in den südwestlichen Mund- arten vorkommende -e j-3 an (7); c. die Angaben zur Konjugation sind dürftig: neben der bereits ge- nannten 1. PI. Präs. führt er lediglich die erweiterte Form in der 3. PI. Präs. an (9). Es fehlen solche auffallenden Merkmale wie die vom Stokavischen abweichende Bildung des Futurs. Vuks Aufzeichnungen der kajkavischen Merkmale sind zum größten 4 Teil richtig ; bei z d j e (13) is t allerdings die Jekavisierung über- flüssig. Die von Vuk in Stichworten verzeichneten Angaben zum Kaj- kavischen zeigen, trotz der Unvollständigkeit, daß er in die Eigen- arten dieses Dialekts gut eingedrungen war. Sie sind ein zusätzli- ches Zeugnis von seinem Interesse für die Dialekte und seine For- schung auf diesem Gebiet. 1 Vgl. Kopitars Rezension der ”Pismenica" in KKS I, S. 314ff. 2 Vgl. ausführliche Analyse bei P. Ivic, О Vukovom Rječniku.., S.57f 3 B. Kopitar, Grammatik der slawischen Sprache in Krain, Kärnten und Steyermark, Laibach 1809. - Ein ausführlicher Vergleich der beiden Grammatiken kann hier nicht unternommen werden. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access I. Die deutsche Übersetzung von Vuks serbischer Grammatik I. Die deutsche Übersetzung von Vuks serbischer Grammatik 1824 erschien in Berlin und Leipzig "Wuk״s Stephanowitsch1 klei- ne serbische Grammatik verdeutscht und mit einer Vorrede von Jacob Grimm". Das Buch enthielt außer der Grammatik und der 54 Seiten um- fassenden Vorrede von Grimm auch noch eine ausführliche Abhandlung über die serbischen Heldenlieder von J. S. Vater. Bei dieser Übersetzung handelt es sich um Vuks Grammatik von 1818; es is t seine zweite Grammatik, die gegenüber der "Pismenica" von 1814 eine neue und selbständige Arbeit darstellt. Der Zeitraum zwischen 1814 und 1818 war Vuks Heranreifen zum selbständigen Wissen schaftie r durch Zusammenarbeit mit Kopitar. Die angereicherten Kennt nisse und Kopitars Ratschläge1 haben es Vuk ermöglicht, sich von Mrazoviè-Smotrickyjs Grammatiksystem zu lösen und die erste Gramma- tik zu schaffen, aus der man das gesamte Deklinations- und Konjuga- tionssystem der serbischen Sprache in seinen Hauptzügen ersehen kann Vuk hat auch aus Kopitars Grammatik3 zumindest Anregungen geschöpft, wie es die folgenden Beispiele zeigen: Kopitar S. 294: Vuk S. LIIIs Mòsh, ki je per meni b il; Čovek, što je bio kod mene; Shêna, ki si jo per meni v id il; Žena, što smo je vifleli. Vuk S. LIIIs Čovek, što je bio kod mene; Žena, što smo je vifleli. Die deutsche Übersetzung der "Srpska gramatika" war eine der ersten Unternehmungen Vuks, die ohne Kopitars Rat oder Hilfe ent- standen is t. Vuk wollte die Grammatik offenbar bereits 1818 in deut- scher Sprache erscheinen lassen, wie aus seinem Brief an Mušicki her vorgeht: "Od Srpskoga Rječnika vec se pečata T... poslije predgovora 1 Vgl. Kopitars Rezension der ”Pismenica" in KKS I, S. 314ff. 2 Vgl. ausführliche Analyse bei P. Ivic, О Vukovom Rječniku.., S.57f 3 B. Kopitar, Grammatik der slawischen Sprache in Krain, Kärnten und Steyermark, Laibach 1809. - Ein ausführlicher Vergleich der beiden Grammatiken kann hier nicht unternommen werden. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access bice и njemu i jedna kratka Srpska gramatika na Njemačkom jeziku (šta mislite o tom?).11 (Prepiska II, 227). Vuks Kenntnisse der deut- sehen Sprache waren nicht ausreichend, um die Übersetzung selbst an- zufertigen; Kopitar maß diesem Plan von Vuk derzeit offenbar keine Bedeutung zu, wie er noch am 3. Juli 1823 schrieb: "Die Zeit, wo man nach illirisch e n Grammatiken fragt, is t noch nicht da." (Prepi- ska I, 222) . 1 Vgl. Leitzmann I, S. 427. - Zu Grimms Beschäftigung mit der serbo- kroatischen Sprache existierte eine handgeschr. Diss. von J. Bogi- čevic: Jacob Grimms Tätigkeit auf dem Gebiete der serbischen Spra- che (Zagreb 1921), die M. Mojaševic, Grimov prevod i primerak Ka- ridžiceve gramatike. Anali FF V, S. 163, erwähnt, die aber nach Auskunft der Philosophischen Fakultät Zagreb verlorengegangen is t. 3 Vgl. Vasmer, S. 5. p g 2 Vgl. Leitzmann I, S. 427f. 1 Vgl. Leitzmann I, S. 426ff. 2 Vgl. A. Sauer, Aus Jacob Grimms Briefwechsel mit slavischen Ge- lehrten. In: Prager deutsche Studien VIII, 1908, S. 612. 3 Vgl. dazu M. Mojaševic, Grimov prevod.., S. 164. 4 Vgl. S. Novakovič, Srpska bibliogrāfijā za noviju književnost 1741 - 1867, 1869, S. 234. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access I. Die deutsche Übersetzung von Vuks serbischer Grammatik Sein plötzlicher Meinungswechsel ist wohl unter dem Eindruck von Vuks Leipziger Liederausgabe entstanden: Die Uber- Setzung sei "unentbehrlich um Deutsche fähig zu machen, sich unsrer Literatur zu freuen, anzunehmen, und sie zu feyern und zu verbreiten. (Prepiska I, 233). Vuk war aber bereits auf dem Wege nach Kassel, um bei Jacob Grimm Hilfe zu suchen. Vermutlich wurde er dazu ermutigt durch Grimms Rezension des Wörterbuchs, in der Grimm den Wert der serbischen Sprache und die Bedeutung der Hilfsmittel zu ihrer Er- lernung hervorgehoben hatte (GKS IV, 104) . Vuk hat die Übersetzung, die für ihn D. Tirol angefertigt hatte, Grimm zur Durchsicht gegeben und ihn gebeten, einen Verleger zu fin- den. Es war offenbar nicht schwer, Grimm für diese Sache zu gewinnen, wie aus seinem Brief an Lachmann zu ersehen is t: "Meine alte nei- gung zu der spräche und den liedern wachte auf, die Verdeutschung der grammatik schien mir sehr nützlich..." Einen Verleger fand Grimm auch sofort und der Druck begann früher als es ihm in Anbetracht 2 der zu erledigenden Arbeit recht sein konnte . Die Übersetzung von Tirol erschien Grimm auf den ersten Blick brauchbar, wie er an Kopitar schrieb: "Meine aufrichtige Ansicht von der Sache is t folgende: die mir von Herrn Vuk vorgelegte, in Ungarn gefertigte Version scheint genau und richtig; in den deutschen Aus- drücken muß ihr ein wenig nachgeholfen werden. Das kann leicht ge- schehen."3 Eine ganz andere Meinung über Tirols Übersetzung äußerte Grimm während der Beschäftigung mit ihr in einem Brief an Lachmann: "Die Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ganze zeit bin ich tüchtig zerstreut worden durch treiben des serbi- sehen, wozu mich ein neugedruckter band voll trefflicher lieder und Vuks besuch wieder aufgeregt hat. Es geschah ihm ein großer dienst, daß ich eine in Ungarn gemacht elende und unverständige Übersetzung seiner serbisch geschriebenen grammatik durchsähe und enderte... ein völlig unbrauchbares manuskript, das ein Ungar gemacht, der we- der deutsch versteht, noch eine idee von grammatik hat."1 Auch an Vuk schrieb er: "Der gute Ungar verstand nicht viel Deutsch und scheint sich einigemahl übereilt oder den Text misverstanden zu ha- ben." (Prepiska II, 6). ganze zeit bin ich tüchtig zerstreut worden durch treiben des serbi- sehen, wozu mich ein neugedruckter band voll trefflicher lieder und Vuks besuch wieder aufgeregt hat. I. Die deutsche Übersetzung von Vuks serbischer Grammatik Es geschah ihm ein großer dienst, daß ich eine in Ungarn gemacht elende und unverständige Übersetzung seiner serbisch geschriebenen grammatik durchsähe und enderte... ein völlig unbrauchbares manuskript, das ein Ungar gemacht, der we- der deutsch versteht, noch eine idee von grammatik hat."1 Auch an Vuk schrieb er: "Der gute Ungar verstand nicht viel Deutsch und scheint sich einigemahl übereilt oder den Text misverstanden zu ha- ben." (Prepiska II, 6). Grimm spricht von Tirol als von einem Ungarn. Auch an Dobrovskÿ schrieb er, daß die "wörtliche Übersetzung eines ungarischen Studen- ten, der aber weder deutsche Sprache noch serbische Grammatik ver- 2 stand. .. zusammengelegt, zugeschnitten, ergänzt wurde." Offenbar ha- ben die Fehler in der Übersetzung, vor allem die von Grimm erwähn- ten Mißverständnisse des Originaltextes, ihn zu der Meinung geführt, Tirol sei ein Ungar. Dimitrije P. Tirol (1793 - 1857) wird in der Vuk-Forschung als der Übersetzer der Grammatik von Vuk genannt. Der Verlust des Ma- nuskripts3 hat es der Forschung erschwert, ein zuverlässiges Urteil über den Wert seiner Übersetzung abzugeben. Die negativen Äußerungen von Grimm und eine Bemerkung von Vuk über Tirols schlechte Kenntnis- se der serbischen Sprache lassen ihn in einem ungünstigen Licht er- scheinen. Er war Lehrer, Übersetzer, Schriftsteller und Herausge- ber der Zeitschriften Banatski Almanah (1828-29) und Uranija (1837- 38) und ordentliches Mitglied des Društvo srbske slovesnosti und 4 der Gesellschaft für Geschichte und Altertümer von Odessa . Als Freund von Vuk setzte er sich auch aktiv für Vuks Reformpläne ein, die er auch mit dem von ihm 1828 gegründeten Društvo lju b ite lja l i - terature srpske unterstützen wollte (Prepiska IV, 602f.). Vuk nannte ihn in einem Brief an Kopitar seinen einzigen Anhänger in Temišvar und fügte hinzu: " ... on istina ne zna ni sam dobre Srpski a li je veliki junak и našoj p a rtiji." (Prepiska I, 263). Bei der Bewertung von Vuks Meinung über Tirols Sprachkenntnisse muß Vuks ablehnende Haltung gegenüber der Vojvodiner Sprache berücksichtigt werden. Ti- rols Briefe an Vuk aus dieser Zeit zeigen nur kleinere Verstöße ge- gen die serbische Sprache vor allem in der Syntax, durch die allzu häufige Endstellung des Verbes. Allerdings verwendet er oft die älte- ren Instrumental- und Lokativforem ohne -ma, die Vuk für falsch h ie lt (Prepiska, IV, 581, 586) . 1 Seine Schüler hat er nach eigenen Worten auch Vuks Orthographie gelehrt (Prepiska IV, 617). 2 Novakovic, Srpska bibliogrāfijā, a.a.O., verzeichnet 18 Titel von T irol. 3 Tirol bat Vuk, der sich um die Herausgabe kümmerte, um eine Vor- rede und um etwaige Einwände und Verbesserungen (Prepiska IV, 589, 593, 595). 4 Vgl. Novakovic, Srpska bibliogrāfija.., S. 144. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. Vasmer, S. 5 I. Die deutsche Übersetzung von Vuks serbischer Grammatik Seit etwa 1827 zeigen Tirols Briefe eine allmähliche Rückkehr zur alten Orthographie, die er als Lehrer o ffiz ie ll verwenden mußte*, und auch mehr Slavenoserbismen als vor- her. Als Schriftsteller war Tirol ziemlich produktiv, aber wohl wenig 2 originell . Eine große Anzahl von seinen Schriften ist den prakti- sehen Zielen, dem Schulunterricht, gewidmet; sie haben nur einen kul- turhistorischen Wert. Tirol hat auch drei Grammatiken verfaßt: eine 1827 in Wien mit Vuks Orthographie gedruckte "slavische" Grammatik3, die Saf^afîk eine ”werthlose Compilation aus Dobrowsky In s titt. ling. 4 slav.” genannte hat ; eine deutsche Grammatik für serbische Jugend von 1830 und eine 1851 in Novi Sad erschienene ”Kleine Grammatik der serbischen Sprache für Deutsche", die er nach eigenen Worten auch in seinem Unterricht an der serbischen Schule verwendet hat (Prepiska IV, 618). In dieser Grammatik führt er ein 35 Buchstaben enthal- tendes Alphabet an, in dem sowohl ksl. Zeichen für beide reduzierte Vokale, y , e , j e , j u , j a als auch Vuks Zeichen für Zf, ô und dz zu finden sind. Die Flexionsparadigmata sind serbisch; die Komposition der Grammatik zeigt gewisse Parallelen zu Vuks Grammatik. Unter Tirols Werken existiert leider keines in deutscher Spra- che aus der Zeit unmittelbar vor oder nach seiner Übersetzung der Grammatik von Vuk, aus dem wir zuverlässige Schlüsse über seine Kenntnisse der deutschen Sprache ziehen könnten. Es existiert aber eine Bemerkung von ihm selbst, die in diesem Zusammenhang inter- essant is t. Am 13. Mai 1847 schrieb er an Vuk in Bezug auf die "Serbische Revolution"von Ranke , dem Vuk bekanntlich Materialien geliefert hat: als Serbe dürfe er den S til nicht beurteilen, "al bi 3 Tirol bat Vuk, der sich um die Herausgabe kümmerte, um eine Vor- rede und um etwaige Einwände und Verbesserungen (Prepiska IV, 589, 593, 595). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM 4 Vgl. Novakovic, Srpska bibliogrāfija.., S. 144. ja tek tek samo rekao, da je gotovo takav, kao i moj, kad što s Ne- mačkoga prevodim." (Prepiska IV, 613). Tirol h ie lt noch 1847 seine Sprachkentnisse und übersetzerfähigkeiten nicht für vollkommen, ob- wohl er sich in der Zwischenzeit mit der deutschen Sprache befaßt hatte. ja tek tek samo rekao, da je gotovo takav, kao i moj, kad što s Ne- mačkoga prevodim." (Prepiska IV, 613). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access I. Die deutsche Übersetzung von Vuks serbischer Grammatik Tirol h ie lt noch 1847 seine Sprachkentnisse und übersetzerfähigkeiten nicht für vollkommen, ob- wohl er sich in der Zwischenzeit mit der deutschen Sprache befaßt hatte. Durch den Verlust des Manuskripts von Tirol is t das Problem ent- standen, wie Grimms Anteil an der Übersetzung festgestellt werden kann. Hier kann zumindest unterschieden werden zwischen demjenigen Teil des Ubersetzungstextes, der mit Vuks Original übereinstimmt, und dem vom Original abweichenden Teil. In dem ersteren können über Grimms Anteil an der Übersetzung nur Vermutungen angestellt werden. Was die Abweichungen der Übersetzung vom Original b e trifft, da kann Tirols Urheberschaft mit Sicherheit ausgeschlossen werden. Es besteh nirgendwo ein Beleg darüber, daß Vuk Tirol ermächtigt hätte, irgend- etwas an seiner Grammatik zu ändern; im Gegenteil, er sprach über Tirols Arbeit nur als von einer Übersetzung der Grammatik. Da uns die deutsche Übersetzung von Vuks Grammatik vor allem in ihrem vom Original abweichenden Teil interessiert, wollen wir die Frage nach der Abgrenzung von Grimms Arbeit gegenüber Tirol nur in diesem Zusammenhang behandeln. Aus Vuks und Grimms Briefwechsel is t deutlich ersichtlich, daß Vuk Grimm völlig frei gelassen hat, in der Grammatik überall dort zu korrigieren und zu ändern, wo es ihm nötig erscheinen sollte (Prepiska II, 9, 18, 30, 38, 40). Außerdem sollte die Grammatik auf Grund von Vuks Materialien ergänzt und mit einer Vorrede von Grimm eingeleitet werden. Grimm sah am Anfang seine Rolle als die eines Korrektors und Vermittlers an. Zu weitergehenden Aufgaben fühlte er sich nicht be- rufen, wie er an Kopitar schrieb: "Sinke ich da nicht in ein für mich noch bodenloses Erdreich über Hals und Kopf oder wenigstens do ramena ein, wo ich mir hernach nicht heraushelfen werde? ...Zu- sätze, Umstellungen, Vergleichungen (wozu mich meine Halbkenntniß zwar führen, aber auch irreführen könnte) muß ich unterwegen lassen" Ganz anders jedoch entwickelte sich seine Einstellung zu dieser Arbeit im Verlaufe der Beschäftigung mit ihr. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1. Die Abweichungen der Übersetzung vom Original 1. Die Abweichungen der Übersetzung vom Original Die deutsche Übersetzung der Grammatik von Vuk zeigt so viele Abweichungen gegenüber dem Original, daß es richtiger wäre, von der deutschen Fassung zu reden. Auf der einen Seite sind Erweite- rungen des Originals durch neue Kapitel und Zusätze im Text, auf der anderen wiederum Kürzungen, Umstellungen und Umformulierungen des Originaltextes vorhanden. Es erhebt sich die Frage nach dem Wert dieser Änderungen, wie auch nach ihrem Urheber. Ihre Klärung ließe Schlußfolgerungen sowohl über Vuks und Grimms Zusammenarbeit als auch über den Umfang von Grimms Kenntnissen und Tätigkeit auf dem Gebiet der serbischen Sprache zu. a. Die Erweiterung der Grammatik Bei der Untersuchung der Erweiterung des Originaltextes is t uns Vuks Briefwechsel eine große Hilfe. Auf Grund der daraus entnomme- nen Angaben können wir zwischen denjenigen Ergänzungen der Gramma- tik , die nach dem von Vuk und Grimm in Kassel besprochenen Plan durchgeführt worden sind, und denen, die Grimm im Laufe der Arbeit sebständig vorgenommen hat, unterscheiden. b. Die von Vuk und Grimm gemeinsam geplanten Ergänzungen Vuk hatte von Anfang an die Absicht, die deutsche Fassung sei- ner Grammatik durch eine S y n t a x zu erweitern (Prepiska I, 210). Dem Übersetzer Tirol hat er aber die versprochene Syntax nicht ge- lie fe rt. Als er Ende September 1823 das übersetzungsmanuskript Grimm zur Korrektur gab, hatte er diesen Plan noch nicht aufge- geben. Da der Druck der Grammatik eher als erwartet begann, und er mit der Herausgabe seiner Lieder beschäftigt war, bat er Kopi- tar um Hilfe :״Predgovor, a osobito о ortografiji valjalo bi da mu [Grimm] Vi pokažete; a malo sintaksisa, valjalo bi da Vi upra- vo načinite, pa da mu pošljete, takoder i о narječijama (ali ovo pošljednje makar mu i ja načinio)." Prepiska I, 237). Da aber Ко- pitars Hilfe ausblieb, und der Druck in schnellem Tempo voranging, erschien die Grammatik ohne die geplante Syntax. 1. Die Abweichungen der Übersetzung vom Original Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Es war auch von vornherein geplant, die Grammatik mit einem neu- en Kapitel über die P a r t i k e l n zu ergänzen- Während aber der Plan von einer Syntax aus dem Jahre 1814 stammt1 und Vuk ihn noch wäh- rend der Übersetzungsarbeit von Tirol durchführen wollte, werden die Partikeln zum erstenmal nach Vuks Besuch bei Grimm erwähnt. Vuk schrieb nämlich am Schluß seiner Grammatik von 1818, daß die Parti- kein im Wörterbuch verzeichnet sind, weshalb ihre Erörterung in der Grammatik überflüssig sei. Nach dem Besuch bei Grimm meldete er Ko- pitar: "[Grimm] još iste da mu posijem što i od partikéin (adver- bia, praepositionen, etc.), a to и тепе ništa nije gotovo! - Sad sam se zbunio sa sviju strana." (Prepiska I, 239). Das Kapitel "Von den Partikeln", mit dem die deutsche Fassung der Grammatik endet (Anh. I, 225ff. ), is t offenbar auf Grimms Idee und Betreiben entstanden. Dafür spricht auch Grimms Interesse für Präpositionen im Zusammenhang mit der Entwicklung und Reduktion der Flexion, "über den trieb und das wesen der bildungen oder zusammen- Setzungen insgemein gibt uns hingegen das adverbium desto reicheren u. erwünschteren aufschlusz, wir sollen auf alle fingerzeige achten, die uns hier der gang und die analogie der sprachen bietet..." (GKS VI, 303). Aus den Interjektionen, in denen nach seiner Meinung größtenteils die ursprünglichen Wurzelwörter bewahrt geblieben sind, deren Bedeutung aber verdunkelt und zum bloßen Ausruf reduziert wor- den is t, erhoffte er wichtige Schlüsse zur Erforschung der Mytholo- gie. Er kannte bereits aus den serbischen Volksliedern solche dem Volk unverständliche Wörter, die meistens in den Refrains vorkom- men. In der Rezension von Vuks Liedern schrieb er: "Lado die göttin wird in der dem volke längst unverständlich gewordenen interjec- tion lado, oj lado angerufen... etwa wie der altdeutsche gott Irmin im Hilderbrandslied übrig is t, oder pol, aede pol, hercle von den Römern, als man sich lange dabei nichts dachte, fortgebrauch wurde, wer weisz wie viel andre dunkle interjectionen in allen spra- chen aus alten götternamen zu deuten sind." (GKS IV, 220). Wahr- scheinlich hat Grimm mehr von solchen Beispielen erwartet, die Vuk ihm aber nicht geben konnte. 1 Vgl. auch Kopitars Rezension der"Pismenica" (KKS I, 317) Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1. Die Abweichungen der Übersetzung vom Original Deshalb hat er im letzten Absatz der Kapitels die ihm bekannten und in Vuks Wörterbuch verzeichneten L a d o i l e l j o i k o l a l o m i aufgeführt (Anh. I, 229 ); dieser Absatz stammt mit Sicherheit von ihm, denn Vuk rechnete diese Wörter nicht zu Interjektionen. Vera Boji - 9783954793044 1 Vgl. auch Kopitars Rezension der"Pismenica" (KKS I, 317) Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 117 - Wenn man die genannte Briefstelle von Vuk liest, drängt sich die Frage auf, warum er nicht einfach das hierher gehörende Wortma- terial aus seinem Wörterbuch Grimm zugeschickt hat• Aus seiner Be- Schreibung der Arbeitsweise am Wörterbuch (GPS I I I , 66) wissen wir, daß er alle Wörter mit deutscher Übersetzung von Kopitar auf Zet- teln besaß. Schon der erste Vergleich dieses Grammatikkapitels mit den entsprechenden Angaben im Wörterbuch gibt eine Antwort auf die- se Frage: in der Grammatik sind die Präpositionen nach der Rektion um die betreffenden Kasus gruppiert. Vuks Wörterbuch von 1818 gibt aber die Rektion nur in einigen Fällen explicite an; sie is t zwar aus den angeführten Beispielen größtenteils ersichtlich, es gibt aber auch Fälle ohne Textbeispiel (z.B. meãu, n a s u p v o t , u n a to Ö ) . Außerdem sind die Beispiele nach der jeweiligen Bedeutung der be- treffenden Präposition und nicht nach der Rektion geordnet (vgl. z.B. 3a, wo die Beispiele in fünf Gruppen eingeteilt sind ohne An- gäbe der Rektion, unter 2) solche, die die Präposition sowohl mit Akk. als auch Instr. belegen). Es war offenbar Grimms Idee, daß die serbische Grammatik für Deutsche diese Angaben zu Präpositionen enthalten müßte, und auch Vuk wird spätestens zu diesem Zeitpunkt eingesehen haben, daß sie auch sein Wörterbuch hätte verzeichnen sollen. Das ist in der zwei- ten Auflage des Wörterbuchs von 1852 nachgeholt worden. Aus diesem Grunde mußte Vuk selbst die Materialien für dieses Kapitel zusam- menstellen, denn es steht außer Zweifel, daß Grimm nicht in der La- ge gewesen wäre, dieses Problem in einer so kurzen Zeit zu lösen. Vuk selbst hat es, wie wir sehen werden, auch nicht auf die beste Weise gelöst. Als Vuk am 6. Nov. 1823 seine ”Materialien" an Grimm schickte, entschuldigte er sich wegen der "unvollständigen und ohne Ordnung gestellten Notizen" (Prepiska II, 11). 1. Die Abweichungen der Übersetzung vom Original Als er später das von Grimm hergestellte Manuskript las, schrieb er: "Auch über Partikeln Sie haben viel besser gemacht, als ich Ihnen Materialien geschickt ha- be." (Prepiska I I , 40). Auch wenn man Vuks Bewunderung des gramma- tischen Genies von Grimm bedenkt, so kann doch aus diesen Zeilen geschlossen werden, daß Grimm die Materialien von Vuk zumindest geordnet und systematisiert hat, daß die Gliederung des Kapitels von ihm stammt. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Die Analyse des Kapitels "Von den Partikeln" und der Vergleich des darin enthaltenen Materials mit den Angaben in Vuks Wörterbuch hat folgendes ergeben: 1. Die Grammatik enthält neues Wortmaterial, das im Wörterbuch fehlt- Das sind Präpositionen: u k r a j , a a v r , 8 onu 8 t r a n u ; Adver- bia: d o v l e , d o n l e , I j e , nobom, n a j p o e l i j e , n a j z a d , o n d a j , 0 8 t r a g A s p o l j a , t u d a я z o r o m ; Konjunktionen: a l (zu a l i ), p re m d a , t a k o û ç r ; Interjektionen: a a ! 3 d e d e r i , n u d e r ! Alle diese Wörter (mit Aus- nähme von nobom, a l und a a ! ) sind in die zweite Auflage des Wörter- buchs von 1852 aufgenommen worden. Sie gehören offensichtlich zu dem nach Erscheinen des Wörterbuchs von 1818 neugesammelten Wör- tervorrat, den Vuk leichter überblicken und in seine "Materialien" aufnehmen konnte, als den umfangreichen Wortschatz seines Wörter- buchs. 2. In dem Grammatikkapitel is t bei weitem nicht alles aus dem Wörterbuch ausgenützt worden. Es fehlen z.B. 1 Zu n a a u p r o t und u n a t o c gibt das Wörterbuch keine Textbeispiele an, aber Vuk selbst hat n a a u p r o t mit Dativ gebraucht. - Vgl. T. Maretic, Gramatika hrvatskoga i l i srpskoga književnog jezi- ka, 3.Aufl. 1963, S. 581. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM i f 1. Die Abweichungen der Übersetzung vom Original In der Grammatik fehlen die in der Volkssprache gängigen Adverbia, die das Wörterbuch enthält, wie z.B.: b a r , d e a n o , (wäh- rend l e v o angegeben wird), d o n e k l e , d o a l i j e , i z b l i z a , i z d a v n a , j e d - v a , k a k o , k a k o mu d r a g o , k a k o t a k o , k a a n o , k u d ( a ) g o d , kuda mu d r a g o , m a lo k a d , m a lo po m a lo , n e , n e g d a , n e k a d ( a ) , n i k a k o , o d a n - d e , o d a n l e , o d a v n o , o d o n d a , o d o n u d a , o d o z g o , o d a e l e , o a e l e , o d - e v a k l e , o d s v a k u d a , o d v e ò , o d v i a e , o n o m l a n i , o n o m v e c e r i , a v a g d a , s v a g d e , a v a k a d , a v a k u d a , t o b o z é , u v e o e , u v i j e k , v a z d a ( n ) , veoma, v r l o , usw. In den Ausführungen über die Konjunktionen wäre eine Vollstän- digkeit und Gliederung nach Satzarten wünschenswert gewesen. 1. Die Abweichungen der Übersetzung vom Original die Präpositionen zum Genitiv: n a v r , oda (z u o d i oda šta je to; es fehlen überhaupt die Angaben der Bedeutung a l s in Vergleichen, und a n : an einer Krank- heit leiden, sterben; es wird lediglich die Übersetzung von ohne Beispiele angeführt); p o v r ; 8 in temporaler Verwendung: s večera; u s r e d ; v a n ; z b o g . Uber den Dativ folgert Grimm auf Grund des ihm zugesandten Materials ausdrücklich, daß es zu diesem Kasus nur ei- ne Präposition gebe: k , k a , obwohl das Wörterbuch auch n a e u p r o t , n a s u s r e t und u n a t o c verzeichnet1. Beim Akkusativ vermißt man c r e z t k r o z a (zu k r o z : kroza me, kroza te); p o d in temporaler Ver- wendung (pod starost, pod noe); uza (zu u z , beide auch in der Be- deutung von n e b e n : stao uza me); beim Lokativ fehlen die Präpo- sitionen p re m a und ргг. Trotz diesen Auslassungen is t in den Ausführungen über die Präpositionen das Wichtigste gesagt worden. Weniger gelungen er- scheint dagegen der Abschnitt über die Adverbia. Es wäre zwar kaum sinnvoll, die zahlreichen Adverbia in der Grammatik vollständig anzuführen. Wenn aber neben der Grundstufe auch Komparative, (al- lerdings als solche nicht bezeichnet), an,gegeben werden, wie; 00051004 ־ 119 ־ v i s e , m a n j e , b o l j e (zu dem letzten fehlt die Grundstufe d o b r o ) und gleichzeitig solche für Ausländer schwer verständliche und identi- fizierbare Adverbia wie b a r , ka ko t a k o usw. fehlen, so deutet das auf eine gewisse Ratlosigkeit hin, in die Grimms Forderung Vuk ge- trieben hat. Sein Wörterbuch bezeichnet die Adverbia und Konjunk- tionen selten als solche, weshalb er Grimm nicht darauf verweisen konnte. 1. Die Abweichungen der Übersetzung vom Original solche Abweichungen: Wörterbuch 1818: i z v a n on je izvan sebe n a k r a j nakraj sela n a a re d nasred kuce, nasred sela oko (Beispiele nur für räumliche Verhältnisse) s v r 1) svr čoveka 2) jedno svr drugoga Wörterbuch 1818: Wörterbuch 1818: Grammatik i z v a n on je izvan sebe izvan pam n a k r a j nakraj sela sjedi nakr wohnt unte n a a re d nasred kuce, nasred sela nasred sel oko (Beispiele nur für räumliche Verhältnisse) (hinzugefü rale Verwe Božica skočio svr glave do z s v r 1) svr čoveka 2) jedno svr drugoga Grammatik 1824: izvan pameti sjedi nakraj sela, er wohnt unten im Dorfe nasred sela, polja (hinzugefügt die tempo- rale Verwendung:) oko Božica skočio svr kuce, savr glave do zelene trave Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 120 - 1) ode uz brdo; uz vodu; uz drvo 2) stao uza me 1) na glavi, na konju, na brdu, na Kosovu 2) na nebu 3) na glavu 4) na vojsku 1) otisao и crkvu; bio sam и crkvi и г (keine Beispiele) (Beispiele nur für räumli che Verhältnisse) 5) s večera na и medu p r e d ( а ) 8(a) s to ji na brdu, poginuo na Kosovu, bio sam и crkv - otisao na brdo, ide na Kosovo, idem и crkvu to je medu nama, daj теби nas (hinzugefügt die temporal Verwendung:) pred smrt (die temprale Verwendung fehlt) Aus der Tabelle is t ersichtlich, daß die Grammatik durch die ab- weichenden Beispiele informativer geworden is t als das Wörterbuch (mit Ausnahme von uz 2) und 8 ( a ) 5j). Es erscheint merkwürdig, daß diese neuen Angaben keinen Einfluß auf die zweite Auflage des Wör- terbuchs gehabt haben; von allen diesen Beispielen finden wir dort nur diese zwei: s to ji na brdu, poginuo na Kosovu. Alle diese Abweichungen der Angaben in der Grammatik von denje- nigen im Wörterbuch sind auf Vuk und nicht auf Grimm zurückzuführen, weil Grimm 1. 1. Die Abweichungen der Übersetzung vom Original Sie beschränken sich auf eine Aufzählung, wobei viele, im Wörterbuch verzeichneten Konjunktionen fehlen, wie z.B.: da (bejahend), da a k o , da k a k o , d o k l e , e d a , k a d ( a ) , k a k o , k a n o , k a o , ma, m a k a r, n e k a , o s im , p o e t o , 8amo, a t o , t a , t e , t e k , z a r , z a t o . Auch zu den Inter- jektionen sind nicht alle Angaben im Wörterbuch ausgenützt worden, wie z.B.: ae ! , a i s ! , b e ! , e i , e i e ! , i d i d i ! , i j u ! , j u j ! , o t e ! , c i d , vo cke !. 3. Die Textbeispile zur Rektion der Präpositionen stimmen o mit denjenigen im Wörterbuch nicht überein. Es zeigen sich z.B. solche Abweichungen: 3. Die Textbeispile zur Rektion der Präpositionen stimmen o mit denjenigen im Wörterbuch nicht überein. Es zeigen sich z.B. 1. Fehlübersetzungen. Es sind zwei Arten von Fehlübersetzunge vorhanden : Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1. Die Abweichungen der Übersetzung vom Original kein neues Wortmaterial, und noch dazu mit Beispielen, hätte liefern können und weil 2 ģ eine solche volkstümliche Ausdrucks weise wie: sjedi nakraj sela, wo das Verb e j e d i t i im Sinne von w o h - nen vorkommt, als auch solche Redensarten wie i z v a n p a m e t i nur von Vuk stammen können, Grimm hätte sich nach Vuks Wörterbuch richten müssen. Die sachlichen Angaben aus dem serbischen Sprachmaterial in dem Kapitel "Von den Partikeln" stammen also von Vuk. Die Unvollständig- keit, von der Vuk in dem oben zitierten Brief spricht, b e trifft wohl vor allem die Übersetzung des Kapitels, die er nicht mitgelie- fe rt hat. Sie stammt offensichtlich von Grimm. Dafür sprechen fol- gende Tatsachen, die sich aus der Analyse des Kapitels ergeben haben 1. Fehlübersetzungen. Es sind zwei Arten von Fehlübersetzunge vorhanden : hlübersetzungen. Es sind zwei Arten von Fehlübersetzung Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 121 ־ a. Fehler, die auf dem Mißverständnis der serbischen Wortformen beruhen. So werden z.B. Aoriste mit Präsens übersetzt: "ode posred sela" - er g e h t mitten durchs Dorf; node pod kucu" - er g e h t unter das Haus. Grimm war zwar über den Aorist informiert, er hat aber über sehen, daß bei diesem Verb das formale Präsens und der Aorist gleich- lautend sind, daß aber ein Hauptsatz wie dieser keine Gegenwart be- deutet. Diesen Fehler hätte Vuk nicht begangen, was einer der Bewei- se is t, daß er Grimm keine Übersetzung geliefert hat. Ebenso wurden in den folgenden Übersetzungen die serbischen Formen mißverstanden: " i z n e s i pred kucu״ - er t r ä g t vor das Haus; "m e t n i preda me" - s e t z t vor mich; "o d n e s i za kucu" - t r ä g t hinters Haus; "ne b r i n i se za njega" - k ü m m e rt sich nicht um ihn. Hier hat Grimm übersehen, daß die betreffenden Verben Imperative sind und hat sie mit Präsens übersetzt. Auch bei "nada s e n ■־ über es hat er das Reflexivum übersehen, obwohl er es gekannt und sonst richtig übersetzt hat (vgl u.a. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1. Die Abweichungen der Übersetzung vom Original oben "kümmert stcfc").Aber solche Flüchtigkeitsfehler deuten auf das Fehlen eines sicheren Sprachgefühls, das Vuk als den Urheber völlig ausschließt. Bei einer Übersetzung erkennen wir Grimm an seiner Vorliebe für die Sprachgeschichte. Er schreibt: "svr ( s a v r ) , von o b e n , z.B. sko- cio 8 v r kuce, er sprang oben vom Hause h e r a b " Dagegen steht in Vuks Wörterbuch, das Grimm in diesem Falle offenbar nicht zu Rate gezo- gen hat:"s0 h o c h a l s , altitudine: svr coveka." In Grimms Handexem- piar der Grammatik befindet sich zu diesem Wort seine handschrift- liehe Anmerkung, in der er es, unter Hinweis auf Dobrovskys "In sti- tutiones" S. 408 mit dem aksl. s - v e r c h u gleichsetzt. So hat er bei der Präposition s v r die bei Kopitar fehlende Übersetzung erschlossen die allerdings auf das betreffende Textbeispiel nicht z u trifft. b. In dem Kapitel "Von den Partikeln" gibt es außerdem einige unvollständige und falsche Übersetzungen, die, wie die Analyse ge- zeigt hat, durch die unadäquate und unvollständige deutsche Uber- Setzung in Vuks Wörterbuch verursacht sind. Grimm hat beim Verfassen der Übersetzung Vuks Wörterbuch benutzt. Das beweisen u.a. die Fälle für die im Wörterbuch keine Übersetzung, sondern eine längere Er- klärung gegeben wird, die Grimm auch unübersetzt läßt und auf das Wörterbuch verweist, wie z. B. bei m o r e i , p i k i 3 l a d o i , l e l j o i , ko la l o m Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 122 ־ Seine Abhängigkeit vom Wörterbuch zeigt sich besonders deutlich in der Übersetzung der Beispiele mit der Präposition k r a j г "am E n d e , z.B. kraj sela, zu Ende des Dorfes; kraj mora, am U f e r des Meeres"• Hier erscheint k r a j als Substantiv (das E nde) und nicht als die ge- meinte Präposition ( n e b e n , a n , b e i ) . Das Wörterbuch von Vuk gibt zwar den semantischen Unterschied beider Wörter an, bei der Präposition allerdings nur die unvollständige Übersetzung: am U f e r , b e i , der die Volksliedverse:"Bevojka sjedi kraj mora" - "Bevojka je kraj gore stajała" hinzugefügt sind. 1. Die Abweichungen der Übersetzung vom Original Kopitar hat seine Übersetzung am U f e r aus dem ersten Textbeispiel von Vuk gezogen, einem der wenigen Fälle wo die Präposition k r a j in Verbindung mit dem entsprechenden Sub- stantiv, (auch da vorwiegend aus stilistischen Gründen) , mit am U f e r übersetzt werden kann. Die Unvollständigkeit der Übersetzung von Kopitar hat Grimm zum Kombinieren der Angaben zum Substantiv und zur Präposition k r a j geführt, woraus seine unadäquate Übersetzung entstanden is t. Während dieser Übersetzungsfehler durch die Unvollständigkeit der Übersetzung im Wörterbuch indirekt verursacht ist, ist die fal- sehe Übersetzung von i z n a d mit von o b e n h e r a b aus dem Wörterbuch direkt übernommen worden. Es überascht uns dort, daß Kopitars über- setzung auf keines der ihm von Vuk angegebenen Beispiele z u trifft: "prode iznad kuce; ode iznad grada; prolece iznad mene". Offensicht- lich war es für Kopitar nicht einfach, die serbischen Präpositionen zu übersetzen, weshalb er oft ungewöhnliche Konstruktionen gebil- det hat, die Grimm in seiner Übersetzung bemüht war zu beseitigen. So z.B. gibt Grimm zu i e p o d : von u n t e n a n , gegenüber dem von u n te n weg bei Kopitar; zu i a p r e d : v o r n e n , weg gegenüber dem v o r - h in w e g bei Kopitar. Bei der Präposition po gibt Grimm alle im Wörterbuch enthaltenen Übersetzungen, mit Ausnahme der in Vuks Beispielen un- ter 9) po j e d a n , p o d v a angegebenen distributiven Bedeutung j e , die allerdings in Kopitars falscher Übersetzung zu e in e m Unverstand- lieh erscheint. Solche Fälle, wo die deutsche Übersetzung des serbischen Wort- materials unvollständig oder unadäquat is t, sind in Vuks Wörterbuch von 1818 nicht selten. Sie weisen darauf hin, daß Kopitar, der gro- ße theoretische Kenntnisse über slavische Sprachen besessen hat, die serbische Sprache praktisch nur unvollkommen beherrscht hat. Er war Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 123 ־ beim Übersetzen auf Vuks Erläuterungen der Wortbedeutung angewiesen. Es is t auffällig, daß die Mängel in der Übersetzung besonders häufig bei den abstrakten Begriffen erscheinen. b i j e d a u n v e r d i e n t e B e s c h u l d i g u n g , Sacuvaj me, Boze, bijede ne- vidovne; Našla да bijeda na šuvu putu; U starca mlada žena bijeda gotova. Die von Kopitar gewählte Bedeutung dieses Wortes beruht nicht auf den Textbeispielen von Vuk, da sie auf das zweite kaum sinnvoll anwendbar is t. Kopitar hat sich wohl von dem Verb b i j e d i t i к о д а be- einflussen lassen; er hat außerdem übersehen, daß im Wörterbuch mehrfach auf dieses Wort verwiesen wird, z.B. bei b e l a j , d e r U n f a l l , das U n g lü c k und s i j a s e t , U n g e l e g e n h e i t ; alle diese Übersetzungen wären auch für b i j e d a in Vuks Beispielen angemessener als die ange- gebene. Diese Übersetzung is t in die zweite Auflage übernommen wor- den, unter Hinzufügung eines dritten Beispieles, auf das sie noch weniger z u trifft. k r a j am U f e r , b e i n e b e n , b e i , am neuk n.p. konj , n i c h t a b- n.p. covjek, u n e r f a h r e n , r i c h t e t konj, n i c h t a b g e r i c h t e t n e b e n , b e i , am n.p. covjek, u n e r f a h r e n , konj, n i c h t a b g e r i c h t e t k r a j am U f e r , b e i neuk n.p. konj , n i c h t a b- r i c h t e t k r a j am U f e r , b e i neuk n.p. konj , n i c h t a b- r i c h t e t k r a j am U f e r , b e i neuk n.p. 1. Die Abweichungen der Übersetzung vom Original In solchen Fällen hat Kopi- tar oft aus dem ihm von Vuk angegebenen Beispiel entweder eine un- vollständige, nur für den betreffenden Kontext geltende Bedeutung angegeben,oder den Begriff mißverstanden und eine unadäquate über- setzung gemacht. Eine Überprüfung dieser Fälle in der zweiten Auf- läge des Wörterbuchs von 1852 hat gezeigt, daß B. Daničic1 Ergänzun- gen und Korrekturen an Kopitars Übersetzung vorgenommen hat; viele Unvollständigkeiten sind dennoch geblieben. Zur Illustration dieser Behauptung möchten wir einige Beispiele mit Kopitars Übersetzungen 2 anführen und sie mit den Angaben in der zweiten Auflage vergleichen Wörterbuch 1852: u n v e r d i e n t e B e s c h u l d i g u n g , Sačuvaj me Boze bijede ne- vidovne; našla да bijeda na šuvu putu. 1 Wir sind der Meinung, daß Danicic sich auch um die deutsche Uber setzung der 2. Aufl. gekümmert hat. Seine Bemühungen, für die Vuk ihm in der Vorrede zum Wörterbuch öffentlich gedankt hat, sind bekannt. 2 Dieses Problem wird ausführlicher in einem Aufsatz behandelt wer Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM 2 Dieses Problem wird ausführlicher in einem Aufsatz behandelt wer den. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access (übernommen) Dieser Fall macht uns Kopitars Probleme deutlich, aus Vuks kon- kreten Textbeispielen die Bedeutung des Wortes zu erschließen. n u d i t i n ö t h i g e n (zum Essen) a n t r a g e n , a n b i e t e m Kuma Я k i C š N Dieser Fall macht uns Kopitars Probleme deutlich, aus Vuks kon- kreten Textbeispielen die Bedeutung des Wortes zu erschließen. n u d i t i n ö t h i g e n (zum Essen) a n t r a g e n , a n b i e t e m Kuma пиЯепа kao i Cašcena. Nu- di kao TurÖin vjerom. a n t r a g e n , a n b i e t e m Kuma пиЯепа kao i Cašcena. Nu- di kao TurÖin vjerom. In diesem Falle haben Kopitar wahrscheinlich seine Kenntnisse des Altkirchenslavischen (n ç d i t i , n ö t i g e n ) und der anderen slavi- sehen Sprachen zur falschen Übersetzung dieses serbischen Wortes verführt. Daničic hat sie verbessert. s l o b o d a d e r M u th 1) d e r Muth 2) Moja kuca, moja F r e i h e i t 1) d e r Muth 2) Moja kuca, moja sloboda F r e i h e i t 1) d e r Muth 2) Moja kuca, moja sloboda F r e i h e i t s l o b o d a d e r M u th Zu diesem Wort hat Vuk kein Beispiel geliefert, so daß nicht ent schieden werden kann, ob er an der unvollständigen Übersetzung mit- verantwortlich is t. Kopitar könnte von dem Verb s l o b o d i t i кода be- einflußt worden sein. Daničic hat hier ergänzt. 2. In dem Kapitel "Von den Partikeln" gibt es außerdem einige vom Wörterbuch abweichende Übersetzungen, die deutlich auf Grimm hinweisen. Das is t z.B. b i j e d a konj , n i c h t a b- r i c h t e t Hier hat Kopitar aus dem Beispiel von Vuk die Bedeutung des Wor- tes erschlossen, die es nur auf Tiere bezogen haben kann. Daničič führt sie erst an zweiter Stelle an. 1 Wir sind der Meinung, daß Danicic sich auch um die deutsche Uber setzung der 2. Aufl. gekümmert hat. Seine Bemühungen, für die Vuk ihm in der Vorrede zum Wörterbuch öffentlich gedankt hat, sind bekannt. 2 Dieses Problem wird ausführlicher in einem Aufsatz behandelt wer den. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access s o v i e l a u f e i n m a l h e r v o r - s c h i e ß t , wenn man m e l k t , daher t r i mlaza krvi udari- Še iz njega, mulctus? m la z Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access (übernommen) die Wiedergabe von n o ô a s , n o ö a ske durch h e i n t , an Stelle von d i e s e N a c h t im Wörterbuch, über solche Bildun- gen in der deutschen Sprache hat Grimm bereits 1822 in seinem Auf- satz "über die adverbia heute, heint und heuer" geschrieben (GKS VI, 302 - 306) und dabei Beispiele aus den slavischen Sprachen, auch aus dem Serbischen, herangezogen. Diese Bildungen hat er als eine Verschmelzung des Demonstrativums mit dem Zeitadverbium erläutert, "die slavischen mundarten setzen ein demonstratives s nicht vor, son dern suffigieren es, heute heiszt ihnen dnes (böhm., russ...), dzis (poln.), danas (serb.), h e in t:... notschas; jutros (serb... von ju tro ...); heuer heiszt letos... 1jetos von leto... (serb. sinoć, diese nacht),.." (ebd., 306). Da er die Adverbia als besonders wich- tig für die Sprachforschung h ie lt, ersetzte er Kopitars Umschreibung des n o ô a s mit d i e s e N a c h t durch die entsprechende deutsche Adverbial bildung h e i n t . Ähnlich verhält es sich mit der Abwandlung der über- Setzungen zu: z i m i , im V i n t e r als: W i n t e r s ; I j e t i , im S om m er,als: Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Sommerei r a n o , f r ü h , des M o r g e n s , als: f r ü h m o r g e n s ; n o b u , nobom, b e i N a c h t , als: N a c h ts (neben b e i N a c h t ) , Auch die Verbesserung der Übersetzung von: s i n o b , g e s t e r n A b e n d s in: g e s t e r n A b e n d ; s a d , i t z t in: j e t z t ; n i j e , i s t n i c h t (von Vuk als Adverb angegeben) in: n e i n , stammt zweifellos von Grimm. Die Umformulierung der Übersetzung von o a l a ! 1 Vgl. S. 145ff. dieser Arbeit (übernommen) t r ü h r t d i e T ro m m e l, als: d i e T rom m el g e r ü h r t , wobei durch das Part. Perf. der Befehl verstärkt wird, weist deutlich auf Grimm hin. Die vorangegangene Analyse zeigt, daß Vuk das im Kapitel "Von den Partikeln" enthaltene serbische Sprachmaterial geliefert und Grimm die Übersetzung angefertigt hat. Aus den wenigen Ubersetzungs- fehlem kann in bezug auf Grimms Kenntnisse der serbischen Sprache geschlossen werden, daß er die aus dem Kontext herausgelösten Wörter Wortformen und -Verbindungen nicht immer verstehen konnte, daß seine Sprachkenntnisse also nicht vollkommen waren. Zu Vuks ursprünglichen Plänen gehörte auch eine Vorrede, die er zusammen mit Kopitar verfassen wollte, zur Zeit als Tirol die Gram- matik übersetzte (Prepiska I, 210). Als Grimm dann die Vorbereitung der Übersetzung zum Druck begann, Übernahm er auch die Anfertigung der V o r r e d e ^ , zu der Vuk ihm "literarische Notizen" schickte (Pre- piska I I , 11). Außer der Vorrede und dem bereits besprochenen Kapitel "Von den Partikeln", die von Grimm und Vuk von vornherein geplant waren, hat Grimm während der Durchsicht der Übersetzung neue Kapitel und Absätze in den Grammatiktext eingeführt, die im folgenden erörtert werden sollen. c. Grimms Ergänzungen der Grammatik Tirols mangelhafte Übersetzung und die methodische Seite von Vuks Grammatik als auch Grimms "alte neigung" zur serbischen Spra- che führten ihn zur Vertiefung in die Grammatik. Die Frage des gei- stigen Eigentums an dem Werk spielte für beide, Vuk und Grimm, kei- ne wichtige Rolle. In dem Briefwechsel nennen sie es meistens "unsere Grammatik" (Prepiska II, 8, 9, 13, 14, 16, 26, 33, 42); der Ein- druck ist beherrschend, daß sie es als ein gemeinsames Werk ansahen. Daraus erklären sich auch Grimms Handhabung des Manuskripts und die Änderungen,die er vorgenommen hat.Vuk munterte ihn immer wieder dazu Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access auf: "Mir is t leid, wenn sie zu wenig ausgestrichen, verbessert und geändert haben; denn das habe ich für unsere serbische Leser, wel- chen noch der wahre Begriff von der Grammatik fehlt, geschrieben; ausser dem Sie sind ganz anderer Grammatiker als ich.--" (Prepiska I I , 18). Auch Grimms Antwort darauf is t bezeichnend: "Um auf unsere Grammatik zukonmen. Sie meinen ich hätte mehr ändern sollen. Ich that es nicht 1) weil es zu schnell gieng; Reimer wollte sogleich drucken lassen, wie ich verlangt, weil Sie es wünschten... 2) weil es Ihre grammatik is t und bleiben soll; ich durfte also Ihren plan nicht ändern." (Prepiska II, 26). Bemerkenswert is t die Reihenfolge der Gründe, die Grimm anführt. Wenn er mehr Zeit zur Verfügung gehabt hätte, wäre in Vuks "Plan" mit Sicherheit noch einiges von Grimm eingegangen; durch die Einführung neuer Kapitel war die Grammatik ohnehin bereits verändert. Dafür spricht auch Grimms Brief an Vuk nach Erscheinen der Grammatik: "Jetzt hinter her f ä llt mir mancher- le i ein, pada mi na um, aber zu spät; so z.b. möchte ich Ihnen Ihre lehre von dem gen. sing, bestreiten, der nach dva, t r i bei masc. und neutr. gelten soll. Deila Bella und Voltiggi stellen zwar die nämliche regel. Aber haben Sie dabei bedacht, dass auch die adj. und participia in solchem fa ll das -a zeigen, z.b. dva se brata milova- la; sastala se č e tiri tabora etc. etc? hier kann doch milovala, sa- stala kein gen. sing, sein? was ist es aber? c. Grimms Ergänzungen der Grammatik entweder alte, übrige dualform, die dann freilich auf die drei- und vierzahl mit erstreckt worden is t, oder die neutrale pluralform." (Prepiska II, 44). Hätte er noch die Möglichkeit dazu gehabt, so hätte diese seine Erkennt- nis sicher ihren Platz irgendwo in der Grammatik oder zumindest in der Vorrede gefunden. Die erste größere Ergänzung hat Grimm in dem Kapitel "Von der Declination" unternommen, dem er ein neues Kapitel Von dem Ge- s c h l e c h t e d e s Nomens vorangestellt hat (Anh. I, S.207ff.). Dem deutschen Leser erklärt Grimm, wie man das Geschlecht der serbischen Nomina erkennen soll. Dabei geht er ausführlich auf kon- sonantische Feminina ein, die er in drei Gruppen e in te ilt: 1. sol- che, bei denen man das Geschlecht weder nach der Bedeutung, noch dem Endkonsonanten erkennen kann, von denen er eine große Anzahl verzeichnet; 2. die abgeleiteten fern. Abstrakta auf - o s t , und 3. die Kollektiva auf - a d . Bei der langen Aufzählung der Feminina der ersten Gruppe folgt Grimm ziemlich genau dem Wörterbuch von Vuk, mit Ausnahme von zwei Wörtern, die dort nicht vorhanden sind: p o d g o r , Gegend unterm Berge, und аЪ, etwa: Närrchen. Wahrscheinlich hat Grimm selbst diese Wörter in einem Text gefunden, worauf die zweifellos von ihm stammende Übersetzung hindeutet1. In den übrigen Fällen übernimmt er die Übersetzung aus dem Wörterbuch. Dort wo Ko- pitar die deutsche Übersetzung nicht geliefert hat, wie z.B. beim wort m u a r , gibt Grimm mit Hilfe der lateinischen Angabe die deut־ sehe Übersetzung M u h a r k le e an. In zwei Fällen hat er Kopitars Deutsch verbessert: zu raS gibt er Roggen statt R o cken an; die über- setzung von p r o s t r i c i am Rocke, Mantel, die Öffnung um die Hand durch zu stecken, weist durch die stilistische Verbesserung: am Rocke, oder Mantel die Öffnung um die Hand durchzustecken, auf 2 Grimm hin . In Grimms Aufzählung fehlen so gängige Wörter wie: j e s e n , r a v a n u.a., während die selten vorkommenden wie: m u a r, p r o e t r i z angeführt werden. Das beweist, daß Grimm diese Wörter aus dem Wörterbuch selbst ausgeschrieben und dabei viele geläufige übersehen hat. 1 Vuks Wörterbuch gibt p o d g o r j e , Gegend unterm Berge, und a c i t i 8e gedehnt sprechen, an; die 2.Aufl. von 1852 verzeichnet P o d g o r als ON und a c , ein Närrchen. , 2 Grimms Verbesserungen sind in der 2. Aufl. des Wörterbuchs nicht berücksichtigt worden. c. Grimms Ergänzungen der Grammatik Auch die Definition der Kollektiva auf - a d als Bezeichnung der jun- gen Lebewesen ־mit Ausnahme von d r v l j a d und b u r a d - kann nur von Grimm stammen, da sie unvollständig ist. Das Kapitel von dem Geschlechte des Nomens sowie die übrigen Zusätze zur Deklination der Substantive sind u. E. Grimms selbstän- diger Beitrag zu Vuks Grammatik. Einen Beweis dafür lie fe rt auch der Briefwechsel. Grimm schrieb in seinem ersten Brief nach Vuks Besuch, daß er die ersten 40 Seiten des Manuskripts für den Druck vorbereitet hat und sie am nächsten Tag abschicken w ill. Diese 40 Seiten umfassen die gesamte Deklination der Substantive. ״Sie wer- den sehen, wenn Sie sich das ms. zeigen lassen, daß ich es sorgsam und genau durchgangen, auch nach vermögen gebessert habe." (Prepiska II, 6). Zu dieser Zeit hatte Grimm noch nicht einmal die "Notizen", die Vuk ihm erst am 6. Nov. schickte (Prepiska II, 11). 1 Vuks Wörterbuch gibt p o d g o r j e , Gegend unterm Berge, und a c i t i 8e gedehnt sprechen, an; die 2.Aufl. von 1852 verzeichnet P o d g o r als ON und a c , ein Närrchen. , 2 Grimms Verbesserungen sind in der 2. Aufl. des Wörterbuchs nicht berücksichtigt worden. , 2 Grimms Verbesserungen sind in der 2. Aufl. des Wörterbuchs nicht berücksichtigt worden. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Grimm kam wohl erst während der Durchsicht der Übersetzung auf den Gedanken, dieses ergänzende Kapitel einzuführen. Die nähere Be- schäftigung mit Vuks Darstellung der Substantivdeklination wird ihn zu der Ansicht gebracht haben, dem deutschen Leser Hinweise zur Bestimmung des Genus geben zu müssen. Vuks Einteilung in vier Deklinationstypen is t nämlich durch die Auslassung des h verwir- rend geworden? es kommen darin solche Maskulina auf - a und - e vor wie: o r a (statt: o r a h ) und 8 l u g a , m i j e (statt: m i j e h ) und S t a n o j e , deren unterschiedliche Deklination dem Uneingeweihten völlig unbe- greifbar sein muß. In seinem neuen Kapitel erläutert Grimm die- se "vokalischen" Maskulina und leistet damit einen Beitrag zur Voll- ständigkeit der Grammatik von Vuk. c. Grimms Ergänzungen der Grammatik Sein Vermögen zu systematisie- ren zeigt sich deutlich in den einleitenden Ausführungen über die Deklinationen und in seinen Definitionen der ersten und zweiten Denklination, die klarer und zutreffender sind als bei Vuk (Anh. I, S. 210 ) . Von den übrigen Ergänzungen im Kapitel über die Deklination der Substantive is t diejenige zur vierten Deklination von besonderer Bedeutung. Grimm schrieb darüber an Vuk: "pag. 39 habe ich so gut ich konnte, nöthiges zugesetzt; ich wollte Sie zählten alle neutra, welche theils e n , theils e t , theils e s einschalten, vollständig auf? die mit es fehlten ganz und Ihr Wörterbuch gibt zwar bei seme das -ena, bei t e l e das - e t a an, nicht aber bei n e b o , t e l o , k o l o , das vermuthliche - e s a ? [dazu Anmerkung:] vermuthl. lautet der gen. lieber n e b a , t e l a , k o l a , c u d a , als n e b e s a , t e l e s a , k o l e s a , b u d e s a ? und nur im plur. schieben einige es ein. Im altslav. und kraini- sehen waltet dies es häufiger.” (Prepiska II, 6). Grimms Darstellung der stammerweiternden Neutra is t Übersicht- licher und vollständiger als bei Vuk. Vuk behandelt die Stammer- Weiterung durch - t - nur in einer Anmerkung (S. XLI). Unter seinen Paradigmen is t zwar ein Beispiel für die Erweiterung durch - n - , er geht aber in seinen Ausführungen nicht näher darauf ein und läßt die Stammerweiterung durch -s- sogar völlig unerwähnt. Aus dieser einen Anmerkung von Vuk macht Grimm zwei in sich geglieder- te Absätze, in denen er alle drei Arten der stammerweiternden Neutra erfaßt und mit Beispielen belegt. Daß er dabei das Kollektivum auf - a d bei Wörtern für Sachen ausschließt, is t auf Vuks Versäumnis zurückzuführen. c. Grimms Ergänzungen der Grammatik Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Es sind darüber hinaus kleinere Ergänzungen vorhanden, wie z Ē В.: zu p e e t o - paS oad fügt er p asce hinzu, von dem das Kollektivum p a e c a d gebildet ist* Diese Angabe wie auch die von Vuks Grammatik abweichende Erläuterung des Wortes rame hat er aus dem Wörterbuch geholt. Vuks Regel zum Vok. Sing. der Fern, auf - i c a führt er kon- sequent durch, indem er sie auch auf die Eigennamen anwendet, was Vuk versäumt hatte (Anh. X, S.210 ). Diese Vokative konnte er in den Volksliedern feststellen. Weitere Ergänzungen, die Auslassungen, Umstellungen und Umfor- mulierungen des Originaltextes nach sich gezogen haben, hat Grimm im Kapitel über die Adjektive vorgenommen, das vielleicht am besten zeigt, wie sehr er Vuks Grammatik auch als eigene Angelegenheit be- trachtet hat. In dem bereits erwähnten Brief schrieb er an Vuk: ”Wegen des adjectivs muss ich fragen, was ich auf dem herzen habe: sollte man nicht die erste hälfte der seite XLII (des originals) ich meine, die eintheilung des adj. in drei classen weglassen? Alles was da an- gemerkt wird, gehört in die (nicht abgehandelte) lehre von der Wortbildung, geht eigentlich die declination nichts an. Sie fahren zwar fort; "die adjectiva der ersten classe (p ril. imena reda) en- digen sich in masc. doppelt" d.h. sind entw. apocopata, oder in- tegra. Von der zweiten und dritten is t aber hernach gar keine re- de. Sind diese letztem überall nur apocopata, so würde das besser in einer anmerkung am schlusse der adj. declination gesagt. Ich bin übrigens ihrer meinung,dass die bennenung abgeschnitten und ganz unpassend sei und werde dafür setzen indefinita und defini- ta." (Prepiska II, 6). Die betreffenden Ausführungen von Vuk beziehen sich auf die Bildung der Adjektive und ihre Bedeutung. Im Rahmen der kurzen Grammatik, die sich hauptsächlich auf die Flexionen beschränkt,ste- hen sie iso lie rt und zusammenhangslos, weshalb sie Grimm beanstan- dete und weglassen wollte. Aber Vuk antwortete: "Wie wohl ich be- kenne,dass sie auch in Betreff des Adjectivs ganz recht haben,., aber ich wünschte sie doch nicht auslassen; und zwar aus folgenden Ursachen: a) In der altslavische Sprache können alle adjectiva diese doppelte Endung haben,., b) Die Böhmen haben diese doppelte Endungen bei keinem von ad jectiven.. . 1 Vgl. S. 160f. dieser Arbeit. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access c. Grimms Ergänzungen der Grammatik v) Ich habe vor Herausgabe Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access dieser Grammatik einem serbischen Professor aus Ungarn öffentlich in Zeitungen der Unterschied zwischen sveti und svet beweisen müssen (denn auch in bisherigen (d.h. bis Dobrowski) altslawischen Gramma- tiken stehet kein Unterschied in Bedeutung). Aus diesen Ursachen glaube ich das sollte bleiben; übrigens machen Sie wie Sie für Besste finden, denn ich bin auch in diesem ein Fuscher gegen Sie." (Prepiska II, 9). Vuk kam es also vor allem auf die Hervorhebung der beiden Adjek- tivformen und ihre semantische Abgrenzung voneinander an. Der be- treffende Text, d.h. die ersten drei Absätze H0 imenu prilagatel- nom" erfüllen aber dieses Ziel kaum: nur ein Kenner des Problems begreift Vuks "zweite Klasse" als die Adjektive, die nur in unbe- stimmter Form Vorkommen, und die "dritte Klasse" als diejenigen, die nur die bestimnte Form haben. Grimm löst dieses Problem dadurch, daß er den ersten Absatz völlig ausläßt, da er sich auf die Deklination der Adjektive nicht bezieht, während er die Beispiele aus dem zweiten und dritten Ab- satz in die A n m erkungen zu den A d j e c t i v - D e o l i n a t i o n e n übernimmt, mit denen er Vuks Text im Anschluß an die Darstellung der Adjektiv- deklination erweitert (Anh. I, S.214 ). Hier erscheinen Vuks Bei- spiele zur Adjektivbildung in einem völlig neuen Kontext als Bele- ge für die ausschließlich unbestimmte oder bestimmte Adjektivform. So e rfü llt Grimm einerseits Vuks Wunsch, dieses Material beizube- halten, andererseits verwirklicht er aber auch die eigene Vorstel- lung von der Gliederung dieses Kapitels. Das ganze Adjektivkapitel, die Darstellung der beiden Deklina- tionsklassen ausgenommen, is t bei Grimm stark verändert, umformu- lie rt und ergänzt (Anh. 1.212 ). Entsprechend seiner historisch- vergleichenden Methode zieht er eine Parallele zwischen dem serbi- sehen und aksl. Adjektiv, erläutert die gängigen Termini und ver- bleibt selbst bei der üblichen Benennung "ganz" und "abgeschnitten" nach seiner Meinung sei in allen slavischen Grammatiken "die lehre vom unbestimmten und bestimmten adj. nicht glücklich behandelt" (Prepiska II, 6). Er befaßte sich mit einem eigenen Deutungsver- such, den er in seiner Vorrede vortrug1. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access c. Grimms Ergänzungen der Grammatik Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Grimms Ausführungen konzentrieren sich um die beiden Adjektiv- formen und seine "Anmerkungen" schließen sich dem konsequent an. Das ganze Kapitel is t systematischer, vollständiger und übersichtli- eher als bei Vuk durchgeführt, so daß Grimms Eingriff in das Ori- ginal als ein Gewinn für die Grammatik angesehen werden kann. In dem Kapitel über die Verben hat Grimm die meisten Änderun- gen und Ergänzungen vorgenommen, die u.a. aus seiner Unzufrieden- heit mit Vuks Behandlung der Konjugationen resultieren (Anh. I, S. 216f f .) . Schon der Anfang dieses Kapitels zeigt starke Abweichungen von Vuk. Vuk definiert zuerst die transitiven, reflexiven und Zustands- verben, polemisiert dann gegen die aus den griechischen und lateini- sehen in die slavischen Grammatiken übernommenen Termini und Ein- teilungen in passive und verba deponentia, zieht in seiner Polemik auch die Fehler der slavischen Grammatiker in bezug auf die Adjek- tive und Pronomina heran und greift dann den Streit mit seinen Geg- nern um die "Hirtensprache" auf. Seinen Ausführungen mangelt es methodisch an Wissenschaftlichkeit. Dieser Mangel is t in Grimms Be- arbeitung beseitigt. Die nicht hierher gehörenden Angaben über das Pronomen e t o weist Grimm dem Kapitel über die Pronomina zu (Anh. I, S. 216 ) und läßt die übrigen Angaben über die Adjektive und Prono- mina aus dem Kapitel über die Verben weg. Dann geht er kurz auf das Problem der Einteilung der Verben in der serbischen und anderen slavischen Sprachen ein,erklärt die reflexiven, transitiven und in- transitiven Verben und hebt die Einteilung der slavischen Verben in perfektive und imperfektive hervor; bei den trans./intrans. Ver- ben s te llt er die semantischen, bei den p f./ip f. die morphologischen und syntaktischen Kriterien in den Vordergrund. In den weiteren Ausführungen über die perfektiven und imperfek- tiven Verben folgt Grimm dem Text von Vuk. Bei der Übersetzung gibt er das historische Präsens mit dem Präteritum wieder, was seine Ein- fühlung in die Eigenart der serbischen Sprache beweist. Er war vom slavischen Verbalaspekt fasziniert; daher erscheint hier, abwei- chend von Vuk, der Hinweis, daß der Deutsche so etwas nicht auszu- drücken vermag und daß die slavischen Sprachen in diesem Punkt über einen großen Reichtum verfügen. c. Grimms Ergänzungen der Grammatik Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vuks Darstellung der Tempora ist bei Grimm ebenfalls verändert. Wenn wir Vuks Terminologie in die heutige umsetzen, rechnet er zur Konjugation: Präsens, Futur, Aorist, Imperfekt und Perfekt, letzte* res mit Einschränkung, weil das Verb nicht fle k tie rt wird; das Plus- quamperfekt hat er aus der Konjugation ausgelassen. Auch Grimm versteht die Konjugation als Flexion im engeren Sinne; er geht ei- nen Schritt weiter als Vuk, indem er meint, daß man das umschriebe- ne Futur ( j a bu r a d i t i ) "aus der eigentlichen Flexionslehre ausē schließen" könnte, während das zusammengesetzte (r a d i b u ) eher als das Perfekt in die Konjugation gehöre (Anh. I, S.220 ) Er führt ei- gene Terminologie ein: Präsens, Futurum, Imperfectum, (einfaches) Präteritum (= Aorist), zusammengesetztes Präteritum (= Perfekt), mit dem Hinweis, daß DobrovskJ das Imperfekt "iteratives Präteritum" nennt. Im Anschluß an die Ausführungen über das Passiv fügt Grimm ei- nen neuen, bei Vuk fehlenden Absatz über die drei aktiven P a r t i z i p i a hinzu, in dem er die Bevorzugung des Aktivs gegenüber dem Passiv in der serbischen Sprache erläutert. Grimms Ausführungen stützen sich auf Vuks Angaben aus den über zwei Seiten verteilten Anmer- kungen unter dem Text. Bei einer guten Gliederung hätte Vuk diesen Angaben eine gewichtigere Stelle gegeben. Er erläutert darin, daß die beiden Partizipia zwar meistens als Gerundia gebraucht werden,dć ihre adjektivische Verwendung durchaus im Geiste der Volkssprache sei, da auch das Volk einige so gebrauche, vor allem das Part. Präs. Diese wichtige Ausdrucksmöglichkeit is t der heutigen serbi- sehen Sprache verlorengegangen. Grimm ergänzt Vuks Angaben durch das Part. Perf. akt. und stellt die Anmerkungen in einem gesonderten Absatz im Anschluß an die Be- handlung der Tempora und Modi zusammen (Anh. I, S.221 ). Die adjek- tivisch-substantivische Verwendungsmöglichkeit wird durch die deut- sehe Übersetzung von Vuks Beispielen zusätzlich hervorgehoben (z.B. i g r a j ü b i - d e r S p i e l e n d e ) . Die umfangreichste Ergänzung im Kapitel über die Verben gibt Grimm anschließend mit der Darstellung der P e r s o n a l e n d u n g e n aller Tempora (Anh. c. Grimms Ergänzungen der Grammatik I, S.222f.)• Er schrieb darüber an Vuk, daß er "etwas über Personen Kennzeichen eingeschaltet habe, was ... nicht unnütz schien." (Prepiska 11* 16). Auch ohne die genannte Briefaussage könnte man in der Heranziehung der Sprachgeschichte Grimms Arbeit ar dieser Stelle erkennen. Er erläutert unter Hinweis auf das Altkirch« Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access slavische, daß in der bei Vuk vDkalisch auslautenden 1. Pers. Sg. ein h fehlt. Dieser Zusatz is t in Vuks Granunatik sehr nützlich; dagegen is t die Erläuterung, daß im Altkirchenslavischen in der 3. Pers. Sg. Präs. die Endung -t war, überflüssig. Das ist ein historisch-vergleichender Ansatz, der in Vuks synchronischer Be- trachtungsweise etwas unorganisch wirkt. slavische, daß in der bei Vuk vDkalisch auslautenden 1. Pers. Sg. ein h fehlt. Dieser Zusatz is t in Vuks Granunatik sehr nützlich; dagegen is t die Erläuterung, daß im Altkirchenslavischen in der 3. Pers. Sg. Präs. die Endung -t war, überflüssig. Das ist ein historisch-vergleichender Ansatz, der in Vuks synchronischer Be- trachtungsweise etwas unorganisch wirkt. Grimm hat für diese Ergänzung seine Kenntnisse des Altkirchen- slavischen* und Vuks Granunatik verwendet. Auf Grund von Vuks An- gaben fügte er die Erläuterung der Akzentunterschiede bei den gleichlautenden Formen im Aorist und Imperfekt hinzu (Anh. I, S. 223). Durch die Auslassung des h haben bei Vuk nämlich alle drei Personen des Aorists im Sing., dazu oft die 1. Pers. Sg. Imper- fekt , gleiche Lautung. Deshalb meinte Grimm, auf die Unterscheid dung durch den Akzent hinweisen zu müssen. Seine Schlußfolgerungen sind aus Vuks Akzent- und Konjugationsdarstellung logisch abge- le ite t, aber auch mit derselben Unzulänglichkeit behaftet: mit dem Zeichen A ("d ritte r Akzent") meinte Vuk, (Grimm konnte es auch nicht anders), sowohl den langfallenden Akzent als auch die unbetonte Länge. Grimms Erläuterung des Akzentunterschieds ist dennoch in Vuks Grammatik sinnvoll. Grimms Darstellung der Personalendungen ist im ganzen nützlich, weil sie die Übersicht über die von Vuk unzulänglich und kompli- ziert dargestellte Konjugation erleichtert. Vuk selbst war sich der Mängel seiner Arbeit in diesem Punkt bewußt, als er meinte, daß bei einer günstigeren Gelegenheit die Einteilung der Verben in die Konjugationsklassen besser gemacht werden könnte. Grimm drückte seine Unzufriedenheit in einer von Vuk abweichenden Formulierung aus: "Mit der Zeit wird es der Serbischen Grammatik schon gelingen, in die Gründe dieser Abweichungen tiefer einzudringen und sie als- dann zweckmäßiger zu ordnen." (Anh. I, S. 1 Grimm hat Dobrovskys "Institutiones" gelesen und eine Rezension geschrieben (GKS IV, 186 - 196). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM I Vgl. P• Ivic, 0 Vukovom Rječniku.., S. 64ff. c. Grimms Ergänzungen der Grammatik 224 ) Die Bemühung, Vuks Mängel so weit wie möglich zu beheben, ver- anlaßte Grimm, die Beispiele in Gruppen zu ordnen, neue Untergrup- pen einzuführen (z.B. die Verben auf - n u t i ) t und die fehlenden In- fin itive zu ergänzen. Dadurch wird die Darstellung etwas übersieht- licher, was zweifellos ein Gewinn für die Grammatik ist. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 134 ־ Grimm hat seine Ergänzung vor allem dort vorgenommen, wo Vuks Grammatik methodische Mängel enthielt (z.B. in dem Adjektivkapi- te l); dabei leistete er weitreichende Ergänzungen des Inhalts (z. B. zur Substantivdeklination und Konjugation). Er konnte die Un- zulänglichkeit von Vuks Grammtik nicht völlig beseitigen; dennoch is t die Grammatik durch Grimms Eingriff vollständiger und Übersicht- licher geworden• Die Behauptung, daß die deutsche Fassung der ser- bischen Grammatik gegenüber dem Original an wissenschaftlicher Qualität gewonnen hat, scheint daher nicht übertrieben. d• Grimms Kürzungen, Umstellungen und Umformulierungen des Textes von Vuk Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access d• Grimms Kürzungen, Umstellungen und Umformulierungen des Textes von Vuk Schon der Anfang der deutschen Fassung ist anders als bei Vuk. Vuk beginnt seine Grammatik mit einer Aufzählung der Laute der ser- bischen Sprache, für die er auch seine endgültigen Zeichen angibt. Sein Zeichensystem umfaßt 28 Laute; es fehlen / und h , obwohl sie im Wörterbuch verwendet werden (Beispiele mit / nehmen zwei Spalten ein, während h vereinzelt vorkommt, z.B. a h a ! a r h i m a n d r i t ) Am Ende der Grammatik ist eine Tabelle "Alphabeti Serborum", in die Vuk die beiden Zeichen aufnimmt mit der Anmerkung, daß sie aus dem kyrillischen Alphabet stammen, daß die bezeichneten Laute aber in der serbischen Sprache nicht existieren. In der Tabelle wird auch ъ angegeben, das im Wörterbuch ebenfalls verwendet wird, unter dem Hinweis, daß es keinen Lautwert besitzt, sondern dem silbischen ג׳י zur Unterscheidung vom konsonantischen beigefügt wird. Auf den ersten Blick erscheint es verwunderlich, daß Vuk in seiner kurzen Grammatik das Alphabet zweimal behandelt. Diese Tatsache wird verständlich, wenn man bedenkt, welchen großen Wert er der Orthographie beigemessen hat. An den Anfang s te llt er näm- lieh sein phonetisches Alphabet, in dem er für jedes Phonem der serbischen Sprache ein Zeichen gibt. In der Tabelle am Ende gibt er auch die Zeichen für die nach seiner Meinung fremdsprachigen Laute und ein graphisches Zeichen ohne Lautwert (ъ)1. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 135 ־ Grimm verfährt hier anders.Er s te llt an den Anfang eine Tabel ־ le mit Vuks phonetischem Alphabet, also ohne f , h und ъ (Anh.I, S. 203). Seine Tabelle is t auch sonst nicht mit jener von Vuk iden- tisch: neben Vuks Kyrilliza und den entsprechenden lateinischen Zeichen gibt er Ausspracheerläuterungen für den deutschen Leser an, die sich zwar auf Vuks Tabelle stützen, jedoch ausführlicher sind (vgl. zu Ô) . Grimm ließ die drei Zeichen aus, weil er wohl die Ausführungen im Grammatiktext, den er durch Beispiele mit bewahrtem h ergänzte, für ausreichend hielt. In der Grammatik geht Vuk immer wieder auf Probleme und Prin ־ zipien seiner Reform der Orthographie und der Schriftsprache ein. Er schrieb seine Grammatik zur Zeit der schärfsten Auseinandersetzun- gen mit seinen Gegnern, weshalb sein Werk viel P o le m ik enthält. Stellenweise gerät die Polemik in den Vordergrund und verdrängt den zu behandelnden Gegenstand. d• Grimms Kürzungen, Umstellungen und Umformulierungen des Textes von Vuk An solchen Stellen hat Grimm viel gekürzt und umformuliert, denn er war gegen Polemik in einem wis- senschaftlichen Werk, das als Kodex der serbischen Sprache gelten sollte. Er hatte Vuks grundsätzliches Einverständnis dazu, wie u. a. auch der folgende Brief zeigt: ”Ich habe, wie Sie sehen werden, viel ausgestrichen,., und einiges umgesetzt, theils um das Ganze wirklich deutsch zu machen, theils weil die Definitionen bekann- ter grammat i scher Begriffe und hin und wieder Polemik gegen Sia- venisten füglich wegbleiben. - Letztere wird die gute Sache an sich zum Schweigen bringen oder bekehren, ohne dass es viele Worte bedarf. Sie waren ja mündlich selbst von der Notwendigkeit solcher Abkürzungen überzeugt... Anderes geänderte und die Ursachen warum? werden Sie selbst finden." (Prepiska II, 15f.). Die Auslassung und Umformulierung der polemischen Stellen sind für die Grammatik im allgemeinen vorteilhaft. Allerdings sind da- bei an einigen Stellen Vuks Gedanken über die Schrift und die Ortho- graphie verlorengegangen. In der Anmerkung zur "Verwandlung" des к z.B. greift Vuk die Verfechter der etymologischen Orthographie an, (er nennt sie "nadriknjige" und "fušeri književni") und führt ihre Forderung * nach der Wiedergabe des Wurzelkonsonanten(z.B. mit Hilfe von ь: b r a t b a , p r u t b e , usw.) ad absurdum,indem er die Beispiele oc (zu o c e ! ) , kn e z (zu k n e z e ! ) usw. konstruiert, um die Unmöglichkeit einer solchen Forderung zu dokumentieren. Mit einem Exkurs in die Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM i f 00051004 ־ 136 ־ Bibel und der Berufung auf Quintilian w ill Vuk sein phonetisches Prinzip der Orthographie untermauern: der erste und einzige Dienst, den die Buchstaben leisten sollen, sei, die Wörter graphisch so darzustellen, wie sie gesprochen werden, und nicht etwa Hinweise zu deren Etymologie oder semantischen Beziehungen zu anderen Wör- tern zu geben. Das alles fehlt bei Grimm (Anh. I, S.205 ) Die Tilgung von Vuks Entgleisung gegenüber seinen Gegnern, (denn die Formulie- rung "nadriknjige" und "fuieri" kann man in einer Grammatik wohl nicht anders sehen), is t zweifellos gut für die Grammatik. Den prinzipiellen Gedanken Vuks über die Schrift hätte Grimm umformu- lieren und beibehalten sollen. Ähnlich verhält es sich mit der Kürzung der allgemeinen An- merkung über die Konsonantenassimilation. Vuk spricht von der "Verwandlung der Konsonanten" und setzt sich von denjenigen Gram- matikern (u.a. d• Grimms Kürzungen, Umstellungen und Umformulierungen des Textes von Vuk von Adelung) ab, die über die stellenbedingte Ver- änderung der Aussprache reden. Während für Adelung b vor t als p ausgesprochen wird, wird es bei Vuk verwandelt, es is t p. Vuk w ill hier eines der Grundprinzipien seiner phonetischen Orthogra- phie betonen. Grimm kürzt die Anmerkung gerade an dieser Stelle, so daß auch dieser Gedanke von Vuk verlorengeht (Anh. I, S.204 ). Bei der Streichung von polemischen Äußerungen läßt Grimm an noch einer Stelle einen wichtigen Satz aus. Es handelt sich um Vuks Ablehnung, die Bezeichnung von Tonquantität und -qualität in seine Orthographie einzubeziehen; die Bezeichnung der Länge durch Vokalverdoppelung nennt er das "Flicken" der serbischen Sprache nach deutschem Vorbild: "Bekoji naši spisatelji pišu raana, podraaniti, vooz i t.d. Ovo... je Njemačko-Srpski: zašto su oni viöeli de Nijemci ovako krpe i natežu и njiovom jeziku... Ovo je četvrta služba, koja se ište od slova, t . j . da pokazuju i razliku glasa и riječma!" (Anh. I, S. 206 ). Die Auslassung der sog. prosodischen Zeichen, die auch zu sprachlichen Phänomenen mit distinktiver Funktion gehören (z.B. s è n a : z b n a usw.), is t in Vuks phonologischer Orthographie die Inkonsequenz von größtem Ausmaß, da sie jedes Wort b e trifft. Vom pragmatischen Standpunkt aus gesehen is t sie die vernünftigste 00051004 ־ 137 ־ Lösung, da sonst aus einer der leichtesten eine der schwierigsten Orthographien geworden wäre, die nur speziell ausgebildete Lingui- sten völlig beherrschen könnten1. Man muß sich die Frage stellen, warum Grimm so oft Vuks Ge- danken über die Orthographie mißachtet hat. Die Antwort liegt u. E. in seiner Einstellung zu diesem Problem zur Zeit der Grammatik- Übersetzung: er maß der phonetischen Orthographie keine so große 2 Bedeutung wie Vuk und Kopitar bei . Als Grimm aber die Arbeit am "Deutschen Wörterbuch" begann und selbst einen Versuch der Orthogra- phiereform unternahm,vertrat er eine ähnliche Meinung wie Vuk3. Außerdem war Grimm wohl der Ansicht, daß diese theoretischen Exkurse die Lernzwecke der Grammatik beeinträchtigten; er lehnte 4 aus diesem Grund eine Grammatik in Tabellen ab . Auch seine Er- gänzungen sind darauf gerichtet, dem deutschen Leser das Lernen zu erleichtern. Grimms Kürzungen haben bei noch einem Problemkreis z. T. Ver- änderungen des Sinnes herbeigeführt. Es handelt sich um den von Vuk immer wieder hervorgehobenen Gegensatz zwischen der Sprache des einfachen Volkes und der Schriftsteller, oder der städtischen und der Dorfsprache. 1 Vgl. hierzu P. Ivic, 0 Vukovom Rječniku.., S. 29f. 2 Er schrieb an Kopitar am 27. Okt. 1829: "Die Menschen schreiben sicher nicht für Etymologen, aber streng genommen auch nicht ein- mal, um die Aussprache rein darzustellen, sondern um Gedanken mitzuteilen." . Vasmer, S. 68. 3 Vgl. dazu S. 36f. dieser Arbeit. 4 Vuk schloß sich Grimm an trotz Kopitars Vorschlag von den Tabel- len, was den starken Einfluß Grimms auf Vuk dokumentiert (Pre- piska I, 232f., II, 15). 4 Vuk schloß sich Grimm an trotz Kopitars Vorschlag von den Tabel- len, was den starken Einfluß Grimms auf Vuk dokumentiert (Pre- piska I, 232f., II, 15). 1 Vgl. hierzu P. Ivic, 0 Vukovom Rječniku.., S. 29f. d• Grimms Kürzungen, Umstellungen und Umformulierungen des Textes von Vuk Zur Zeit als er die Grammatik schrieb, vertrat Vuk die Meinung, daß das einfache Volk in den Dörfern überall richtig spricht, während die Sprache der Städter durch den Ein- fluß der Schriftsteller verdorben sei. Zu den "Fehlern" rechnete er auch die älteren Kasusformen. In Grimms Übersetzung der Anmerkung über den Dat. PI. auf - о т (Anh. I, S. 210 ) fehlt Vuks Angabe "in den Städten", und die Formulierung, daß man es auch in "der Rede" (u govoru) hören kön- ne, übersetzt Grimm als: "Man hört sie auch unterm dem Volke". So wird Vuks Entgegensetzung der städtischen und der Dorfsprache g j 2 Er schrieb an Kopitar am 27. Okt. 1829: "Die Menschen schreiben sicher nicht für Etymologen, aber streng genommen auch nicht ein- mal, um die Aussprache rein darzustellen, sondern um Gedanken mitzuteilen." . Vasmer, S. 68. 4 Vuk schloß sich Grimm an trotz Kopitars Vorschlag von den Tabel- len, was den starken Einfluß Grimms auf Vuk dokumentiert (Pre- piska I, 232f., II, 15). 4 Vuk schloß sich Grimm an trotz Kopitars Vorschlag von den Tabel- len, was den starken Einfluß Grimms auf Vuk dokumentiert (Pre- piska I, 232f., II, 15). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access verwischt. Ähnlich läßt Grimm in den Ausführungen über den Lok. PI. ohne -ma Vuks Anzweiflung, daß die einfachen Leute so reden, weg. Beide Auslassungen bedeuten eine Korrektur von Vuks Irrtü - mern in bezug auf die Volkssprache. Grimm nahm sie wohl aus dem Grunde vor, weil ihm Vuks Entgegensetzung der Sprache des ein- fachen Volkes und der Gebildeten mißfiel. Seine Kürzungen zeigen auch sonst die Tendenz, den von Vuk betonten Gegensatz zwischen dem Volk und den Schriftstellern zu mildern oder ganz zu löschen. Das is t z. B. der Fall in der Übersetzung der Anmerkungen zu j e d a n a e s t und t r i e s t (Anh. I, S.215 ). Diese Tendenz entspringt aus Grimms hoher Einschätzung der Rolle der Dichter in der Sprach- bildung, obwohl er in bezug auf das Serbische eindeutig für Vuk Partei ergriffen hatte. Bei den sonstigen Auslassungen von polemischen Stellen, wie etwa der Streichung von Vuks Formulierung über die "Flickerei" des aksl. Alphabets, an deren Stelle bei Grimm von der "Unvoll- kommenheit des altslavischen Alphabets" die Rede ist (Anh. I, S. 207), is t für die Grammatik inhaltlich nichts verlorengegangen. d• Grimms Kürzungen, Umstellungen und Umformulierungen des Textes von Vuk Außer der Polemik läßt Grimm auch solche Exkurse aus, die in- haltlich in eine Grammatik nicht hineingehören oder zu dem be- handelten Thema nichts beitragen. So entspringt z.B. der letzte Satz von Vuks Anmerkung über die besonderen Laute é, è und d z , wo er über die komische Aussprache der serbischen Mönche erzählt, sei- ner Liebe zur Folklore, hat aber mit dem grammatischen Problem nichts zu tun; ebenfalls der Vergleich der Unterschiedlichkeit des Wohllautes verschiedener Sprachen mit unterschiedlichen Ge- schmäckern unter den Menschen; auch die Angaben über die Beschaf- fenheit des Lazar-Diploms, die in einem anderen Kontext von Inter- esse wären, tragen zur Erörterung der grammatischen Frage nichts bei (Anh. I, S.204, 213). Einige Auslassungen betreffen Vuks Wiederholungen, wie z.B. die folgende, über die Grimm an Vuk schrieb: "p. 55 des msp. strei- che ich unter dem Satz, der das schon Vorgetragene nochmals vor- trägt, aber sich auch p. XLVIII. des Orig, eingeschlichen hat, wo sogar die Ziffer 1. falsch is t, indem 1. bereits pag. XLVI stehet! p. XLVIII des Orig, is t mir die note hinter selä undeutlich, we- nigstens undeutlich abgefaßt; ich habe sie getilgt; irre ich, so bleibt Ihnen Zeit (und Macht überall) zu bessern.” Grimm meint die Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 139 ־ Wiederholung der Ausführungen über die Zahl jedan, in denen er auch einen Fehler von Vuk korrigiert: *1pag. 53. des msp. setze ich, dass jedan abgeschnitten flectiere, obgleich Sie im Texte p. XLVII kao cijelo sagen." (Prepiska II, 15f.). Die von Grimm gemein- te Anmerkung zu seta ist ein Versehen von Vuk, so daß ihre Auslas- sung sinnvoll ist. Grimms Kürzungen sind notwendigerweise oft mit U m f o r m u l i e r u n g e n verbunden. Im Kap. über das Zahlwort z.B. läßt er den ersten Satz und alle Definitionen der einzelnen Klassen aus und nennt statt- dessen die Zahlwortklassen am Anfang des Kapitels, wobei er Vuks terminologische Schwierigkeiten durch Einführung von lateinischen Termini löst (Anh. I, S.215 ). Desgleichen streicht er Vuks um-־ ständliche Charakterisierungen der einzelnen Pronominalarten und setzt an deren Stelle die allgemein verständlichen lateinischen Termini (Anh. I, S. 216 ). Auch im Kapitel Über das Verbum streicht er die überflüssigen Erörterungen des Unterschieds zwischen De- klination und Konjugation (Anh. I, S. 218 ). d• Grimms Kürzungen, Umstellungen und Umformulierungen des Textes von Vuk In dem Kapitel über die Verwandlung der Konsonanten führt Vuk zu m - v und n - nj Beispiele für die Dissimilation der Konsonan- tengruppe mn an (wie: tavnica, mlogi) und nennt diese Erscheinung eine "alte etymologische Veränderung". Grimm streicht diese Be- Zeichnung und schränkt in seiner Formulierung die Konsonantendissi- milation ein , indem er auf die Existenz der Formen mit dieser Konsonantenverbindung hinweist (Anh. I, S. 205 )• Vuks Angaben zum aksl. o t , in denen er u.a. erläutert,daß die vielen aksl. Abkürzungen aus praktischen Gründen gepflegt wurden, um schneller zu schreiben und um Pergament zu sparen, (wobei er den serbischen Lesern erklärt, was Pergament is t ) , kürzt und formuliert Grimm um und macht einen Verweis auf Dobrovskÿs "Insti- tutiones" (Anh. I., S.206 )- Grimms Umformulierungen tragen im allgemeinen zur Klarheit des Textes von Vuk bei. Das zeigt sich besonders deutlich im Falle von Vuks Ausführungen in der Anmerkung zum aksl. ь, wo die gewählten Belege seinen Gedankengang nicht klar dokumentieren. Grimms Umfor- mulierung beseitigt diese Unklarheit (Anh. I, S. 207 ). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 140 ־ An einigen Stellen sind Grimms Umformulierungen von seiner Sprachauffassung bestimmt. So z.B. am Ende des Kapitels über die Ordinalia, wo er die Bildungen p rv o m usw. (gegenüber p r v i p u t usw.) ”die wahren einfachen Ausdrücke" nennt (Anh. I, S.215 ), weil nach seiner Auffassung in den alten Sprachen für jeden Be- g riff ein neues Wort geschaffen wurde, während die neueren Spra- chen die Tendenz zu Zusammensetzungen zeigen. Seine Ansicht von der ursprünglichen Vollkommenheit des Sprachorganismus zeigt sich z.B. in der Formulierung über die Komparative ohne j nach einem palatalen Konsonanten wie t e s i , wo er meint, daß die ursprüngli- chen vollständigen Formen (t e z j i ) verlorengegengen sind (Anh. I, S. 214 ) Aus demselben Grunde ersetzt er Vuks Beispiel n e s r e t n j i c a durch das im Wörterbuch angegebene n e s v e b n i c a , das ihm wohl "orga nischer" zu n e s r e b a erschien (Anh. I, S.210 ). d• Grimms Kürzungen, Umstellungen und Umformulierungen des Textes von Vuk An einigen Stellen versucht Grimm, Vuks S til wissenschaftli- eher zu machen. So z.B. dort, wo Vuk die Verwendung des dativus ethicus als "kao za neko čudo" beschreibt. Grimm bleibt der Vor- läge inhaltlich treu, formuliert aber geschickter, wenn er sagt, daß diese Dative als eine Art "interjectio admirantis" eingeschal tet werden. Ebenfalls ist die Umformulierung von Vuks Eingeständ- nis, er erinnere sich an keine Beispiele mehr zu m i , in: "Die Bei spiele von mi sind seltener" stilistisch vorteilhafter (Anh. I, S. 216 ). Grimm hat auch mehrere U m s t e l lu n g e n im Text des Originals vor genommen. In unserer Analyse des Kapitels über die Adjektive und die Verben wurde auch diese Frage berührt. Grimm übernimmt Vuks Ausführungen aus dem "Dodatak", die in vier Punkten verschiedene Themen behandeln, in den Grammatiktext an den entsprechenden Stel len. Der erste Punkt, der sich auf die Konsonantenassimilation be zieht, is t am Ende des Kapitels über die Verwandlung der Konsonan ten eingefügt worden (Anh. I, S. 205 ). Den zweiten Punkt aus dem "Dodatak", in dem Vuk über die Auslassung des s vor der Personal- endung der 1. Pers. PI. Aorist in Sumadien berichtet, übernimmt Grimm in seine Ausführungen über die Personalendungen, und zwar als Anmerkung zur 1. Pers. (Anh. I, S.223 ). Den dritten Punkt nimmt er als die neunte Anmerkung am Schluß der Darstellung der zweiten Konjugationsklasse. Den vierten Punkt, der sich Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 141 ־ auf die Beweglichkeit des Akzents bei der Deklination der Substanti ־ ve bezieht, fügt er am Schluß des Kapitels über die Akzente hinzu (Anh. I, S.207 ). Ebenso bringt Grimm Vuks Anmerkung zu den verba deponentia mit den Angaben zum Verb c u t i in einen neuen Kontext, zu den Ausführun- gen über die perfektiven und imperfektiven Verben (Anh. I, S.219 ). Vuks Anmerkung zur zweiten Deklination der Substantive mit den Anga- ben über die Maskulina auf -a übernimmt er in sein Kapitel "Von dem Geschlechte des Nomens" (Anh. I, S.209 ). Die Ausführungen über das Pronomen c t o im Kapitel über das Verbum versetzt er in das Kapitel über die Pronomina, nachdem er sie ergänzt und umformuliert hat (Anh. I, S.216 ). Die Deklination des Interrogativpronomens c i j i is t ebenfalls völ- lig umgestellt worden (Anh. I, S. 1 Vgl. Vuks Grammatik S. LIf.,und das Wörterbuch von 1818, wo nur c i j , c i j a * <5ije angegeben wir ā ģ, $ i j i und b t fehlen. 2 Vgl* S. 160f. dieser Arbeit. d• Grimms Kürzungen, Umstellungen und Umformulierungen des Textes von Vuk 216 ). Bei Vuk steht an erster Stel- le die längere Form c i j i , danach werden die kürzere c i j und die zu- sammengezogene c t genannt. Grimm setzt die kurze Form c i j als die üblichere an, die längere führt er in Klammern auf, die zusammengezo- gene läßt er aus. Dazu hat ihn wohl die Tatsache bewogen, daß Vuk sonst entweder nur die kurze Form nennt oder ihr den Vorrang gibt1, so daß Grimm sie als die üblichere annahm. Außerdem wird ihm das auf einen Konsonanten auslautende Maskulinum c i j neben m o j , t a j usw. als ursprünglicher und organischer erschienen sein. Die Deklination c i j y č i j ega usw. entspricht somit der von Grimm konstruierten Ad- jektivdeklination, wonach das "ursprüngliche" z u t , z u t o g a usw. neben v 2 dem z u t i , z u t a usw. steht. Grimm war hier nicht auf dem richtigen Wege, die Analogie der beiden Fälle weist auf ihn als den Urheber hin. Aus dieserAnalyse is t ersichtlich, daß die Kürzungen, Umstellun- gen und Umformulierungen, die Grimm im Text des Originals vorgenommen hat, das Aussehen der Grammatik von Vuk nicht weniger verändert ha- ben als die Ergänzungen . Was die Kürzungen b e trifft, die sich auf die Polemik, die Wiederholungen und Vuks Freude am Erzählen beziehen, so kann behauptet werden, daß sie Vuks Grammatik positiv verändert haben. Die Umstellungen und Umformulierungen sind im allgemeinen Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access vorteilhaft: sie tragen zur Übersichtlichkeit und Klarheit der Dar- Stellung des Stoffes bei*. e. Die Mißverständnisse des Originaltextes und die übersetzungs- fehler e. Die Mißverständnisse des Originaltextes und die übersetzungs- fehler An einigen Stellen zeigt die Übersetzung inhaltliche Mißverständ־ nisse des Originals und Übersetzungsfehler; wir möchten versuchen, ihren Urheber festzustellen. Der Text, der dem ersten Punkt des "Dodatak" entspricht (Anh. I, S.205 ), enthält eine Fehlübersetzung. Vuk sagt, daß die Konsonan- tenassimilation auch an der Wortgrenze stattfindet, wie z.B. 8 n j i m , bea p o e t a usw. Er selbst gibt in seiner Grammatik dem morphologi- sehen Prinzip den Vorrang und nimmt diese Assimilation in seine Re- geln nicht auf. 1 Einige der Ergänzungen, Kürzungen und Umstellungen sind, ohne Analyse, von P. P. Bordevic (GPS XI, 241ff.) zusammengestellt. d• Grimms Kürzungen, Umstellungen und Umformulierungen des Textes von Vuk Er meint aber, daß, wenn jemand in Zukunft so schrei- ben möchte, es ihm erlaubt sein müsse, da so gesprochen wird und Dositej so geschrieben habe. Die Übersetzung besagt dagegen: Vuk hoffe, daß es ih m und den anderen in Zukunft erlaubt werden müsse, "der allgemeinen Aussprache gemäßer zu schreiben". Der knappe und klare S til an dieser Stelle, sowie die logische Schlußfolgerung, daß Vuk, der sich auch sonst nach der Aussprache richtet, es auch in diesem Falle tun w ill, lassen vermuten, daß dieses Mißverständnis der Vorlage auf Grimm zurückgeht. Eine weitere Fehlübersetzung befindet sich am Schluß des Kapi- tels über die Akzente, und zwar in dem Absatz, der dem vierten Punkt von Vuks ”Dodatak" entspricht (Anh. I, S. 207 ). Hier sind die Aus- führungen über m a a lo und v e e l o völlig verfehlt. Bei Vuk heißt es, daß v e a l o im Plural den gleichen A k z e n t wie im Gen. Sg. hat, wäh- rend in der Übersetzung von der Gleichheit der E n d u n g e n die Rede is t. Das Wort E n d u n g, das im Original an dieser Stelle gar nicht vorkommt, steht in der Übersetzung hier im Sinne von K a a u a . Diese Fehlübersetzung stammt u.E. nicht von Grimm, sondern von Tirol, und zwar aus folgenden Gründen: Das Kapitel über die Akzente zeigt in der Übersetzung eine ein- Das Kapitel über die Akzente zeigt in der Übersetzung eine ein- apitel über die Akzente zeigt in der Übersetzung eine e Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 143 ־ heitliche Terminologie: dort, wo Vuk über "glasoudarenije", "udare- nije glasa” spricht und die Zeichen für die Akzente erläutert, wo er also die Akzente meint, steht für g l a s der Terminus A k z e n t , sonst in den übrigen Fällen T o n . Vuks inkonsequente Verwendung von 8 1 0 V 0 und 8 l o g nebeneinander als Träger des Akzents is t einheitlich mit S i l b e übersetzt. In dem letzten, aus dem "Dodatak" übernommenen Absatz wird plötzlich für g l a s der Terminus L a u t (= Akzent) einge- führt. 1 Vgl. D. Tirol, Kleine Grammatik der Serbischen Sprache für Deut- sehe, Novi Sad 1851, S. 17. 2 Es ist im allgemeinen feststellbar, daß Vuk sich dem Problem der Akzentuation mit weniger Hingabe als sonst gewidmet hat.Die Akzent- bezeichnungen im Wörterbuch von 1852 sind wohl Daničics Verdienst, der noch 1851 über Vuks Fortgebrauch von nur drei Akzentzeichen klagte. - Vgl. A. Beli&, Vukova borba.., S. 162f, Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access aber genauer als diejenige von Vuk war. Tirols Vorlage erscheint auch in der Übersetzung der Beispiele: "ja sam za putom", ich bin zur Reise bereit, und "sto ji pod putom", er is t unter Wegs (Anh. I, S.210 )• Das erste Beipsiel is t in Vuks Wörterbuch unter za übersetzt als: ich muß abreisen. Grimm wäre als Übersetzer dem Wörterbuch gefolgt, oder er hätte die üblichere, kon- krete Bedeutung angegeben; deshalb meinen wir, sie Tirol zuschreiben zu dürfen1. Die Übersetzung des zweiten Beispiels is t sprachlich falsch und unverständlich: es is t unklar, ob sie ”unterwegs", (das wäre allerdings eine Fehlübersetzung), oder "unter dem Wege" meint. Jedenfalls kann sie in dieser Form nicht von Grimm stammen, er hat sie offenbar übersehen; in einem Brief an Vuk klagt er, daß er die "serbische Currentschrift" von Tirol schwer lesen kann (Prepiska II, 15). Die Übersetzung zeigt an mehreren Stellen die Beibehaltung der abhängigen Kasus aus dem Original, die in den deutschen Text nicht hineinpassen. Solche Fehler stammen von Tirol, wie wir aus Grimms Brief an Vuk schließen können: " ... der Ungar hat oft ganz sinnlos gearbeitet, z.b. pag. 42 zeile 3 des msp. den serbischen Instrumen־ 2 talis cijelim stehen lassen, der bei das deutsche [sic!] verbum genannt gar nicht past!" (Prepiska II, 15). Grimm hat nicht alle solche Fehler korrigiert, z.B. in dem Absatz über die Ordinalia die Genitive Sg. s t o t i n e und i l j a d e ; im Kap. über die aksl. Buchstaben den Lok. PI. s r b u l j a m a ; in den Erläuterungen zur dritten Deklina- tion den Gen Sg. m a t e r e , den er als Plural verstanden und deshalb al! d i e M ü t t e r übersetzt hat. Von den festgestellten Fehlern können also zwei Grimm zugeschrie ben werden: o s im m a t e r e - a u ß e r d i e M ü t t e r , wo es sich um ein Miß- Verständnis der von der Präposition abhängigen Genitivform handelt; wir haben bei der Analyse des Kapitels "Von den Partikeln" gesehen, daß Grimm die aus dem Kontext isolierten Wortformen leicht mißver- stehen konnte. Der zweite Fehler liegt in den Ausführungen über die Konsonantenassimilation an der Wortgrenze (8 n j i m ) vor, wo Vuks S til volkstümlich und für einen Ausländer schwer verständlich ist: "... 1 Diese Redewendung scheint eine Eigenart der Vojvodina zu sein (in der heutigen Sprache von Vuks Heimat konnte ich sie nicht fest- stellen); Vuk hat sie in der 2. Aufl. des Wörterbuchs gestrichen ; Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access d• Grimms Kürzungen, Umstellungen und Umformulierungen des Textes von Vuk Wenn man Grimms Arbeitsweise kennt, wird man die Möglichkeit ausschließen, daß er die eigene Terminologie so mißachten würde. Außerdem verwendet Grimm im weiteren Text den Terminus Kasus und nicht E n d u n g , wie das hier der Fall is t. Tirol dagegen schreibt, die serbische Sprache habe " ... in jeder Zahl ... sieben Endungen"*. heitliche Terminologie: dort, wo Vuk über "glasoudarenije", "udare- nije glasa” spricht und die Zeichen für die Akzente erläutert, wo er also die Akzente meint, steht für g l a s der Terminus A k z e n t , Grimm hat diesen Punkt aus dem "Dodatak" offenbar nachträglich in das Kapitel über die Akzente verwiesen, wobei er eine gründliche Korrektur und Anpassung an den übrigen Text versäumt hat. Mit dem Kapitel über die Akzente hat er sich aber gründlich beschäftigt. Er war mit Vuks Lösung dieses Problems nicht zufrieden, wie aus sei- nem Brief an Vuk hervorgeht: "Was p. 22. 23. des ms. steht, sollte deutlicher ausgedrückt seyn, doch habe ich es gelassen, weil das original p. XXXVI ebenso lautet" (Prepiska II, 7). Er fand Vuks Be- Zeichnung verschiedener Akzente mit denselben Zeichen als verwirrend, 2 was sie in der Tat auch war . Grimms Ausführungen über den "vierten" Akzent von Vuk (A) ver- dienen ein besonderes Interesse. Vuk bezeichnete bekanntlich mit diesem Zeichen sowohl den langsteigenden als auch langfallenden Akzent, wenn ihnen eine lange Silbe folgt (z.B. im Gen. Pl.). Vuks Erläuterungen waren praktisch nur für die Angehörigen der neuštoka- vischen Akzentuation verständlich und nachvollziehbar. Grimm entging es nicht, daß es sich hier um zwei qualitativ verschiedene Akzente handelt. "Der accent des vierten Zeichens (л) is t insofern doppelt, als jene Wörter schon im sing, zweierlei accente haben." Er schlug eine synthetische Akzentbezeichnung vor (d&ga (iridium) dóga (tabu- larum), Prepiska II, 8), die keineswegs einfach und vollkommen, ־ 144 ־ aber genauer als diejenige von Vuk war. aber genauer als diejenige von Vuk war. aber genauer als diejenige von Vuk war. a drugi ко и napredak ако cedbude..." (Anh. I, S.210 ). 1 Diese Redewendung scheint eine Eigenart der Vojvodina zu sein (in der heutigen Sprache von Vuks Heimat konnte ich sie nicht fest- stellen); Vuk hat sie in der 2. Aufl. des Wörterbuchs gestrichen ; Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 145 ־ Die Übersetzungsfehler, die u.E. von Tirol stammen, zeigen, daß er zu dieser Zeit keine Erfahrung im übersetzen ins Deutsche besaß, (vgl. Beibehaltung von Instrumental und Lokativ, die die deutsche Sprache gar nicht hat), und daß er die deutsche grammatische Termino- logie nicht beherrschte? sein Deutsch war fehlerhaft (vgl. ”unter Wegs"). Das Vorhandensein dieser Fehler weist aber auch auf Tirols Anteil an der endgültigen deutschen Fassung der Grammatik hin. Man kann sich ein Bild von dem druckfertigen, verlorengegangenen Manu- skript auf Grund von Grimms Brief machen, in dem er sich über Ti- rols Handschrift beklagt: "Er schreibt das Vjedi, ich weis nicht warum, nicht v sondern... [es folgen drei Zeichen] welches ich ihm zu gefallen auch thue, damit der Setzer nicht durch zweierlei Zei- chen gestört wird." (Prepiska I I , 15). Die Übersetzung von Vuks Gram matik wurde von Tirol und Grimm geschrieben? die endgültige Fassung hat ihre Gestalt durch Grimms Hand erhalten. von den maßgeblichen Lexika verzeichnet nur das Wörterbuch der SANU einen Beleg aus der Übersetzung der Odyssee von P. Papakosto- pulos von 1950. 2 Im deutschen Text der Prepiska sind zahlreiche Druckfehler ent- halten. 1 Vgl. Vasmer, S. 5. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access von den maßgeblichen Lexika verzeichnet nur das Wörterbuch der SANU einen Beleg aus der Übersetzung der Odyssee von P. Papakosto- pulos von 1950. von den maßgeblichen Lexika verzeichnet nur das Wörterbuch der SANU einen Beleg aus der Übersetzung der Odyssee von P. Papakosto- pulos von 1950. 2 Im deutschen Text der Prepiska sind zahlreiche Druckfehler ent- Vera Boji - 9783954793044 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" Als Grimm mit der Korrektur der Grammatik auch die Anfertigung der Vorrede übernahm, meinte er in einem Brief an Kopitar: "Was kann ich in einer Vorrede anders thun, als was von Ihnen und Ändern ge- sagt is t über Geschichte und Werth der Serbischen Sprache wiederho- len, und literarische Notizen, die mir Vuk geben w ill, mittheilen."* Ganz anders schrieb er an Vuk nach der intensiven Beschäftigung mit der serbischen Sprache: "In der vorrede werde ich mir einige äusse- rungen erlauben und Ihnen einiges bestreiten... Kopitar muntert mich auf, meine ansichten auszukramen. Irre ich, so verzeihen mir die Slavisten." (Prepiska II , 26). Die erwähnten literarischen Notizen schickte Vuk am 6. November mit der Entschuldigung, daß er "mit so vielen und doch unvollstän- digen Notizen" seine teuere Zeit raube. Grimm möge daraus "das Beste und Brauchbare aussuchen." Zugleich verwies er ihn auf Kopitars Grammatik und Rezensionen (Prepiska I I , 11). Was diese Notizen von Vuk enthielten, is t unbekannt. Aus seinen Briefen erfahren wir. von den maßgeblichen Lexika verzeichnet nur das Wörterbuch der SANU einen Beleg aus der Übersetzung der Odyssee von P. Papakosto- pulos von 1950. p 2 Im deutschen Text der Prepiska sind zahlreiche Druckfehler ent- halten. 1 Vgl Vasmer S 5 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access sind zahlreiche Druckfehler ent- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 146 ־ was er sich von Grimms Vorrede erhofft hat. Zehn Tage vor der Absen- dung der Notizen schrieb er an Kopitar : "Gledaj te i Vi štogod te mu y. site, molim Vas. Ja sam mu obecao literarische Notizen za predgovor, pa i sad iste, a ja upravo ne znam, šta ču mu poslati, nego оси da ispisem desto iz moga predgovora к rječniku... i da да uputim пека cita Vaše recenzije о rječniku i o Dobrovskoga gramatici. Da осе štogod dobro о o rto g ra fiji kazatiî" (Prepiska I, 239). Er bat Grimm, "etwas Gutes und Gewisses" über die Orthographie und besonders über die Notwendigkeit des Zeichens j zu sagen (Prepiska II, 13). "In der Vorrede bestreiten Sie mir alles, nur d, l j , n j, und j nicht." (ebd. 31). 1 So nannte ihn Kopitar, vgl. Vasmer, S. 6. 2 Vgl. H. Peukert, Bemerkungen über Jacob Grimms "Vorrede" zu Vuks ,Kleiner serbischer Grammatik1. In: Anali FF 4, 1964, S. 358ff. 3 Die römischen Zahlen in diesem Kapitel beziehen sich auf Grimms Vorrede, die in der Neuausgabe von Mojaševic/Rehder (s. Literatur- Verzeichnis) zugänglich is t. Sie is t auch in GPS I I , 432 461 ־ abgedruckt. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" Vuk kam es zu dieser Zeit vor allem darauf an, daß seine Ortho- graphiereform von dem anerkannten Gelehrten und "unbefangenen Aus- länder"1 Grimm befürwortet wird. was er sich von Grimms Vorrede erhofft hat. Zehn Tage vor der Absen- dung der Notizen schrieb er an Kopitar : "Gledaj te i Vi štogod te mu y. site, molim Vas. Ja sam mu obecao literarische Notizen za predgovor, pa i sad iste, a ja upravo ne znam, šta ču mu poslati, nego оси da ispisem desto iz moga predgovora к rječniku... i da да uputim пека cita Vaše recenzije о rječniku i o Dobrovskoga gramatici. Da осе štogod dobro о o rto g ra fiji kazatiî" (Prepiska I, 239). Er bat Grimm, "etwas Gutes und Gewisses" über die Orthographie und besonders über die Notwendigkeit des Zeichens j zu sagen (Prepiska II, 13). "In der Vorrede bestreiten Sie mir alles, nur d, l j , n j, und j nicht." (ebd. 31). Vuk kam es zu dieser Zeit vor allem darauf an, daß seine Ortho- graphiereform von dem anerkannten Gelehrten und "unbefangenen Aus- länder"1 Grimm befürwortet wird. Es is t bereits bemerkt worden, daß Grimm bei der Anfertigung sei 2 ner Vorrede weitgehend sich selbst überlassen war . Kopitar zeigte sich ziemlich unbeteiligt und Vuk besaß ein großes Vertrauen in Grin wissenschaftliche Fähigkeiten: " ... machen Sie wie Sie für Beste finden... ich zweifle nicht, daß Sie alles recht gut gemacht haben." (Prepiska II, 9, 38). Grimm hat die Angaben für seine Vorrede von verschiedenen Seiter zusammengetragen: aus Kopitars, Vuks und Dobrovskys Schriften und seinem Briefwechsel mit ihnen; die im Laufe der Jahre anläßlich meh- rerer Rezensionen gesammelten Kenntnisse bildeten bereits eine gute Grundlage. Es entsprach Grimms Arbeitsweise, das ihm Gebotene vom eigenen Standpunkt aus darzulegen. Er scheute dabei nicht, sogar Dobrovsky zu widersprechen, obwohl er der Meinung war, daß "dessen kleiner Finger mehr slawisch versteht, als mein ganzes Ich". (Pre- piska I I , 36). Sein Grundsatz war: " ... mein Fehlschlagen schadet... nicht und mein Treffen h ilf t der Wahrheit, die wir alle suchen." (XXXI)3. Die Vorrede zeigt trotz der Anlehnung an die benutzten Quellen eine weitgehende Eigenständigkeit und is t Grimms ausführlich ste slavistische Arbeit. 1 So nannte ihn Kopitar, vgl. Vasmer, S. 6. 2 Vgl. H. Peukert, Bemerkungen über Jacob Grimms "Vorrede" zu Vuks ,Kleiner serbischer Grammatik1. In: Anali FF 4, 1964, S. 358ff. 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" 3 Die römischen Zahlen in diesem Kapitel beziehen sich auf Grimms Vorrede, die in der Neuausgabe von Mojaševic/Rehder (s. Literatur- Verzeichnis) zugänglich is t. Sie is t auch in GPS I I , 432 461 ־ abgedruckt. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Grimms Vorrede is t inhaltlich und äußerlich in zwei Teile geglie- dert: einen kultur- und sprachgeschichtlichen Bericht über die Sia- ven allgemein, besonders aber über die Serben, und einen sprachwis- senschaftlichen Teil, in dem er den Sprachforschern seine eigenen ״*Bemerkungen" m itte ilt, die ihm während der Arbeit an der Gramma- tik eingefallen seien (ebd.). In dem ersten, ausführlicheren Teil berichtet Grimm zunächst über die Verbreitung der Slaven in Europa und weist auf die Bedeutung der Sprachforschung für die Erkundung ihrer Siedlungsgeschichte hin. Er selbst gibt einige Beispiele slavisch-gotischer Lehnbeziehungen an: "Der Gothe des vierten Jahrh. gebraucht slavische Wörter, die sich in keinem Dialecte des innern Deutschlands nachweisen lassen, z. B. d u l g (altslavisch d i g , d o l g ) , das Gothische p l i n s j a n (saltare) is t ohne Zweifel das altslavische p l ç s a t i , russisch p l j a s a t , ׳pol- nisch p l ç s a ô ; em akka (altslavisch s m o k v a ) , wogegen der Slave e t k l o (s t i k l s ) u.a. vom Gothen borgte, wahrscheinlich mzda (m i z d ö )... Das gothische s i p ö n e i s . . . war bisher in der deutschen Sprache unerklär- lieh; wie wenn es das gleich dunkle slav. г и р а п ъ wäre?-., die Süd- slaven haben г и р а п ъ (croat. s p a n , woraus ungar. i s p a n . . ; die West- slaven allein p a n ... vgl. poln. p a n n a ... litth . p ö n a s (Herr), poná- t i s (junger Herr), ѣ и р о п п е (Frau); altpreuß. s u p u n i (Frau)... Der litthauische Sprachstamm hat zwar eignen Grund, aber auch Gemein- schaft mit Gothen und Slaven, die auf sehr frühe Zeit weist; einen Beleg bietet das letzte Beispiel." (II). 1 Vgl. M. Vasmer, Etymologisches Wörterbuch der russischen Sprache I - I I I , 1953-1958. - A. G. Preobraženskij , Êtimologiceskij slovar russkogo jazyka, 1959. 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" Die etymologischen Versuche von Grimm enthalten zum größten Teil richtige Angaben; die spätere Sprachforschung hat einige korrigiert (aksl. smoky als Entlehnung aus dem got. s m a k k a ; Ablehnung des Zu- sammenhangs zwischen z u p a n und s i p o n e i a ) , oder nur eine Urverwandt- schaft festgestellt Ы ъ і д ъ , m bzda) . Wir haben festgestellt, daß Grimms Etymologien einen Einfluß auf das bekannte altslavische Wörterbuch von Miklosich, damit auch auf spätere Etymologen, ausgeübt haben. Miklosich verweist bei mbzda und ги р а п ъ ausdrücklich auf Grimm; wir finden bei ihm auch Grimms Be- hauptung vom got. d u l g als dem slav. Lehnwort und dem got. p l i n s j a n 1 Vgl. M. Vasmer, Etymologisches Wörterbuch der russischen Sprache I - I I I , 1953-1958. - A. G. Preobraženskij , Êtimologiceskij slovar russkogo jazyka, 1959. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access als dem slav. p l ę a a t i (das letztere wird auch heute so gesehen). Mi- klosich beruft sich in den genannten Fällen allerdings nicht auf Grimms Vorrede, sondern auf die "Deutsche Grammatik" und die "Ge- schichte der deutschen Sprache", in die Grimm diese Etymologien über nommen hat1. als dem slav. p l ę a a t i (das letztere wird auch heute so gesehen). Mi- klosich beruft sich in den genannten Fällen allerdings nicht auf Grimms Vorrede, sondern auf die "Deutsche Grammatik" und die "Ge- schichte der deutschen Sprache", in die Grimm diese Etymologien über nommen hat1. Grimm zählt in seinen weiteren Ausführungen die slavischen Volke 2 auf und g re ift Kopitars und Dobrovskÿs Einteilung der Slaven in ei- ne südliche und eine nördliche Gruppe (Dobrovsky rechnete die Russen zu der südlichen, Kopitar zu der nördlichen Gruppe) auf:"Dem südli- chen Dialecte fallen Slovenen, Serben und Bulgaren, dem nördlichen Böhmen, Polen und Russen zu." Er weist aber auch auf die Verwandt- Schaft des Russischen mit dem Südslavischen hin. 1 Wieviel Miklosich sonst von Grimm geschöpft haben könnte, wäre im Rahmen einer Arbeit über den Slavisten Grimm zu untersuchen; Grimm gesamtes Werk enthält viel Material aus den slavischen Sprachen. 2 Vgl. dazu Peukert, a.a.O., S. 359ff. 3 Kopitar schrieb ihm am 17. Dez. 1823: "Die Russen, die überall in- triguiren,haben sich auch unter die Südslaven hinein in trig u irt, indem Dobrowsky nur ihre Schriftsprache kennt, die gemacht ist aus der alten und neuen. Reipsa sind sie Nordslawen..." - Vgl. Vasmer, S. 11. 4 Vgl. P. J. Safarik, Geschichte der slavischen Sprache und Literatu nach allen Mundarten, 1826, 2,Abdr. 1869, S. 32. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM i f 1 Wieviel Miklosich sonst von Grimm geschöpft haben könnte, wäre im Rahmen einer Arbeit über den Slavisten Grimm zu untersuchen; Grimm gesamtes Werk enthält viel Material aus den slavischen Sprachen. 2 Vgl. dazu Peukert, a.a.O., S. 359ff. 3 Kopitar schrieb ihm am 17. Dez. 1823: "Die Russen, die überall in- S 4 Vgl. P. J. Safarik, Geschichte der slavischen Sprache und Literatu nach allen Mundarten, 1826, 2,Abdr. 1869, S. 32. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 2 Vgl. dazu Peukert, a.a.O., S. 359ff. 3 Kopitar schrieb ihm am 17. Dez. 1823: "Die Russen, die überall in- triguiren,haben sich auch unter die Südslaven hinein in trig u irt, indem Dobrowsky nur ihre Schriftsprache kennt, die gemacht ist aus der alten und neuen. Reipsa sind sie Nordslawen..." - Vgl. Vasmer, S. 11. 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" Diesen Gedanken formulierte er noch deutlicher in seiner Rezension von Dobrovskys "Institutiones", nämlich "dasz nicht nur eine lin ie zu ziehen sei, welche die Verwandtschaft des russischen mit dem südöstlichen stamme darstellt, sondern auch eine andere, welche es dem nordwestlichen zuweist, das russische steht folglich zwischen beiden hauptstämmen; ursprünglich dem westlichen näher, neigte es sich später dem südli- chen zu; durch den einflusz der kirchensprache..." (GKS IV, 190). Mit dieser Ansicht steht Grimm der späteren Einteilung der slavische Sprachen in drei Gruppen näher als die derzeit bekanntesten Slavi- sten Dobrovskÿ und Kopitar. Er erhielt wohl von Kopitar eine Anre- ג­gung dazu, die er mit kritischer Distanz verarbeitete . Grimms Vorstellung von den drei slavischen Sprachgruppen hat kei nen unmittelbaren Einfluß in der Slavistik ausgeübt. Sie ist jedoch offensichtlich nicht ganz ohne Wirkung geblieben. P. J. Safarik geht 4 nämlich in seiner 1826 erschienen "Geschichte", ohne Grimms Namen zu nennen, auf diesen Einwand gegen DobrovskJ ein und s te llt die Fra ge, ob eine d ritte , dazwischenstehende Gruppe einzusetzen wäre, ver- bleibt aber zunächst bei Dobrovskys Klassifikation. Sieben Jahre S. 11. 4 Vgl. P. J. Safarik, Geschichte der slavischen Sprache und Literatu nach allen Mundarten, 1826, 2,Abdr. 1869, S. 32. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access später zieht áafarík in seinem bedeutenden Werk "Serbische Lesekörner1 eine Grenze zwischen der "illyrischen" Sprachgruppe, zu der er die "serbische, chorwatische und windische Mundart" rechnet, und der russisch -bulgarischen1• Obwohl er der letzteren Gruppe auch das A lt- kirchenslavische und Neubulgarische zuwies, bedeutete sein Werk nach Jagifcs Worten einen Schritt vorwärts, indem es Dobrovskÿs und Kopi- 2 tars Klassifikationen korrigierte . Es scheint uns, daß áafarík für seine Einteilung die erste Anre- gung von Grimm erhielt• Sie standen im Briefwechsel miteinander und der folgende Brief Safariks aus dem Jahre 1834 zeigt seine große Be- wunderung für Grimm; er schrieb, "...daß seit ein Paar Jahren, be- sonders seit dem genauem Studium Ihrer früher mir ganz unbekannten Werke, eine gewaltige Revolution in meinem Innern hervorgegangen is t"3. In seinem Werk "Serbische Lesekörner" is t ein unverkennbarer Einfluß von Grimms Gedanken feststellbar, sowohl in der Zielsetzung der Sprachforschung als Erhellung der Vorgeschichte und Völkerkunde, der Auffassung der Mundarten als auch in seiner Terminologie: er ver- wendet die Termini "Sprachtrieb", "schaffender Sprachgeist" und so- gar den Grimmschen Begriff vom "Gesetz der Ausnahme" . 1 Vgl. P. J. Safarik, Serbische Lesekörner,.. 1833, S. 116. 2 Vgl. V. Jagic, Isto rija slavjanskoj filo lo g ii, 1910, S. 273. 3 Vgl. Vasmer, Bausteine I, S. 64. 4 Vgl. P.F. Safarik, Serbische Lesekörner,.. S. 4f., 113. - Eine eingehende Untersuchung von Safariks Schriften würde vermutlich viele Berührungspunkte mit Grimm aufdecken. 5 Grimm v e rtritt hier Kopitars Auffassung vom pannonischen Ursprung der aksl. Sprache. Später wandte er sich von dieser Theorie ab und wies auf die Unzulänglichkeit der Argumente Kopitars hin (GKS V, 234ff.), während der bekannte Slāvist Miklosich sie noch lange danach für ausreichend hielt• 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" Im weiteren Verlauf seiner Vorrede erörtert Grimm die Christia- nisierung der Slaven, die Folgen der Kirchenspaltung auf ihrem Bo- den und den Einfluß der Kirchensprache auf die Entwicklung von Schriftsprachen5. Dabei " ... übte die erstarrende Kirchensprache, so weit sie sich erstreckte, bei dem gänzlichen Mangel aller Weltlite- ratur, fre ilich bedeutenden Einfluß auf Volksmundarten aus und mach- te sie dadurch einander ähnlicher; den bedeutendsten auf das Russi- sehe, welches gleichsam wider seine Natur südslavisch gestimmt worder ist, geringem auf das Polnische, wo der lateinische Ritus bald über־ g p 5 Grimm v e rtritt hier Kopitars Auffassung vom pannonischen Ursprung der aksl. Sprache. Später wandte er sich von dieser Theorie ab und wies auf die Unzulänglichkeit der Argumente Kopitars hin (GKS V, 234ff.), während der bekannte Slāvist Miklosich sie noch lange danach für ausreichend hielt• Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access wog." (X).Dem ursprünglichen Vorteil der Einführung des Slavischen in die Kirche setzt er das spätere Verharren bei der Kirchensprache als negativ entgegen. "Die slavische Kirchensprache hingegen... hat die freie Entwicklung der Vulgardialecte, da, wo sie herrschte, ohne Frage vervortheilt. Das war der Fall in Rußland, bis endlich seit hundert Jahren die weltliche Sprache ihr Recht nach und nach geltend machte und glücklich gediehen is t." (Xlf.). An diesem Punkt seiner Vorrede wendet sich Grimm dem Serbischen zu. Seine Verwendung dieses Begriffs is t von Kopitar und Vuk auf ver schiedene Weise beeinflußt worden. Grimm hat Kopitars Schriften als Quelle für seine Vorrede be- nutzt. Wie bereits bemerkt wurde, hat Kopitar die Benennung " illy - risch" durch "serbisch" ersetzt1. In Grimms Ausführungen über die Ausdehnung des serbischen Sprachraumes sind Kopitars Angaben deut- 2 lieh erkennbar , indem er die Grenzen "... oben die Kulp, unten Cat- taro, hinten Timok" nennt (XXVf.). Grimm spricht auch über die Kroa- ten, die "lateinischen Illy re r", die seit Jahrhunderten ihren Dia- lekt in der Literatur pflegen (XVI) und nennt ihre Wörterbücher (XIX Der Nachdruck liegt bei ihm auf der Sprache, die auf dem gesamten Gebiet dieselbe is t, für die er den serbischen Namen vorzieht: "... der lebendige Name serbisch ist besser als der gelehrte und halbtodt illyrisch , besser als der von einigen (frühem) Illyrern gebrauchte bosnisch." (XXV). 1 Vgl. KKS I, 2 3f., 44, 294; Spisi 11,1. knj., 6. 2 Vgl. dazu Peukert, a.a.O. S. 359ff. 3 Er beruft sich dabei auch auf Dobrovskÿ, der in der Slovanka 1814, S. 165, diesen Namen für geeignet hielt, die Benennung aller Sia- ven zu sein. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" "In der That aber scheint der Serbe die beste Be- nennung, mit der man alle diese Völker einer Abkunft und Sprache 3 grammatisch umfassen könnte." (XXIII). Diesen Argumenten fügt er die kulturellen Gemeinsamkeiten hinzu, wobei er die Angaben aus Vuks Brief benutzt (Prepiska II, 23, 28), daß nämlich die Lieder über die serbischen Helden Marko Kraljeviò und Fürst Lazar auch "in Dalma tien, Bosnien, Herzegowina wie im innern Serbien" gesungen werden. "Überhaupt auch die Ausbreitung aller Volkssagen in diesen Ländern verbürgt ihre genaueste Genossenschaft..." (XXV). Es ist deutlich zu sehen, daß Grimm in Vuks Bemühungen um die Kodifizierung der Schriftsprache auch den Weg zur Bildung einer Na- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 151 - tion auf dem betreffenden Sprachgebiet sah, was im Einklang mit sei- ner Auffassung von der einigenden Funktion der Schriftsprache steht. Mit diesem Aspekt seiner Vorrede g riff er der Illyrischen Bewegung und dem späten Vuk vor, der sich z. Zt. seiner "Serbischen Gramma- tik " auf das sprachliche Problem bei den Serben eingeschränkt hatte. Andererseits haben aber Grimms Autorität und seine Stellungnahme zum serbischen Namen sicher einen Einfluß auf Vuks Verhalten in der spä- teren Diskussion um den illyrischen Neimen gehabt. In seinen Ausführungen über die drei serbischen Dialekte (XXVIIff.) folgt Grimm Vuks Angaben aus der Vorrede zum Wörterbuch und der ge- rade erschienenen Leipziger Liederausgabe (z.B. bei der Bestimmung der Grenzen der Ressaver Mundart). Er nimmt aber auch das auf, was Vuk ihm brieflich mitgeteilt hat, daß in Sumadien "eve, ene statt evo, eno" gesprochen wird, daß bez und b r e z nebeneinander gelten und bemerkt: "Wenn die illyrischen Wörterbücher uho (auris) muha (musca) suh (siccus) schreiben, Wuk aber uvo, muva, suv, thut das noch keine wirkliche Dialektverschiedenheit zwischen Dalmatien und Serbien dar: jenes kann ungenaue, der gelehrten Sprache zu Gefallen gewählte Fassung der Aussprache s e i n 1(XXX; Prepiska II, 22, 28). Er greift auch auf Kopitars Rezension des zweiten Liederbandes von 1815 zurück und te ilt mit: "Einzelne Wörter, die nur in der einen oder ändern Gegend Vorkommen, begründen keine wahre dialektische Ver- schiedenheit, z.B. außerhalb Bosnien und der Herzegowina wird man kaum das mit devojka gleichbedeutende cura... hören. Nicht wenige Aus- drücke erhalten sich fast allein in den Liedern, die gemeine, täg- liehe Rede hat ihrer vergessen, z.B. moma (puella), utva (ein zauber- hafter Wasservogel, vgl. russ. 1 Zu beachten ist, daß Grimm die Termini "Dialekt" und "Sprache" nicht von einander trennt. 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" utka), (Ente), riznica (conclave) etc. S tulli und Deila Bella haben sie nicht aufgenommen." (XXX). Hier ist Grimms Auffassung, daß die Literatur die Sprache festigt und bewahrt, erkennbar, die auch einer der Beweggründe für seine Unterstützung von Vuks Bemühungen um die serbische Schriftsprache war. Grimm nimmt den bei Vuk 1818 fehlenden ikavischen Dialekt in einer Anmerkung auf: "Die Schokzen in Slavonien geben dem i den Vor- zug und sprechen vira, mira, rie, divojka, vrime." (XXIX). Vuk hat Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ihn darum gebeten: da "die Rede von der Sprache überhaupt ist, so mü sen Sie auch über den Slawonischen (Schoktzischen) Dialekt etwas er- wöhnen (das is t am leichtesten zu bestimmen:vira, mira, rie, divojka vrime ect.)( ״Prepiska II, 13f.). ihn darum gebeten: da "die Rede von der Sprache überhaupt ist, so mü sen Sie auch über den Slawonischen (Schoktzischen) Dialekt etwas er- wöhnen (das is t am leichtesten zu bestimmen:vira, mira, rie, divojka vrime ect.)( ״Prepiska II, 13f.). A. Belic hat bemerkt, daß Grimm Vuks damaligen Kenntnissen des Ikavischen nicht genügend Rechnung getragen hat1. Das ist zeifellos richtig, da Vuks "Dodatak к Sanktpeterburgskim sravniteljnim rjeeni- cima" von 1822 (GPS II, 179 - 214) mehr Material über das Ikavische enthält. Nur der Vorwurf t r i f f t nicht Grimm, der alle ihm zugeschick ten Angaben, sogar in derselben Reihenfolge anführt. Die Knappheit der Darstellung entspricht allerdings seiner Auffassung, daß im Zuge der Ausbildung einer Schriftsprache die nahverwandten Mundarten sich in größere Einheiten zusammenschließen. Aus diesem Grunde schlug er an Stelle von Vuks Einteilung in vier, die Einteilung in zwei Mund- arten vor, eine s ü d w e s t l i c h e mit dem herzegowinischen und bosnischer und eine n o r d ö s t l i c h e mit dem sirmischen und ressavischen Dialekt. Belic hat richtig bemerkt, daß Grimm einen Einfluß auf Vuks endgül- v v v 2 tige Dialekt in j u ž n o , i s t o e n o und za p a d n o n a r j e e i j e ausgeübt hat . Grimms Einfluß is t besonders daran erkennbar, daß Vuk neben den ei- genen Termini hin und wieder auch diejenigen von Grimm gebraucht (GPS II, 167). 1 Vgl. A. Belic, Vukova borba.., S. 143f. 2 Vgl. ebd., S. 174ff. 3 Vuk schrieb an Grimm: "Sie sind das größte Glück unserer Lieder, unserer Sprache und unserer Literatur." "Wahrhaftig, auch die Feinde unserer Sprache lesen gerne solche Sachen und bekehren sichi" (Prepiska II, S. 12, 48). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" Aber auch Grimms Ansichten über die Entstehung der Schriftsprache durch Reduzierung der Mundarten haben zumindest eine Klärung dieser Frage bei Vuk eingeleitet, der zu dieser Zeit mit den Widersprüchen rang, die sich aus seiner Zulassung aller Dialekte in der Literatur ergaben. Grimm geht in seiner Vorrede auch auf das spezielle serbische Sprachproblem ein (Xllff.) Seine Stellungnahme zugunsten der Volks- spräche is t eindeutig und sie geschieht nicht nur zu propagandisti- sehen Zwecken, obwohl sie auch in diesem Sinne eine große Wirkung gehabt hat3. Das Festhalten an der Kirchensprache beruht nach Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Grimms Meinung ,,auf völligem Verkennen dessen, was todte und lebendi- ge Sprache seyn können und sollen". Auch an Dobrovskÿ schrieb er: "Auf welchen ändern grund kann aber eine serbische literatur gebaut werden, als auf das von Vuk befolgte verfahren; in diesem dialect, sei er wie er wolle, müssen geistreiche Serben schreiben, denn nur in ihm werden sie sich frei und ungezwungen gehaben können."* Die to- te Kirchensprache, ,,sie sey noch so wohllautend, reich und gewaltig, in weltlichen Dingen is t sie unbeholfen, arm und versagend. Ich bin fern davon zu glauben, daß in dem Umfang des Cyrillisch-altslavischen die ganze Fülle der altslavischen Zunge enthalten seyn könne. Der Wörter und Wurzeln zu geschweigen, selbst gewisse Flexionen, Bildun- gen, Ableitungen, z.B. Diminutiva, Augmentativa, vor allem eine Menge natürlicher poetischer Wendungen, deren das Leben nicht entrathen mag, die ganze comische Kraft, müssen ihr mangeln, da sie ihre Würde beeinträchtigt hätten. Solche weltliche Wörter und Bildungen leben aber in der Volkssprache, die Hohes und Niederes für alle Bedürfnis- se in sich trägt und duldet. Dieses Vorraths kann weder die Dicht- kunst entbehren, noch die Geschichte." (Xlllf.) Ein provenzalischer Troubadur oder ein deutscher Minnesänger hätten nie in der Kirchen- Sprache singen können. Das Bemühen der serbischen Schriftsteller, in einem "buntlappigen" Stil zu schreiben, verglich er mit dem "unmün- digen Stammeln oder trostlosem Ermatten einer göttlichen Fähigkeit." Die geistige Fortbildung der Sprache bedarf nach Grimms Auffas- sung der lebendigen Pflege sowohl in der Sprachgemeinschaft als auch in der Literatur. Die serbische Sprache, "eine der ausgezeichnetsten, lieblichsten Mundarten lebt und webt unter dem Volke fort, nur ihre Literatur liegt in tiefstem Schlummer, aller unberechenbaren Vor- theile, die aus der geistigen Sprachbildung für Volksbildung hervor- gehen, muß der Serbe bis jetzt entbehren.( ״XII). 1 Vgl. A. Sauer, Aus Jacob Grimms Briefwechsel.. S. 619. 1 Vuk nahm Grimms Rezension der Lieder von 1824 in die Vorrede zum 1. Bd. der Leipziger Liederausgabe auf. 2 Bekanntlich hat Ivar Aasen in Norwegen einen ähnlichen Versuch un- ternommen, die traditionelle dänisch-norwegische Schriftsprache (bokmal) durch die Volkssprache zu ersetzen. Das Resultat ist der schriftsprachliche Bilinguismus (bokmal und landsmal). - A. Gallis sieht als einen der Gründe darin, daß es Aasen nich gelungen ist, wie Vuk eine ”zeitgenössische Tradition" für seine Schriftsprache zu schaffen. Vgl. A. Gallis, Vuk Karadzic i Ivas Aasen, "norveski Vuk” . In: Anall FF 4, 1964, S. 100f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" Die Sprach- und Geistesentwicklung sind nach Grimms Sprachauffassung unzertrennlich und er sah in der serbischen Sprachsituation ein Hemmnis der kulturel len Entwicklung. Aus dieser Sicht bewertete und unterstützte er Vuks Bemühungen als einen Schritt, "aus solcher Schmach und Ertödtung Serbien und die serbische Sprache zu retten". "Die Wahrheit hier Aus Jacob Grimms Briefwechsel.. S. 619. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access früher als andere auszusprechen ist Beruf des Grammatikers." (XXIII) Nach Grimms Auffassung hat der Grammatiker eine aktive Rolle in der Geistesgeschichte seines Volkes; Vuk hat sie in der Praxis auch aus- geübt. Grimm hebt die Bedeutung von Vuks Liederausgaben für die Durch- Setzung der Volkssprache in der Literatur hervor: "Für die Sprache is t durch die Sairanlung nicht weniger geleistet worden, als für die Poesie.. .Braucht noch die Thorheit: es gebe keine serbische Sprache, mit weitern Gründen bestritten, die Klage: es gebräche an Mustern edler Schreibart, weggeräumt zu werden?” (XXI). Grimm sah aber die Sprache der Lieder als ein Ubergangsstadium in der Entwicklung der Schriftsprache als Trägerin der künftigen Kultur: ” ... sobald sich Serbien zur cuitur emporarbeitet, wird eine mundart die ändern überwältigen; dann wird auch die zeit dieser epischen dichtungen vorüber sein...” (GKS IV, 200). Grimms Ansicht von der Volksdichtung als dem Beweis für die Lite raturfähigkeit der serbischen Volkssprache hat zweifellos bedeuten- den Einfluß auf Vuk ausgeübt*. Vuk hat es richtig begriffen, daß seine Liedersammlungen einen wichtigen Beitrag zum Sieg seiner Sprac 2 konzeption darstellen . Grimm greift auch die ihm durch Kopitar und Vuk bekanntgewordene Diskussion um die Beziehung der serbischen zur ksl. Sprache auf. In Bezug auf die grammatische Struktur der zu kodifizierenden Schriftsprache vertritt er eindeutig ihre Eigenständigkeit gegenüber der ksl. Sprache. Die Abweichungen im Serbischen lassen sich ”nicht durch die bloße Annahme des allmählichen Sinkens eines und desselben Dialects wegräumen..." (XIV). Er wandte sich auch gegen Dobrovskys Ansicht von den ”verdorbenen" Formen im Serbischen: ”Das beweist mir zuviel, nämlich auch gegen das böhmische, polnische. Worin be- steht also das Vorrecht dieser letztem? bloß darin, daß sie seit 00051004 - 155 - mehrern jahrh. eine literatur und bûcher besitzen, wodurch die vor- theile oder nachtheile ihrer mundart befestigt worden sind."1 mehrern jahrh. eine literatur und bûcher besitzen, wodurch die vor- theile oder nachtheile ihrer mundart befestigt worden sind."1 Zum Problem der Entlehnungen aus dem Kirchenslavischen hat sich Grimm nicht ausführlich und eindeutig geäußert. 1 Vgl. A. Sauer, Aus Jacob Grimms Briefwechsel.., S. 619. 2 Das gibt Vuk an in der Ankündigung des Wörterbuchs von 1818 (GPS I, 94). 1 Vgl. A. Sauer, Aus Jacob Grimms Briefwechsel.., S. 619. 2 Das gibt Vuk an in der Ankündigung des Wörterbuchs von 1818 (GPS I, 94). 3 Hervorhebung von V.B. 4 Grimm trennt die Termini Buchstabe - Laut nicht klar von einande 3 Hervorhebung von V.B. 4 Grimm trennt die Termini Buchstabe - Laut nicht klar 4 Grimm trennt die Termini Buchstabe - Laut nicht kla 2. Grimms Vorrede zur "Kleinen serbischen Grammatik" Er meinte, daß die Spuren, die die Kirchensprache auch im Serbischen hinterlassen ha- ben muß, "nicht ganz zu verwischen sind". Ihm war das Spezifische in der slavenoserbischen Sprache offensichtlich nicht ganz klar, worauf seine wiederholten Äußerungen über die tote Sprache hindeuten. Das Slavenoserbische war mit dem toten Latein nicht vergleichbar, da es von der gebildeten Schicht zumindest teilweise auch gesprochen 2 wurde . Die Vertreibung der Kirchenslavismen, Russismen und Germa- nismen für die in der Volkssprache fehlenden Begriffe wäre jedoch nicht im Sinne von Grimms Sprachauffassung? er hat ähnliche puristi- sehe Versuche in Deutschland bekämpft. In der Vorrede äußert er sich in diesem Sinne über die Turzismen: eine "gründliche Sprachfor- schung erkennt auch die Nothwendigkeit und Natürlichkeit der Ein- mischung einiger fremden Bestandtheile in beinahe jeder Sprache an. Sie sind festigender Mörtel und füllen L ü c k e n aus, ja sie dienen wohl dem A u s d r u c k e zur Färbung und Schmeidigung.- ." 3 (XV). Grimm sieht in einer mäßigen Anzahl von Fremdwörtern sowohl eine Bereiche- rung des Wortschatzes als auch der stilistischen Möglichkeiten der Sprache. In dieser Frage unterscheiden sich seine und Vuks Ansichten. Grimm kommt Vuks Wunsch in bezug auf die Orthographie entgegen. 4 "Dem Wohllaut der serbischen Buchstabenverhältnisse kann kein Un- befangener Gerechtigkeit versagen; wir würden die feinen Modificatio- nen der Consonanten weniger erkennen, wenn der Verfasser mehr die etymologische Wurzel festgehalten, als die Abstufungen der wahren Aussprache beachtet hätte." (XXII). Er betont die Vorteile der pho- netischen Orthographie und die Notwendigkeit der von Vuk eingeführ- ten Zeichen. Grimm vertrat aber zu zwei orthographischen Fragen eine von Vuk abweichende Meinung. Die eine betraf die Graphie: Grimm wäre eine 1 Vgl. A. Sauer, Aus Jacob Grimms Briefwechsel.., S. 619. 2 Das gibt Vuk an in der Ankündigung des Wörterbuchs von 1818 (GPS I, 94). 1 Vgl. P. Ivic, О Vukovom Rječniku.., S. 26, 108. 2 Dieser wichtige slavistische Beitrag von Grimm is t in der bisheri- gen Forschung nicht ausreichend gewürdigt worden. Lj. Stojanovic nennt es in der Aufzählung der Themen von Grimms Vorrede, geht abe: dem Ziel seines Buches folgend, nicht näher darauf ein. Die Übrigei Forscher behandeln bei der Untersuchung verschiedener Teilaspekte von Vuk-Grimm-Beziehung diese Frage nicht. Vgl. P. Ivic, О Vukovom Rječniku.., S. 26, 108. 3 Hervorhebung von V.B. 4 Grimm trennt die Termini Buchstabe - Laut nicht klar von einander• Schon in der kurz vor der Vorrede entstandenen Rezension von Dobrovskys Institutiones vermißte Grimm eine "genauere einsicht in Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 157 ־ die beschaffenheit dieser halbvocale"1; er wandte sich dieser Auf- gäbe selbst zu• An Vostokovs und Dobrovskÿs Erkenntnisse über die russ.. о und e an Stelle der inlautenden reduzierzen Vokale an- knüpfend meinte Grimm: ”Ich gehe noch einen Schritt weiter und be- haupte 1) diese о und e waren früherhin wahrscheinlich kurzes и und г. 2) ъ und ь am Ende der Wörter deuten nicht weniger auf ein frühe- res и und г. In beiden Fällen sind ъ und ь geschwächte, unbestimmter gewordene Vocale, denen in der Mitte, zwischen schützenden Consonan- ten noch voller, am Ende hingegen, wo sie von einer Seite bloß деде- ben sind, nur halber Vocalgehalt bleibt. Dieser halbe Vocal w irft sich auf den vorausstehenden Consonanten und a ffic ir t ihn, d.h. nach- dem ihm ein halbes и oder І folgt, empfängt er eine dickere (härte- re) oder dünnere (weichere) Aussprache. Auslautende ъ und ь sind folglich Bildungsvocale, die sich geschwächt haben, gleichsam nur noch nachhallen und im Verlauf der Zeit völlig zu Grunde gehen. Die scheinbare Wahrnehmung.., daß die altslavische Sprache keinen reinen Consonanten zu Ende des Wortes leide, muß demnach dahin berichtigt werden: daß sie noch kein nacktes Wort kenne, vielmehr jedes mit einem (in ъ, ь nur halblauten) Vocal der Ableitung bekleide... Zur Zeit.., wo wir sie zuerst kennen lernen, sind bereits ihre schlie- ßenden Flexionsconsonanten verschwunden, z. B. jenes -s des Nom. Sing. Masc." (XXXIVf.). Seine Ansichten bekräftigte er mit den Belegen aus verschiedenen idg. Sprachen: Zuerst für das inlautende ъ, ь: ѵ ъ і к ъ , ѵ і ъ к ъ , griech. Ликос, wie goth, v u l f s , la t. l u p u s lauter Formen desselben Worts; Ы ъ с к а , lat. p u l e x , hochd. f l o h ; pl*bm>,goth .f u l l s ; Ь ъ гд ъ , altnord. t o r g ; v e t b o h ъ, lat. v e t u s ; ^ п ъ к ъ , althochd. 3 Hervorhebung von V.B. 4 Grimm trennt die Termini Buchstabe - Laut nicht klar von einander• Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 156 - einheitliche Graphie innerhalb der europäischen Kulturgemeinschaft lieber gewesen. "Das lateinische schönste und deutlichste Alphabet würde mit verständiger Vermehrung für die eigentümlichen Laute jeder Sprache und Mundart allen europäischen Sprachen wohltätig werden." (XXII). Die zweite Frage, Vuks Weglassung des h , g riff er wohl aus Rücksicht auf Vuk in der Vorrede nicht auf. Das holte er in seiner Anzeige der Grammatik nach: "Aus der reihe der serbischen buchstaben hat vielleicht herr Wuk das h, welches die illyrischen grammatiker beibehalten, zu voreilig ausgestoszen. musz er es doch in den inter- jectionen ah! oh! und in fremdwörtern dulden... durch das h wird nicht nur die flexion des gen. pl.und des imperfects hervorgehoben, sondern auch, der erleichterten etymologien zu geschweigen, jener altslavische umlaut des ch in s und sch für die formenlehre aufge- klärt..." (GKS IV, 226f.). Grimm hat richtig erkannt, daß das Feh- len des h die grammatische Regelmäßigkeit des sprachlichen Systems beeinträchtigt. Die Auslassung des h hat zum Synkretismus der Kasus geführt, der in der modernen Schriftsprache nicht besteht, (vgl. Vuks Paradigma z u t i als Nom., Akk., Vok. Sg. und Nom., Gen., Vok. Pl.), ebenfalls zur Konsonantenalternation 0 : 8 bzw. 8 { o r a : o r a s i , о г а в е ! ) • "Tako je odsustvo h stvorilo anomalij и da se imenske osnove mogu završavati i samoglasnikom (npr. o r a , a n a a , s i r o m a ) . . . " und eine Anhäufung von Vokalen, die die heutige Schriftsprache nicht kennt, z.B.: Gen. o r a a ; и r u u ; j a a o (j a h a o ) usw.1 Den zweiten Teil der Vorrede benutzt Grimm, um seine eigenen sia• vistischen Erkenntnisse vorzutragen (XXXIff.). Im Mittelpunkt stehen seine Ausführungen über die aksl. reduzierten Vokale, deren Etymo- 2 logie und vokalischen Wert er als erster erläutert hat . 1 Bei Dobrovsky S. 22 dienen sie "pro vario soni temperamento". 2 Giimm war nah der etymologischen Lösung des Infixes ־c v - der sog. u-Stämme: "Ich würde auch das Augment - o v auf dieses -u beziehen und z.B. s y n o v e , s y n o v von 81/яъ mit dem goth. s u n j u s , s ü n i v é oder Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Bei Dobrovsky S. 22 dienen sie "pro vario soni temperamento". 3 Hervorhebung von V.B. 4 Grimm trennt die Termini Buchstabe - Laut nicht klar von einander• d u n n i ; з а Ы ъ к о (für З а Ъ ъ Іко ?) , althochd. a p f u l ; д г ъ к ъ , althochd. k r i e h ( g r a e c u s ) ; І ъ п ъ , lat. l i n u m ; l a b b d b ( o y g n u s ) , a lthochd. a l p i z . Dann für die auslautend 2 den: 6 у п ъ л goth. s u n - u s ; medъ { m u ls u m ) , angelsächs. m e d - u ; І ъ п ъ , l i n - u m ; das slav• Supinum auf - t־b, das lateinische auf tu m ; одпъ, lat. i g n - i s ; g o s t ь, althochd. k a s t , PI. k e s t - i ; д и е ъ { a n s e r ) ,althoch g a n s ; Pl. g e n s - i ; litth . é a s - i s ; к а т е п ъ ( l a p i s ) , litth . à k m e n - i s . Aufmerkende werden bei weiterer Vergleichung manche altslav. Nomina 1 Bei Dobrovsky S. 22 dienen sie "pro vario soni temperamento". 2 Giimm war nah der etymologischen Lösung des Infixes ־c v - der sog. u-Stämme: "Ich würde auch das Augment - o v auf dieses -u beziehen und z.B. s y n o v e , s y n o v von 81/яъ mit dem goth. s u n j u s , s ü n i v é oder Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access auf ъ lateinischen auf - и в , und a ltsl. auf ь lateinische auf - І 8 entsprechend finden." (XXXVIf.) auf ъ lateinischen auf - и в , und a ltsl. auf ь lateinische auf - І 8 entsprechend finden." (XXXVIf.) Abgesehen von der Nichtbeachtung der Liquidametathese im Aksl-, weshalb neben den aksl• die aruss. 3 Hervorhebung von V.B. 4 Grimm trennt die Termini Buchstabe - Laut nicht klar von einander• Formen ohne Metathese wie ѵ ь і к ъ und Ьъѵдъ stehen, und der Vermischung des einzelsprachlichen Wort ־ schatzes mit dem aksl• (vgl. nruss. к а т е п ъ , д и в ъ ) , was hinsichtlich des damaligen Standes der Slavistisk verständlich is t, enthalten Grimms Etymologien keine Fehler, sie sind allerdings z.T. unvoll ־ ständig. Der von Vostokov und Dobrovskÿ abgehandelten Vertretung der re ־ duzierten Vokale im Russischen fügt Grimm ihre Entwicklung im Serbi ־ sehen (und z.T. im Čechischen) hinzu; "Der Serbe drückt diese In ־ laute verschiedentlich aus: 1) Іъ oder ъI verschmilzt ihm in u, als ѵ ъ і к ъ , р і ъ п ъ , in v u k , p u n ; ebenso І ь , ъ і , als в і ъ г а in в и г а . 2) Vor oder nach r schreibt er gar keinen Vocal, z.B. t r g , t r n , s m r t für Ьъгдъ, Ь ъ т ъ , в т ъ г Ь ъ , obgleich die Illy re r t a r g , t a r n , s m a r t schrei- ben. 3) In allen anderen Fällen ersetzt er beide durch ein helles a : s a t , l a z , d a n , mac für altslav. ebtb, І ъ г , d b n b , т ъ с . - Der BÖh ־ me unterdrückt neben I und r den Vocal, als w l k , p l n y , s l z a , t r h , t r n , s m r t . Neben anderen Vocalen steht e t l e z , d e n , mec u.s.w." (XXXIV). Grimms Ausführungen, zusammengefaßt und in die heutige Termino- logie übersetzt, lauten: die aksl• ъ und ь sind die zu Halbvokalen reduzierten ursprünglichen Vokale и und г. Diese reduzierten Voka- le haben in starker Stellung einzelsprachlich verschiedene Entwick- lung zum Vollvokal genommen- In schwacher Stellung sind sie geschwun den, ь hat aber einzelsprachlich die Palatalität des vorausgehenden Konsonanten bewirkt. In der Erörterung der Bewahrung oder des Ver- lustes dieser Palatalität zeigt Grimm eine bemerkenswerte Kenntnis der Lautverhältnisse in den einzelnen slavischen Sprachen (XXXVII). domove von domb n it domus vergleichen, wenn nicht auch Masc. auf ь ein augmentatives - e v bekämen (in s titt. p. 468), z.B. k o n e v i Dat. Sing. von к о п ь •" (XXXV). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM 3 Hervorhebung von V.B. 4 Grimm trennt die Termini Buchstabe - Laut nicht klar von einander• Kopitar nannte Grimm einen "Riesen" und wandte sich an ihn auch mit slavistischen Fragen• So bat er ihn um sein "Votum über den wahr scheinlichsten Laut des von Vostokov hervorgehobenen g und ę und ih- rer mit J angelauteten Silben..." "non fuissem ausus, wenn ich nicht Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access wüßte, wie sehr Sie nicht nur das Ganze, sondern auch das Einzelne durchdringen: Beweis Ihre Erklärung unseres ъ und ь."* Diese slavi- stische Erkenntnis Grimms beruht auf seinen reichen Kenntnisse des Sprachmaterials aus verschiedenen Sprachen; das aus seiner Sprach- auffassung resultierende Interesse für den Prozeß des Formenzerfalls hat hier einen produktiven Niederschlag gefunden• wüßte, wie sehr Sie nicht nur das Ganze, sondern auch das Einzelne durchdringen: Beweis Ihre Erklärung unseres ъ und ь."* Diese slavi- stische Erkenntnis Grimms beruht auf seinen reichen Kenntnisse des Sprachmaterials aus verschiedenen Sprachen; das aus seiner Sprach- auffassung resultierende Interesse für den Prozeß des Formenzerfalls hat hier einen produktiven Niederschlag gefunden• Aus dem Bereich der serbischen Sprache greift Grimm zunächst die Verben auf und k ritis ie rt Vuks Einteilung der Verben in die Verbal- klassen nur nach dem Präsensstamm, die dazu geführt hat, daß in die zweite Klasse mit dem Themavokal в sehr unterschiedliche Verben ge- kommen sind. "Eine gute Aufstellung der Conjugation und jeder Flexion überhaupt ist mir nicht die, welche Ähnliches dem oberflächlich 1er- nenden Gedächtnisse zu Gefallen vereinigt, sondern die jede noch so kleine Abweichung ehrt." Er schlug vor, die -nuti-Verben in eine gesonderte Klasse zu trennen (XLIX). Der slavische Verbalaspekt war für Grimm besonders interessant und er entwickelte auch eigene Gedanken darüber. Seine Definition: " ... Erfüllung oder Fortwähren einer Handlung..." (L) klingt durch- aus modern in der Fülle der Definitionsversuche. Er erkannte darin ein morpho-syntaktisches Phänomen: "Die slavische Formenlehre kann also des Unterschieds zwischen Perfectivis und Imperfectivis nicht ganz entbehren, obgleich er strenge genommen die Flexion nichts an- geht und der Syntax zufällt." Den deutschen Leser wies er auf die Präfigierung und Ableitung { - n u t i ) als Mittel der Perfektivierung hin und die Fähigkeit der perfektiven Verben, die griech. Aoriste z.T. zu erreichen. Offenbar is t es ihm aber unklar geblieben, daß die perfektiven Präsensformen im Serbischen nicht das Futur bedeuten wie in den anderen slavischen Sprachen (LI). Vuk hat das zwar un- terstrichen, aber aus seiner Definition des p f. Aspekts ("... 1 Vgl. Vasmer, S. 48, 80. 3 Hervorhebung von V.B. 4 Grimm trennt die Termini Buchstabe - Laut nicht klar von einander• рока- zuju da je posao samo jedan put raden i svršen ( i l i da ce se raditi i s v rs iti). . . )״und aus seiner Benennung der an sich zeitlosen pf. Präsensformen als Präsens konnte Grimm eine analoge Möglichkeit wie im Russischen, Polnischen und Cechischen schließen. Grimm war einer der ersten Gelehrten, der sich dem Aspektpro- blem in der deutschen Sprache zuwandte; als Verfasser der "Deut- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access sehen Grammatik" wird er nicht ohne Einfluß auf die anderen Forscher gewesen sein. Er meinte: "Es is t nicht unmöglich Spuren eines die slavische Sprache so durchdringenden Unterschieds auch in der deut- sehen aufzufinden. Composita mit ver-, be-, hin-, durch- etc. (wie im Slavischen mit po-, do-, na- etc.) böten etwa Perfectiva dar, unzusammengesetzte dagegen Imperfectiva. Kopitar lehrt S. 310, daß auf die Frage: was machst Du? nicht mit dem Präsens eines Per- fectivums geantwortet werden könne. Danach prüfe man auch deutsche Verba. Wirklich is t uns fühlbar, daß von einem Sterbenden, Reisenden Lesenden, Bleibenden nicht gesagt werden dürfe: er verstirbt, verrei durchliest, verbleibt, sondern nur: er stirbt, reist, liest, bleibt. Wogegen es im Prät. unbedenklich heißt: er verstarb, verreiste, ver- blieb, durchlas? verstarb aber und starb unterscheiden sich wie im Serbischen umrije und mrije, d.h. man muß sagen: er sichte und starb gleichsam ein Leben lang; er verstarb gestern an seiner Krank ״ heit. Und wiederum spielt das deutsche Präsens in die Bedeutung des Futurums über: ich verreise morgen etc. Sobald in der Partikel ei- ne Abänderung des Sinnes liegt, hat das Präsens nichts Anstößiges, z.B. ich verachte." Er wies darauf hin, daß die alte deutsche Spra- che in diesem Punkt den slavischen ähnlicher gewesen sein könnte. Desgleichen stellte er eine Parallele zwischen der althochdeutschen und slavischen Sprache in der Unterscheidung zwischen Nom. und Akk. der Wörter für Lebewesen fest (XLf.). Grimm entwickelte auch eine eigene Theorie über die slavischen Adjektive- Er sah,entsprechend der von ihm aufgestellten starken und schwachen Adjektivdeklination in der deutschen Sprache, die kurze, im Nom. konsonantisch auslautende Form des slav. Adjektivs als die ursprüngliche ("starke") an, (wohl wegen der auch sonst herrschenden Genusunterschiede: K o n s o n a n t , -a, -o bzw. 1 Auf diesen Gedanken scheinen ihn Vuks Angaben für das Serbische ge bracht zu haben, wo das unbestimmte Adjektiv neben den nominalen auch pronominale Endungen hőiben kann, wobei der Akzent die Di- stinktion bewirkt. 3 Hervorhebung von V.B. 4 Grimm trennt die Termini Buchstabe - Laut nicht klar von einander• - e ), und die lange Form als die neuere ("schwache"), was auch z u trifft. Sein starkes Adjektiv wird aber nach der sog. bestimmten, das schwache nach der sog. unbestimmten Adjektivdeklination fle ktie rt (also: z u t , z u t o g a usw., z u t i , z u t a usw.)1 (XLII). In seiner Bemühung um die 1 Auf diesen Gedanken scheinen ihn Vuks Angaben für das Serbische ge bracht zu haben, wo das unbestimmte Adjektiv neben den nominalen auch pronominale Endungen hőiben kann, wobei der Akzent die Di- stinktion bewirkt. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Feststellung des ursprünglichen Sprachorganismus meinte Grimm, die slavischen Grammatiker könnten bei der Aufstellung der Adjektivdekli- nation "den Rumpf und das Haupt verwechselt haben" (XLI). Dobrovskÿ hatte zwar schon die auch heute als richtig geltende Erklärung der Adjektivdeklinationen geboten, Grimm fand sie aber nicht zufrie- densteilend.Sein Versuch einer neuen Deutung muß im Zusammenhang mit seiner Sprachauffassung gesehen werden. Er war bemüht, den Prozeß der Ausbildung von Flexionen in der Sprachgeschichte zu erfassen; deshalb stellte er zu Dobrovskÿs Deutung der bestimmten Adjektivde- klination durch Zusammenwachsen mit dem Pronomen die Frage nach dem- jenigen auf, woraus die pronominalen Flexionsendungen zu erklären sind. "Wer uns darauf antwortet, hat uns zugleich die Pronominalform und die adjectivische aufgeschlossen." (XLIV). Grimms Theorie zeichnet sich durch eine logische Gedankenführung aus. Kopitar antwortete auf die Bitte um seine Meinung, daß er sie "derweil mehr bewundern als beurtheilen kann". Seine zusätzlichen Angaben waren mehr verwirrend als klärend : "Wenn Sie noch dazu ahnen, daß die Deklination in g, s[g], nebst ago, omu etc. auch -aago, -uumu etc. hat (in Codd.), so bekämen Sie gar drey Formen." Er тип- terte Grimm auf, Dobrovsk^ ״was zu denken" zu geben. "Also ־da Ihre Gramm, ohnehin für höhere Liebhaber bestimmt is t, so überlassen Sie sich getrost Ihres Genius Eingebungen."1 Vuk hatte auch keine Einwände (Prepiska II, 34f.). Grimm selbst, der die ganze Arbeit an der Grammatik und der Vorre- 2 de innerhalb von dreieinhalb Monaten erledigen mußte, war wegen der eventuellen Fehler besorgt: "Schlimmer wirds mit den blössen stehen, die ich den Slavisten gebe. Wenn ich nur mit heiler haut da- von komme. Sie und Kopitar seid schuld daran, ihr habt mich dazu an- getrieben und mir doch keine zeit gelassen. Mit der zeit wäre ich weiser geworden." (Prepiska II, 44). 1 Vgl. Vasmer, S. 7. 2 Vuk war bei Grimm in Kassel zwischen dem 29. Sept. und 2. Okt. 1823; Grimm hat den letzten Teil des Manuskripts und die Vorrede zwischen dem 21. und 25• Januar 1824 abgeschickt (Prepiska I, 234; 11,35,39). 3 Vgl. Vasmer, S. 5. 4 Vgl. Vasmer, S. 6. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 3 Hervorhebung von V.B. 4 Grimm trennt die Termini Buchstabe - Laut nicht klar von einander• Grimms ursprünglicher Wunsch war, ein ganzes Jahr Serbisch zu studieren und erst dann die Arbeit an Vuks Grammatik zu unternehmen , aber für Vuk und Kopitar war die 4 Grammatikübersetzung ein "dringendstes Bedürfnis" . Man kann es be- 00051004 00051004 ־ 162 - dauern, daß sie es versäumt haben, einen solchen Wissenschaftler wie Grimm die serbische Sprache ein ganzes Jahr intensiv erforschen zu lassen. Das, was er in einer so kurzen Zeit geleistet hat, läßt auf fruchtbare Resultate schließen• dauern, daß sie es versäumt haben, einen solchen Wissenschaftler wie Grimm die serbische Sprache ein ganzes Jahr intensiv erforschen zu lassen. Das, was er in einer so kurzen Zeit geleistet hat, läßt auf fruchtbare Resultate schließen• 3• "Wuk's Stephanowitsch kleine serbische Grammatik verdeutscht und mit einer Vorrede von Jacob Grimm" - ein gemeinsames Werk der beiden Autoren 3• "Wuk's Stephanowitsch kleine serbische Grammatik verdeutscht und mit einer Vorrede von Jacob Grimm" - ein gemeinsames Werk der beiden Autoren 3• "Wuk's Stephanowitsch kleine serbische Grammatik verdeutscht und mit einer Vorrede von Jacob Grimm" - ein gemeinsames Werk der beiden Autoren Die Analyse hat gezeigt, daß die deutsche Fassung der serbischen Grammatik sehr stark von Grimm geprägt is t; obwohl Tirols Anteil an der Übersetzung unbestreitbar is t, is t die endgültige Gestalt der Grammatik ein Werk von Grimm• Die zahlreichen Ergänzungen, Kürzungen Umstellungen und Umformulierungen, die Grimm vorgenommen hat, ha- ben das Aussehen der Grammatik stark verändert• Es wurde ebenfalls gezeigt, daß von den nachgewiesenen Ergänzungen nur das Kapitel "Von den Partikeln" gemeinsam von Vuk und Grimm geplant wurde, wozu Vuk das sprachliche Material lieferte, das Grimm geordnet und übersetzt hat. Die übrigen Ergänzungen dagegen sind Grimms selbstän- diger Beitrag zu Vuks Grammatik. Die Kürzungen, Umstellungen und Umformulierungen hat Grimm, mit Vuks grundsätzlichem Einverständnis, ebenfalls selbständig durchgeführt. Das Resultat von Grimms Eingriff in Vuks Grammatik sind einer- seits die vollständigere Behandlung einzelner grammatischer Fragen, größere Übersichtlichkeit des dargestellten Stoffes durch eine ge- lungenere Komposition und die Beseitigung einiger methodischer Mängel, andererseits aber der Verlust einiger prinzipieller Gedanken Vuks. Die deutsche Fassung der Grammatik is t deshalb als ein ge- meinsames Werk von Vuk und Grimm zu bezeichnen. Grimms Anteil an Vuks Grammatik und seine Vorrede haben den wis- senschaftlichen Wert dieses Werkes erhöht und einen wichtigen Bei- trag zu seiner Verbreitung und Wirkung geleistet• Kopitar schrieb an Grimm, die Grammatik werde "verschlungen"; obwohl sie nicht im voraus angezeigt war, suche man sie "de fama"; erst durch Grimm sei Vuks Grammatik "en vogue" und der Verleger könne mit dem Absatz zufrieden sein. Noch am 6. September 1826 meldet er Grimm: "Ihre Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־־ 163 - Serb. Grammatik hat soeben Bowring von hier bestellt!" Die Wirkung der Grammatik von Vuk und Grimm wird besonders durch die Grammatik von I. A. Brlic veranschaulicht.In seiner Vor- rede schreibt Brliò, daß er Vuks Grammatik und Wörterbuch von 1818 Wuk י s Stephanowitsch И aus Unkenntnis abgelehnt habe, bevor ihm kleine serbische Grammatik von Jacob Grimm... in die Hände kam..." Er habe "einen reichlichen Gebrauch davon machen können und ge- 2 macht." In Brlics Grammatik is t der Einfluß der "Kleinen serbischen Grammatik" auch feststellbar, sowohl in der Komposition als auch in den angeführten Beispielen. 1 Vgl. Vasmer, S. 23, 25, 29, 46. 2 Vgl. I. A. Brlic, Grammatik der Illyrischen Sprache, wie solche in Boßnien, Dalmazien, Slawonien, Serbien, Ragusa etc. von den Illyrie rn in Banat und Ungarn gesprochen wird. Ofen 1833, S. V llf. 3 Vgl. ebd., S. 82f. 4 Vgl. ebd. S. 147ff. -Ein eingehender Vergleich der beiden Gramma- tiken, der zu unseren Aufgaben nicht gehört, würde wahrscheinlich viele zusätzliche Gemeinsamkeiten feststellen• 3• "Wuk's Stephanowitsch kleine serbische Grammatik verdeutscht und mit einer Vorrede von Jacob Grimm" - ein gemeinsames Werk der beiden Autoren In seiner Darstellung der Deklina- tion der Zahlen z.B. werden die einzelnen Probleme in derselben Reihenfolge wie bei Vuk behandelt^. In der Darstellung der Pro- nomina erscheinen Vuks Beispiele, wie aus der folgenden Gegenüber- Stellung zu ersehen is t: Brlic S. 243: vino shto 1smo (shto ,smo да oder koje ,smo) p ili nôx shto ,sam (shto *sam да oder koga ,sam) izgubio pero shto se pìshe (s* njim) brùs, shto se sykira oshtri (s* nj im) Vuk S. LUI: vino, sto smo (да) p ili noz, sto smo danas kupili pero, sto se (njim) piše brus, što se britva ostri Brlic S. 243: vino shto 1smo (shto ,smo да oder koje ,smo) p ili nôx shto ,sam (shto *sam да oder koga ,sam) izgubio pero shto se pìshe (s* njim) brùs, shto se sykira oshtri (s* nj im) Brlic gibt auch eine "Bildungs-Tafel" mit den Personalendungen der Tempora, die vermutlich durch den entsprechenden Zusatz von 4 Grimm angeregt worden is t . Das gemeinsame Werk von Vuk und Grimm hat seine Wirkung als eine Ganzheit ausgeübt. 4 Vgl. ebd. S. 147ff. -Ein eingehender Vergleich der beiden Gramma- tiken, der zu unseren Aufgaben nicht gehört, würde wahrscheinlich viele zusätzliche Gemeinsamkeiten feststellen• Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access II• Grimms und Vuks Plan einer erweiterten Auflage der serbischen Grammatik Grimms Beschäftigung mit Vuks Grammatik f ä llt in die Zeit der Arbeit am zweiten Teil seiner "Deutschen Grammatik". Es is t kein Wunder, daß Vuks Grammatik ihm zu klein erschien und er sie in dem Titel auch so bezeichnete• Er fand sie auch unvollständig; in der Anzeige schrieb er: "Ein werk für den ersten anlauf, nicht einmal vollständige darstellung der serbischen formenlehre, aber aus unge- trübter quelle geschöpft•" (GKS IV, 225). Grimm schätzte Vuks Kennt- nisse der Volkssprache und war der Meinung, daß Vuk eine umfangrei- chere Grammatik ausarbeiten sollte• Gleich nach Erscheinen der kleinen Grammatik schrieb er an Vuk: "Ich hoffe, daß Sie unablässig an erweiterung der Grammatik arbeiten, damit sie bei einer neuen aufl. noch einmal so stark wird; dafür kann die vorrede sehr ge- kürzt werden..• Sammeln Sie doch besonders alle serb. ableitungs- Silben recht vollständig." (Prepiska I I , 49)• Grimm hatte offenbar die Vorstellung von einer vollständigen Darstellung der Morpholo- gie in der neuen Grammatik. Vuk war für alle Anregungen von seiten Grimms empfänglich und antwortete:: "Ich möchte noch einmal nach Ihrem schönen Cassel kom- men und bei Ihnen etliche Monate bleiben; dann würden wir die ser- bische Grammatik erweitern. Kann ich aber so glücklich nicht seyn, dann werde ich Ihnen etwas schicken, und das übrige werden Sie tausendmahl besser machen wie ich." (Prepiska II, 50)• Grimms In- teresse für die serbische Sprache hat während der Arbeit an der Grammatik zugenommen; am 2• November 1823 schrieb er an Vuk:" Wenn ich das serbische mehr studiert habe (was sicher geschehen soll) hoffe ich studiertere Dinge darüber vorzubringen." (Prepiska II, 10) Vuks Idee von einer gemeinsamen Ausarbeitung der serbischen Gramma- tik hat offenbar Anklang bei ihm gefunden; er hat angefangen, Material dazu zu sammeln. Bekanntlich is t es zu einer Neuauflage der serbischen Grammatik nicht gekommen. Die Vorbereitungen, die Vuk und Grimm unternommen haben, sind aber bisher nicht untersucht worden. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 165 ־ 1 Vgl. Vasmer, S. 43. 1. Vuks Vorbereitungen für die erweiterte Auflage seiner Grammatik Vuk hat sogleich angefangen, Materialien für die neue Grammatik vorzubereiten. Kopitar berichtete Grimm darüber: 1*Vuk arbeitet an einer umständlichen serb. Grammatik, wo Sie dann alle Beispiele bei- sammen finden werden.” * Die Verzögerung der Grammatikausarbeitung in den darauffolgenden Jahren is t offensichtlich auf Vuk zurückzu- führen. Er schrieb 1828: "...koje razlicni drugi poslovi, koje že- Ija i nadežda, da obidem najprije sve predjele naroda Srpskoga, jednako me još zadržavaju, da je svršim i na svije t izdam." (GPS II , 292). Vuks gewissenhaftes Verhalten gegenüber der Volkssprache, die er für seine neue Grammatik besser erforschen wollte, is t einleuch- tend. s Arbeit zur Wortbildung der Substantive und Adjektive a. Vuks Arbeit zur Wortbildung der Substantive und Adjektive Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access a. Vuks Arbeit zur Wortbildung der Substantive und Adjektive Zu Vuks Vorbereitungen der erweiterten Grammatik gehört seine Schrift "Glavna svršivanja suštestvitelni i prilagatelni imena u srpskom jeziku", die er in der "Danica” 1828 veröffentlicht hat (GPS II, 292 - 370). Sie is t auf Grimms Anregung, "Ableitungssilben" zu sammeln, entstanden; Vuk selbst bestätigt das in der Einleitung. Bei der Beurteilung dieser Arbeit muß zunächst ein terminologi- sches Problem gelöst werden. In seinem Titel spricht Vuk über die "svrsivanja"; seine Arbeit war die erste auf diesem Gebiet und er konnte, wie auch sonst, nicht auf eine ausgearbeitete Terminologie zurückgreifen. Wenn wir an Stelle dieses seines Terminus die moder- nen Termini "Suffixe" oder "Bildungsmorpheme" in der serbischen Sprache setzen wollten, gerieten wir in einen Widerspruch mit den von Vuk angeführten Beispielen fremdsprachiger Herkunft, wie z.B. I b a r (ON, wahrscheinlich illy r . ) , P e t - a r , M i t - a r (griech. Lehnw.), b u z d o v - a n , b a l v - a n (Turzismen), m a n a s t - i r (lat. Lehnw.), (GPS II, 294, 303, 307f.), wo es sich gar nicht um Ableitungen mit den serbi- sehen Suffixen handelt. Diese Wörter "enden" aber so wie die abge- leiteten serbischen Wörter. In seiner Einleitung erläutert Vuk, daß die Wörter in "korene, proizvodne i složene" eingeteilt werden. Zur Kategorie der Ablei- tungen (proizvodne) führt er zwei Gruppen an: zur ersten Gruppe ge- hören solche, die nur mit einem Vokal abgeleitet seien, wie z.B. z e n - a t p o l j - e usw. Hier trennt Vuk die Wortbildung und Flexion nicht voneinander. Danach würden die Maskulina wie g r a d , g r a d - a usw. ab Genitiv auch zu den abgeleiteten Wörtern zählen, denn z e n - а is t auch nur in der Korrelation mit z e n - e usw. zu sehen. Zur zweiten Gruppe der Ableitungen rechnet er solche, die mit ganzen Silben (citavim slogovima) abgeleitet sind, mit denen er sich in seiner Arbeit beschäftigen w ill (GPS I I , 292f.). Bei dieser Definition is t Vuk ein Fehler unterlaufen, der offensichtlich von Grimms Terminus "Ableitungssilben" beeinflußt is t. 1 Vgl. Vasmer Bausteine I, 26 a. Vuks Arbeit zur Wortbildung der Substantive und Adjektive Vuks "svršivanja" stellen keine Silben in der serbischen Sprache dar, seine Beispiele zeigen es deutlich: z n a n - a c , j u n - a k , k o 8 - a c , b a l - 0 usw. (ebd., 296, 297, 297, 336). (Die Silben sind: z n a - n a c , j u - n a k , k o - 8 a c , b a - l o ) . Vuk hat außerdem in seine Darstellung der Ableitungen viele Wurzelwörter aufgenommen, z.B. g r a d , r o d , g o v o r , t r k usw., (sie seien aus der Verbalwurzel gebildet; ebd., 344ff.). In einem Brief an Grimm sagt er zwar, einige Wurzelwörter "wegen ihre(r) Endungen mit abgeleiteten vermischt” zu haben1; offensichtlich besaß er doch nicht genügend Einsicht in diese Problematik. Er führt z.B. folgen- de Wörter als mit - h abgeleitet an (das h schreibt er zu dieser Zeit nicht): V i a , o r a , p r a , a t r a usw. Diese Wörter sind aber durch Vuks damalige Auslassung des h nur noch ein Rest der ursprünglichen Wurzel und keine Ableitungen. Mit - a j seien abgeleitet: z m a j , r a j , k r a j usw.j mit - e l j : m - e l j (hmelj) usw. (ebd., 305). Vuks "svršivanja" sind dennoch in den meisten Fällen Suffixe, und wir werden diesen Terminus verwenden mit der Einschränkung, die sich aus dem Gesagten ergibt. Abgesehen von den methodischen und einigen sachlichen Fehlern, (zu den angeführten sei noch v i j e n - a c genannt, das vom Prt. Pass, abgeleitet sein soll, ebd., 296), enthält Vuks Arbeit viele rich ti- ge Feststellungen und reiches Material zur Wortbildungslehre. Er führt die nach formalen morphologischen Kriterien festgestellten Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 167 ־ Suffixe auf, zieht aber bei der Aufzählung von Beispielen auch se- mantische Kriterien heran. Er gibt dabei viele feine Beobachtungen, in denen er sich, wie auch sonst, als ein ausgezeichneter Kenner der serbischen Volkssprache zeigt. So sind z.B. 1 Lj. Stojanovic, a.a.O., S. 314, hat u.W. als einziger eine knappe, im wesentlichen richtige Beurteilung dieser Schrift Vuks gegeben. 2 B. Daničic, Osnove srpskoga i l i hrvatskoga jezika, 1876. 3 A. Fick, Vergleichendes Wörterbuch der Indogermanischen Sprachen, Bd. I, 1874. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access a. Vuks Arbeit zur Wortbildung der Substantive und Adjektive seine Ausführungen über die Ableitungen auf - a l o , bei denen er die Bedeutung der nomi- na agentis (b e n e t a l o , n a k l a p a l o usw.), des Mittels (g u d a l o , d r z a l o usw.) und des Ortes der verbalen Handlung (s j e d a l o , c j e p a l o usw.) fe stste llt (ebd., 331f.). Der Hinweis, daß m o t o v i l o aus zwei Verben { m o t a t i und v i t i ) entstanden und daß ѵ т а д - 0 1 - а п (und die übrigen Ab- leitungen zu v r a g ) analogisch zu Ü a v o l a n , (wegen der semantischen Nähe der beiden Wurzelwörter Üavo und v r a g ) , gebildet sind, zeigt Vuks Fähigkeit, bei der Betrachtung der sprachlichen Struktur ver- schiedene Aspekte heranzuziehen• Vom heutigen Standpunkt aus gesehen is t diese Arbeit von Vuk überholt*. Sie muß aber im Zusammenhang mit der damaligen Slavistik, insbesondere der Erforschung der serbischen Sprache gesehen werden. Wenn uns heute Vuks Darstellung überwiegend als eine Aufzählung von Suffixen erscheint, müssen wir uns daran erinnern, daß er als erster diese Suffixe in der serbischen Sprache festgestellt und verzeichnet hat; das allein war schon ein Gewinn für die serbische Sprachfor- schung, die vielen Angaben aus der Semantik kommen hinzu. Vuk hat, wie er in der Einleitung selbst angibt, für seine neue Grammatik die Grammatiken von Dobrovsk^ zum Vorbild genommen. Do- brovsk^s "Institutiones” bieten in dem Kapitel zur Wortbildungslehre auch eine Aufzählung der Suffixe. Allerdings hat er in seine Arbeit verschiedene aksl., serb.-ksl. und russ.ksl. Denkmäler einbezogen; das gibt seinem Werk jene wissenschaftliche Vielseitigkeit. Vuk dagegen nahm nur die lebendige Volkssprache als Quelle. Auch Daničic, der bessere Fähigkeiten im wissenschaftlichen Sy- 2 stematisieren als Vuk besaß, hat mit seinem Werk "Osnove" keinen großen Fortschritt im Vergleich mit Vuk gebracht. Daničic is t in Anlehnung an das bereits überholte Wörterbuch von Fick^ oft von den konstruierten asl. Wurzeln ausgegangen, was den Wert seiner Arbeit beeinträchtigt hat. ־ 168 ־ Eine diachronisch-synchronische Erforschung der serbischen Suffix« is t viel später erfolgt. A. a. Vuks Arbeit zur Wortbildung der Substantive und Adjektive Belic1 hat sie in seiner Studie über die Entwicklung der slavischen Deminutivsuffixe besonders unter dem se- mantischen Aspekt behandelt. R. Boškovic verfolgt u.a. ihre histo- risch-funktionale Entwicklung innerhalb der südslavischen Sprachge- meinschaft• 1 A. Belic, Zur Entwicklungsgeschichte der slavischen Deminutiv- und Amplificativsuffixe. In: AfslPh 23, 1901, S. 134 - 206. 2 R. Boškovic, Razvitak sufiksa и južnoslovenskoj jezičkoj zajednici In: JF 15, 1936, S. 1 - 155. 3 Vgl. Archiv SANU 8552/30(9). 4 Bisher hat и.W. nur Lj. Stojanovic, a.a.O., S. 416, ein knappes, ohne vorherige Analyse getroffenes, negatives Urteil über diese Ar beit abgegeben: sie zeige, daß Vuk dieser Aufgabe nicht gewachsen war. 5 Vuks Materialien über die Verben werden im Archiv SANU unter der Sign. 8552/11-12 aufbewahrt. 6 P. Bordevic hat später diese Arbeit für den Druck in dem nicht er- schienenen IV. Bd. der"Gramatički i polemički spisi" vorbereitet 1 dabei die fehlende Numerierung, u.E. nicht immer in Vuks Sinne, hi zugefügt; außerdem hat er in Vuks Manuskript eigene Anmerkungen ur Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 A. Belic, Zur Entwicklungsgeschichte der slavischen Deminutiv- und Amplificativsuffixe. In: AfslPh 23, 1901, S. 134 - 206. 2 R. Boškovic, Razvitak sufiksa и južnoslovenskoj jezičkoj zajednici In: JF 15, 1936, S. 1 - 155. 3 Vgl. Archiv SANU 8552/30(9). 4 Bisher hat и.W. nur Lj. Stojanovic, a.a.O., S. 416, ein knappes, ohne vorherige Analyse getroffenes, negatives Urteil über diese Ar beit abgegeben: sie zeige, daß Vuk dieser Aufgabe nicht gewachsen war. 5 Vuks Materialien über die Verben werden im Archiv SANU unter der b. Vuks Arbeit über die Verben b. Vuks Arbeit über die Verben Die zweite, auf Grimms Anregung entstandene Arbeit von Vuk, die in den Rahmen seiner Vorbereitungen für die erweiterte Auflage der serbischen Grammatik gehört, b e trifft die Verben. Vuk hat hauptsäch- lieh 1832 daran gearbeitet (Prepiska I, 444), is t aber auch später mit dieser Arbeit beschäftigt gewesen. In seiner Anzeige der zweiten Auflage der Sprichwörter vom 8. Januar 18493 nennt er zehn Arbeiten, die er noch herausgeben wollte, darunter: M8. Naše glagole razraflene od p rilike onako kao sust. i p r il. imena и Danici za godinu 1828." Die Menge an begonnener Arbeit hat ihn an der Durchführung dieses Vorhabens gehindert.Vuks Arbeit über die Verben is t unveröffentlicht 4 und bisher auch unbewertet geblieben . Diese Arbeit von Vuk is t kein druckfertiges Manuskript, sondern eine Sammlung von Notizen. Sie enthalten eine angefangene, nicht be- endete Vorrede, eine Darstellung der Verben auf -a m , -em in der 1. Pers. Sg. Präs., ein gesondertes Manuskript über die Verben auf - i m und ein unvollständiges alphabetisches Verzeichnis der pf. und ip f. Verben (g - z , p r i - 0 )5. Die einzelnen Punkte in seiner Dar- Stellung sind numeriert, jedoch nicht kontinuierlich; dazwischen be- finden sich oft Notizen über das noch zu berücksichtigende Material6 3 Vgl. Archiv SANU 8552/30(9). 4 Bisher hat и.W. nur Lj. Stojanovic, a.a.O., S. 416, ein knappes, ohne vorherige Analyse getroffenes, negatives Urteil über diese Ar beit abgegeben: sie zeige, daß Vuk dieser Aufgabe nicht gewachsen war. 5 Vuks Materialien über die Verben werden im Archiv SANU unter der Sign. 8552/11-12 aufbewahrt. 5 Vuks Materialien über die Verben werden im Archiv SANU unter der Sign. 8552/11-12 aufbewahrt. 6 P. Bordevic hat später diese Arbeit für den Druck in dem nicht er- schienenen IV. Bd. der"Gramatički i polemički spisi" vorbereitet 1 dabei die fehlende Numerierung, u.E. nicht immer in Vuks Sinne, hi zugefügt; außerdem hat er in Vuks Manuskript eigene Anmerkungen ur чпплг Hlnwpisp für den Setzer einaetraaen. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vuk unternimmt in dieser Arbeit eine Klassifikation der serbi- sehen Verben vom Standpunkt der Wortbildungslehre aus. Er nimmt die 1. Pers. Sg. Präs. zum Ausgangspunkt und wendet dabei überwiegend for male morphologische Kriterien an. b. Vuks Arbeit über die Verben Unter diesem Aspekt gesehen, gehö- ren bei ihm die Verben im allgemeinen nicht zur Kategorie der Wurzel- Wörter, da sie immer eines der Flexionskennzeichen aufweisen, die er zu den Ableitungsmorphemen rechnet. Deshalb erscheinen in seiner Klassifizierung, neben den aus Substantiven, Adjektiven usw. abge- leiteten Verben, auch solche, die aus "Silben ohne Bedeutung" abge- le ite t seien (z.B. a r - a m , b a c -a m usw.), bei denen es sich Überwie- gend um die Wurzelverben handelt. Innerhalb der Einteilung in die drei großen Gruppen: -a m , -em , - i m , hat Vuk zahlreiche Unterteilungen vorgenommen, die sich auf rieh tigen Beobachtungen und reichhaltigem Material gründen. Das is t ins- besondere der Fall bei der Gruppierung der sekundären verbalen Ab- leitungen, wo Vuk die Ableitungsverhältnisse sowohl in Bezug auf die Alternation der Konsonanten und Vokale als auch der Akzente beobach- tet und Kategorien vom Typ: nadm-em 8e : n a d im -a m se (mit der Alter- nation 0 : i ) und d o g o v o r - i m 8e : d o g o v a r - a m 8e (mit dem Ablaut о : с in der Wurzel und dem gleichzeitigen Wechsel der Konjugationsklasse) feststellt; viele richtige Beobachtungen des Konsonantenschwundes enthalten seine Ausführungen über die abgeleiteten pf. - n e m / - n u t i - Verben, wie z.B.: v r - n e m , o k r e - n e m usw. (t ; 0 ); z a k i - n e m usw. (d : í k a - п е т usw. (p ; 0 ). (Bei v i - n e m , l i - п е т , k i - n e m , b a - п е т is t Vuks Annahme der Alternation j : 0 , bzw. h : 0 nicht zutreffend). 1 Vuks Bezeichnung der prosodischen Merkmale wird hier wiedergegeben, b. Vuks Arbeit über die Verben Ein sy- stematisches Erfassen der Ableitungsverhältnisse zeigt seine Gruppie- rung der Verben: - ò z i m / - ò z i t i : - azem/ - â g a t i (o b l o z - i m : o b l a z - e m ) - ò c i m / - ò c i t i : - a c e m / - â k a t i (z a m o c -im : za m a c-em ) - ê g n e m / - ê g n u t i : - ê z e m / - ê z a t i { n a p r e g n - e m : n a p r e z - e m ) , wo neben der lautlichen auch eine Alternation des Akzents besteht. Vuk zieht in seiner Darstellung auch sonst prosodische Merkmale heran, die zuweilen eine distinktive Funktion haben, z.B.; z à g le d - a m 8 e , z à g l e d a t i : z a g l e d - a m , z a g l é d - a t ' , Ein weiters Kriterium seiner Einteilung in Untergruppen sind die Konsonantenalternationen zwischen dem Präsens- und Infinitivstamm, worauf verschiedene Typen von Verben beruhen: -ce m : - k a t i (v i c - e m 1 Vuks Bezeichnung der prosodischen Merkmale wird hier wiedergegeben, konsonantischen Verben hat Vuk innerhalb der Gruppen -em : - s t i ; - em : - b i mehrere Unterteilungen vorgenommen: -bem : - s t i (g re b - e m : g r e p - s t i ) ; - dem : - s t i (b o d -e m : b o - s t i ) ; -zem : - s t i (v e z -e m : v e - s t i ) ; - sem : - s t i (p a s -e m : p a - s t i ) . Eine Aufstellung der Hegel zur Konsonantenassimilation hätte die Gegenüberstellung der beiden letzten Gruppen überflüssig gemacht. Die - n e m / - n u t i - V e rben, deren Einordnung zur zweiten Konjugations־ klasse in der Grammatik Grimm in seiner Vorrede k ritis ie rt und deren Trennung in eine besondere Klasse er vorgeschlagen hatte, werden hier als eine besondere Gruppe behandelt und in Untergruppen mit doppel- ten Infinitivformen eingeteilt: - n u t i / - s t i (d e n -e m , ü e n - u t i / ü e - s t i ) ѣ, - n u t i / - b i (v r g - n e m , v r g - n u t i / v v - b i ) . Es is t im allgemeinen festzu- stellen, daß diese Arbeit von Vuk, verglichen mit seiner Grammatik von 1818, ein reichhaltigeres Material bietet und ein besseres Sy- stem in der Darstellung der Verben zeigt. In seiner Klassifikation zieht Vuk neben den morphologischen stellenweise auch semantische Kriterien heran und s te llt fest, daß die verbalen Ableitungen vom Typ - k a m : b a c - k a m / b o c - k a m zu bod-em , l u p - к а т zu lu p - a m , t r c - k a m zu t r c - i m usw. eine deminutivierende Be- deutung haben. 1 Vuks Bezeichnung der prosodischen Merkmale wird hier wiedergegeben, Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 170 ־ v i k a t i ) ; - oem : - c a t i { d o t i c - e m : d o t i c - a t i ) ; -sem : - s a t i (b i t i s - e m b i t i s - a t i ) ; - bem : - t a t i ( k r e b - e m : k r e t - a t i ) ך - a t e m : - s k a t i (i s t - e m und i s b - e m : i 8 k ~ a t i ) . Die Verben, die zu zwei verschiedenen Kon- jugationsklassen gehören, werden als solche berücksichtigt: -am, - I j e m : - a t i (g i b - a m , g i b - l j e m : g i b - a t i ; d o z i v - a m , d o z i v - l j e m ; d o z i v - a t i ) ; ebenfalls werden die aus der e-Vertretung resultierenden Doppelformen der - (j ,Jett-Verben (sm-em, 8 m - e t i : s m i - j e m , s m - j e t i usw ziemlich vollständig angeführt. Die Gruppe von Verben mit der Alter- nation V o k a l : 0 ( b e r - e m : b r - a t i , z o v -e m : z v - a t i usw) wird aus- führlich behandelt. Bei den sog. 1 Vuks Bezeichnung der prosodischen Merkmale wird hier wiedergegeben, Perfektivierungsvorgang deutlich werden, z.B.: a m e s t i : a m ê t a t i ; s p r e m i t i : a p r e m a t i ; a p o m e n u t i : s p o m i n j a t i ; a r o d i t i ae : a r a t a t i s e ; s t u k a t i : s t u k n u t i ; a u a t a t i : s u s t a j a t i ; a u s t i ö i : s u s t i z a t i ; 8 u - a r e a t i : a u a r e t a t i usw. sie durch die Imperfektivierung der p f. Verben entstanden sind; bei den -rtuti-Verben gibt er die wenigen ip f. an. In seinem alphabeti- sehen Verzeichnis der pf. und ip f. Verben sind zahlreiche Unter- Streichungen vorhanden, durch die einerseits die Aspektpaare, ande- rerseits die Ableitungsverhältnisse bei dem Imperfektivierungs- bzw. Perfektivierungsvorgang deutlich werden, z.B.: a m e s t i : a m ê t a t i ; s p r e m i t i : a p r e m a t i ; a p o m e n u t i : s p o m i n j a t i ; a r o d i t i ae : a r a t a t i s e ; s t u k a t i : s t u k n u t i ; a u a t a t i : s u s t a j a t i ; a u s t i ö i : s u s t i z a t i ; 8 u - a r e a t i : a u a r e t a t i usw. Vuks Arbeit weist auch viele Mängel auf. Es wurde bereits auf seinen unklaren Begriff des Suffixes hingewiesen1. Seine Definitio- nen sind oft sehr unbeholfen. Es sind ihm aber auch sachliche Fehler unterlaufen, wie z.B. 1 Vuks Bezeichnung der prosodischen Merkmale wird hier wiedergegeben, Bei den - u j e m / - o v a t i - V e rben, die von Substantiven abgeleitet sind, s te llt er die folgenden semantischen Kategorien auf: jemand sein (a j d u k - u j e m , a j d u k - o v a t i ; r o b - u j e m , v o b - o v a t i usw.), Zeit verbringen oder ein Fest feiern (b o z i b - u j e m , b o z i b - o v a t i ; I j e t - u j e m , I j e t - o v a t i usw.) oder sonst etwas (in der Bedeutung des Substantivs Enthaltenes) tun (a s i k - u j e m , b o l - u j e m , d a r - u j e m usw.). Bei den - u j e m / - i v a t i - V e r b e n gibt Vuk den richtigen Hinweis, daß Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 171 ־ sie durch die Imperfektivierung der p f. Verben entstanden sind; bei den -rtuti-Verben gibt er die wenigen ip f. an. In seinem alphabeti- sehen Verzeichnis der pf. und ip f. Verben sind zahlreiche Unter- Streichungen vorhanden, durch die einerseits die Aspektpaare, ande- rerseits die Ableitungsverhältnisse bei dem Imperfektivierungs- bzw. 1 Aus Vuks Definition der Ableitungen vom Typ b a c -a m ergibt sich die Schlußfolgerung, daß solche Verben durch die Flexionsendung -am überhaupt erst eine Bedeutung erhalten. 2 Vgl. Archiv SANU 8552/30(9). 1 Auf dieses Material in dem "durchschossenen" Handexemplar hat als erster M. Mojaseviè״ Grimov Drevod i orimerak... a.a.O. hinqewie- sen und die drei auf dem Titelblatt befindlichen Notizen er- läutert. Vgl. auch ders., Einleitung. In: Vuk Stefanovic Karadzic, Kleine serbische Grammatik.neu hg. und eingel. von M. Mojaševic und P. Rehder,^1974, S. 10-23. - Vgl. zu diesem Thema auch P. Reh- der, Grimov lieni primerak prevoda Vukove Srpske gramatike. In: Naučni sastanak 4,2, 1974, S. 277-282. - - 1 Vuks Bezeichnung der prosodischen Merkmale wird hier wiedergegeben, die Annahme einer Alternation e : i statt 0 : i bei nadm-em : n a d im -a m a e . Unter den Ableitungen auf - kam be- finden sich einige dazu nicht gehörende, onomatopoetische Verben vom Typ g u - c e m , g u k a t i . Vuks Notizen über die Verben enthalten überwiegend richtige Be- obachtungen und reiches Material. Es is t u.E. als ein Verlust anzu- sehen, daß er sie nicht ausgearbeitet und veröffentlicht hat. Sie hätten zweifellos für die damalige Forschung auf diesem Gebiet ei- nen Schritt vorwärts bedeutet. Es is t interessant zu sehen, daß Vuk diese ArWêit im ekavischen Dialekt geschrieben, die Vorrede jedoch im ijekavischen angefangen hat; auch darin is t die Entwicklung seiner Sprachkonzeption fest- stellbar. Die Vorrede is t später entstanden (Vuk schreibt bereits das h ) , vermutlich in den 40er Jahren. Vuk hatte nämlich seinen Plan von der Herausgabe einer neuen Grammatik noch 1849 nicht auf- gegeben. In der bereits erwähnten Anzeige der Sprichwörter schrieb er: "9. I po treći put gramatiku našega jezika, ako ne onaku kao sto sam negda mislio, a ono barem štogod vecu i bolju od one dru- 2 ge.” Zu dieser Zeit hatte Vuk offensichtlich nicht mehr die Vor- Stellung von einer Zusammenarbeit mit Grimm bei der Anfertigung der neuen serbischen Grammatik. 1 Aus Vuks Definition der Ableitungen vom Typ b a c -a m ergibt sich die Schlußfolgerung, daß solche Verben durch die Flexionsendung -am überhaupt erst eine Bedeutung erhalten. 2 Vgl. Archiv SANU 8552/30(9). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 2 Vgl. J. Grimm, Deutsche Grammatik, 1., 1819 ( 1822, 1840); 2., 1826; 3., 1831; 4., 1837. Grimm hat eine Überarbeitung geplant und viel Material angesammelt, das in der von W. Scherer, G. Roethe und G. Schröder besorgten Neuauflage (1. 1870 und 1893; 2. 1878; 3. 1889; 4. 1898) abgedruckt wurde. 2. Jacob Grimms Materialien in seinem Handexemplar der "Kleinen serbischen Grairanatik" Nach der Beschäftigung mit Vuks Grammatik setzte Grimm seine zeitweise unterbrochene Arbeit an der "Deutschen Grammatik" fort. Er hörte aber nicht auf, sich mit der serbischen Sprache zu beschäf- tigen, obwohl von ihm nach der "Vorrede" keine Sprachwissenschaft- liehe Arbeit zur Serbistik erschien• Sein Handexemplar der "Kleinen serbischen Grammatik" enthält viele handschriftliche Eintragungen des sprachlichen Materials1, das er vermutlich für die neue, erwei- terte Auflage der serbischen Grammatik gesammelt hat. Dieses Material hat Grimm zum größten Teil in seine "Deutsche Grammatik" sowohl un- ter vergleichendem als auch typologischem Aspekt aufgenommen. Grimms Notizen im Handexemplar beziehen sich auf verschiedene grammatische Probleme. Sie lassen einige Schlüsse über seine Vor- Stellung von der neuen serbischen Grammatik und über seine Kenntnis- se der serbischen Sprache zu• Unter ihnen befinden sich aber auch einige, die offenbar nicht zu seinem Grammatikplan gehörten. Das sind vor allem die Anmerkungen, die Vuk und die Rezensionen seiner Werke betreffen (Anh. II, S. 230 ). Grimm vermerkt außer seinen eigenen und den Rezensionen Kopitars die 1824 im "Morgenblatt" er- schienene, lobende Besprechung von J. V. Adrian und die in der "Allgemeinen Zeitung" 1839/40 von P. Adamovic und Kopitar geführte Polemik über Vuks Autorschaft an der Grammatik"*. In Grimms Bemerkung: "die Übersetzung der grammatik is t von Tirol" liegt u.E. der Nach- druck auf dem Namen von Tirol, der ihm bis zu dieser Zeit wohl unbe- kannt war. (Er sprach von Tirol immer als von einem "Ungarn"). , , , 2 Vgl. J. Grimm, Deutsche Grammatik, 1., 1819 ( 1822, 1840); 2., 1826; 3., 1831; 4., 1837. Grimm hat eine Überarbeitung geplant und viel Material angesammelt, das in der von W. Scherer, G. Roethe und G. Schröder besorgten Neuauflage (1. 1870 und 1893; 2. 1878; 3. 1889; 4. 1898) abgedruckt wurde. 3 Vgl. Mojaševic, Grimov prevod i primerak.., S. 165ff. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Von besonderem Interesse is t Grimms Hinweis auf zwei Artikel über Vuk im "Literarischen Conversationsblatt" von 1824, von denen einer durch Böttiger gezeichnet war und der andere, anonyme, nach Grimms Meinung von Kruse1 stammte. Sie gehören zu den ersten Berichten über Vuk, die einem breiten Lesepublikum zugedacht waren, denen später in der zweiten Hälfte des 19, Jhs. die Aufnahme von Vuk in die füh- 2 renden Enzyklopädien und Konversationslexika folgte . In dem Aufsatz von Böttiger wird Vuk ein Apostel der serbischen Literatur und Kultur genannt. 1 Vgl. dazu und im folgendenden die Zusammenstellung von Grimms Quellen im Anhang II, S.230ff. 2 Zu Vuk-Rezeption in Deutschland vgl. H. Peukert, О prihvatanju Vuka Stefanovica Karadzića u drugoj poiovini XIX veka и Nemačkoj. In: Naučni sastanak 3, 1973, S. 309 - 321. 3 Vgl. Literarisches Conversationsblatt Nr. 98, 1824, S. 389. 4 Vgl. ebd., S. 390. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vielleicht stehen diese Notizen im Zusammenhang mit der von der Gö tinger Gesellschaft der Wissenschaften 1835 ausgeschriebenen Preis aufgabe, eine "Geschichte der slavischen Stämme" zu schreiben, "welche sich ehemals im Osten und Norden des jetzigen Deutschlands bis zur Elbe, Saale und Rednitz erstreckten, allmählich aber be- siegt und germanisiert wurden."- Vgl. Gött. Gel. Anz., Register 42 - Zit. nach Vasmer, S. 118, Anm. 2 Vgl. Grimms Vorrede zu Vuks Grammatik S. IV. 2. Jacob Grimms Materialien in seinem Handexemplar der "Kleinen serbischen Grairanatik" Vuk habe eingesehen, "daß nicht durch Berufung deutscher Lehrer nach Serbien, sondern durch Absendung ta- lentvoller serbischer Jünglinge zum Unterricht in unsere Gegende, das Reich des Lichtes und der Wissenschaft in seinem Vaterlande erweitert werden könne." Der Autor würdigt Vuks Bemühen um die Volkssprache, seine Bibelübersetzung und die Liederausgaben. Es werden auch "Grimms Bearbeitung der serbischen Sprachlehre" und Goethes, Vaters und Grimms Unterstützung von Vuk genannt"*. Der zweite Aufsatz ist eine kurze Rezension der deutschen Fassung von Vuks Grammatik, in der Grimms Lob der serbischen Sprache z itie rt wird. Der Autor te ilt außerdem einige biographische Angaben mit, in 4 denen er über "unsern Wuk" spricht . In beiden Aufsätzen zeigt sich die Empfänglichkeit der deutschen Spätromantik für Vuks Bemühungen um die Volkssprache und Volkspoesie. Uber Grimms Vorstellung von der Neuauflage der serbischen Gramma- tik erfahren wir aus seinem Brief an Vuk, daß sie "noch einmal so stark", die Vorrede dagegen gekürzt werden sollte (Prepiska II, 49). Viele seiner Notizen beziehen sich dennoch auf Siedlungs- und Kul- turgeschichte. Sie sind für uns bedeutend als ein Zeugnis über den Umfang von Grimms Interessen,unabhängig davon, ob sie in eine neue Vorrede eingegangen wären. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 2 Vgl. Grimms Vorrede zu Vuks Grammatik S. IV. a. Zur Siedlungs-und Kulturgeschichte a. Zur Siedlungs-und Kulturgeschichte a. Zur Siedlungs-und Kulturgeschichte Grimm hat bereits in seiner Vorrede eine Anregung zur Verbin- dung der Sprachforschung mit der Volkskunde gegeben, ln seinem Hand- exemplar sind viele Stichworte mit Angaben der Quellen zur slavi- sehen Siedlungsgeschichte in Europa enthalten1 (Anh. II, S.231f.). Es handelt sich überwiegend um alte Chroniken Und Urkunden, die viel onomastisches Material bieten. Solche Urkunden sind in dem "Corpus traditionum Fuldensium" von Schannat, den "Regesta" von Lang und der "Politischen Geschichte des Eichsfelds" von Wolf enthalten. Wolf hat auf Grund der Ortsnamen in verschiedenen, überwiegend aus dem 13. Jh. stammenden Urkunden das Vorhandensein von mindestens sechzehn wendischen Siedlungen in dem an der Grenze zwischen Sach- sen und Thüringen liegenden, bis 1803 kurmainzischen Eichsfeld fest- gestellt. In den "Regesta", in denen alte bayerische Urkunden abge- druckt sind, verfolgt Grimm den Gebrauch des lat. s u p a n im Zusammen- hang mit seiner Erklärung des got. s i p o n e i s aus dem slav. z u p a n , die er in seiner Vorrede gegeben hat. Er verfolgt ebenfalls die Ver- breitung des slav. U p ra u d a , als dessen Lehnübersetzung er das lat. 2 J u s t i n i a n gedeutet hatte . Weitere Angaben schöpft Grimm aus der lateinisch geschriebenen annalistischen Chronik Preußens seit 1360 von Johannes von Posilge genannt Lindenblatt (1340 - 1405), die nach seinem Tode ins Deutsche übersetzt und bis 1419 fortgesetzt wurde. Zu der bereits in der Vor- rede angeschnittenen Frage von der Zurückdrängung der slavischen Sprache von der Ostseeküste findet er auch bei Sastrow u.a. die An- gäbe, daß in Hinterpommern noch im 16. Jh. Wendisch gesprochen wurde Unter Grimms Notizen nehmen die Verweise auf die Aufnahme der Slaven in der mittelhochdeutschen Literatur eine besondere Stelle ei Grimm hat sie sicher nicht als historische Quellen gemeint, da er Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access zwischen einer "poetischen” und einer "historischen*1 Wahrheit unter- schied (GKS IV, 74f.)• Es is t zweifellos eine interessante Frage, wie das Slavenbild in der deutschen höfischen Literatur ausgesehen hat. zwischen einer "poetischen” und einer "historischen*1 Wahrheit unter- schied (GKS IV, 74f.)• Es is t zweifellos eine interessante Frage, wie das Slavenbild in der deutschen höfischen Literatur ausgesehen hat. 1 Grimm z itie rt in der Orthographie und nach der Ausgabe von Pez; vgl. dagegen die Ausg. von J. Seemüller in: Monumenta Germaniae historica. Deutsche Chroniken. Bd. V, 1.2, 1890. 2 Vgl. Wirnt von Gravenberg, Wigalois, hg. G.F. Benecke, 1819, Vers 9893f., 11117. a. Zur Siedlungs-und Kulturgeschichte Der erste Verweis bezieht sich auf die berühmteste mittelalter- liehe Reimchronik von Ottokar von Steier (früher Ottokar von Horneck genannt, 1265 - 1320). In dieser Chronik werden sowohl Serben als auch Kroaten mehrmals erwähnt, was aus der geographischen Nähe und historischen Umständen erklärbar is t. Die von Grimm zitierte Stelle befindet sich im Zusammenhang mit der Schilderung der Geschichte Un- gams, Ungarn sei dadurch stark, daß im Süden zur "tatarischen" Gren- ze die "Syrfen”1 ("Sirven" = Serben) wohnen. In Ottokars Chronik ist der historische Hintergrund deutlich erkennbar. In der zwischen 1264 - 1268 entstandenen Dichtung "Biterolf und Ditleib" eines unbekannten Autors wird u.a. Dietleibs Vergeltungs- zug gegen die Wormser Könige mit Hilfe des Hunnenkönigs Etzel ge- schildert. An der von Grimm gemeinten Stelle wird eine Beratung am Wormser Hof beschrieben: der aus dem Niebelungenlied bekannte Held Hagen rät dem Könige, unter dem Vorwand einer Hochzeitsfeier die befreundeten Fürsten mit ihren besten Helden herbeizurufen, um die Verteidigung zu sichern. Unter den einzuladenden Gästen werden "die Durenge und die Surben" (die Thüringer und die Sorben) genannt- In der um 1204 nach französischem Vorbild entstandenen Dichtung "Wigalois" von Wirnt von Gravenberg wird an der von Grimm zitierten Stelle Wigalois* Vorbereitung zum Krieg gegen den Fürsten Namur Lion geschildert. Wigalois' Freunde raten ihm davon ab: "si sprachen: Herre wizzet daz / Im helfent die Sarvie." (=Serben) . Unter den vie- len Kriegern, die der Held Wigalois besiegt, wird namentlich nur 2 der Herzog "von Servie" genannt . In der deutschen höfischen Literatur, die die ritterlichen Kämpfe und Sitten zum Inhalt hat, werden die Slaven vor allem als tapfere Krieger dargestellt. 1 Grimm z itie rt in der Orthographie und nach der Ausgabe von Pez; vgl. dagegen die Ausg. von J. Seemüller in: Monumenta Germaniae historica. Deutsche Chroniken. Bd. V, 1.2, 1890. 2 Vgl. Wirnt von Gravenberg, Wigalois, hg. G.F. Benecke, 1819, Vers 9893f., 11117. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 176 ־ Die übrigen Notizen von Grimm beziehen sich auf die Kultur ־und Literaturgeschichte der Serben (Anh. II, S. 232 ). a. Zur Siedlungs-und Kulturgeschichte Grimms Auswahl von Angaben zeigt auch hier, daß er das Festhalten an der Kirchen- spräche bei den Serben für anachronistisch hielt• Seine Aufzeichnun- gen deuten darauf hin, daß er bemüht war, einen Überblick auch über die serbische Kunstdichtung zu gewinnen, aber die Angaben, über die er verfügte, sind dürftig• Grimms Materialien zu den einzelnen gram- matischen Fragen sind dagegen ergiebiger. 1 Vgl. J. Grimm, Deutsche Grammatik 1. 3• Aufl. 1840, S. 33. 2 Vgl• P. Skok, Etimologijski rječnik hrvatskoga i l i srpskoga jezi- ka 1 - 4, 1971 - 1974, unter d u b o k . Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. J. Grimm, Deutsche Grammatik 1. 3• Aufl. 1840, S b. Zur Lautlehre 235 ). Bekanntlich wurde das ъ nur in der serb. Redaktion des Altkirchenslavischen durch ь ersetzt. Grimm hat eine Reihe von Wörtern gesammelt, bei denen es sich zum größten Teil um die sog. Liquidametathese handelt ( b r a d a , g r a d , t a b u d , m l e k o , p r a s e , ra m e , v l a d a t i , z l a t o ; Anh. II, S. 233 ). Ei ־ nige von ihnen hat er auch in seine "Deutsche Grammatik" aufgenom- men (n a , L a b e , t a b u d )1. Er hat sie dort allerdings als eine Aphäre- sis des Wurzelvokals erklärt ( t a b u d < a l a b u d , o l a b u d , ahd. e l p i z e l a p i z ) . Die Aphäresis is t jedoch nur bei dem balt.־slav. na (awest. 2 a n a , got. a n a ) zutreffend. Grimm gibt in seinen Notizen auch das Stichwort B u c h s t a b e n u m s e t z u n g an, das er vermutlich auch auf die Gegenüberstellung der Beispiele wie b r a d a : p a r t , g r a d : g a r t o be- zieht. Er hat hier offenbar eine Gesetzmäßigkeit bemerkt, die er noch nicht definieren konnte. Einen Definitionsversuch hat er in seinem "Deutschen Wörterbuch" unter a r b e i t gegeben . Grimm verfolgt in seinen Aufzeichnungen auch die Konsonanten- assimilation, wobei er sich besonders mit den einen kombinatori- sehen Lautwandel aufweisenden Formen wie k ő i , (die aus der gleich- zeitigen Wirkung des Assimilations- und Dissimilationsgesetzes ent- standen ist), und ( p i c e l a , z ( i ) b e l a (Wurzel: +Ь ъ к; zb < e b ) * be- schäftigt und einzelsprachliche Beispiele sammelt (Anh. II, S. 233 ) In Grimms Materialien nehmen seine Anmerkungen zu Vuks Darstel- lung der "Konsonantenverwandlung" einen breiten Raum ein, wobei er sich besonders den Gutturalen k , g , h widmet (Anh. II, S. 234f. ). Vuks Behandlung dieser Frage is t rein synchronisch. b. Zur Lautlehre Grimms sprachwissenschaftliche Methode war vergleichend; er hat auch in seiner Anzeige der "Kleinen serbischen Grammatik" hervor ־ gehoben, daß keine der slavischen Sprachen aus sich selbst erklärt werden kann, weshalb sie untereinander v e rg lic h e n werden müssen (GKS IV, 225). Es steht damit völlig im Einklang, daß das von ihm gesam- melte Material sich nicht nur auf die serbische Sprache bezieht. In seinen Aufzeichnungen zur Lautlehre befinden sich zusätzliche Belege für die von ihm aufgestellte Etymologie der aksl. Halbvokale und ein Verweis auf Bopps Vergleichende Grammatik (Anh. II, S•233 ) Bopp hat Grimms Erklärung des ъ < и abgelehnt. Aber Grimm besaß aus- reichende Argumente für eigene Auffassung, die er auch in seine "Deutsche Grammatik" aufgenommen hat: in seiner graphischen Darstel- lung der Vokale, in der die beiden Vokale и und г die Außenstellen zum Konsonantismus hin einnehmen, verweist er auf die beiden Halbvo- kale im Altkirchenslavischen . Grimm hat viele Angaben zur serbischen Entwicklung der reduzier- ten Vokale in Verbindung mit I (в и г а , v u k , p u n . k u n e m ; Anh. II, S.* 234| ) gesammelt. In diesem Zusammenhang beschäftigte ihn besonders das Wort d u b o k , dem er slavische Formen mit wurzelhaftem g l - gegen- überstellte. Es gab vier ursl. Adjektive mit derselben Bedeutung, aber verschiedener Etymologie { * d ^ l b o k - b , * д ъ І Ь о к ъ , * g lç b o k y > , * g Іу Ь о к ъ ) , woraus die unterschiedliche einzelsprachliche Entwicklung resultiert, im serbokroatischen Sprachraum sind sie alle vertreten: kajk, g l i b o k , 2 g l o b o k , čak. g u b o k , štok• d u b o k ) . 1 Vgl. J. Grimm, Deutsche Grammatik 1. 3• Aufl. 1840, S. 33. 2 Vgl• P. Skok, Etimologijski rječnik hrvatskoga i l i srpskoga jezi- ka 1 - 4, 1971 - 1974, unter d u b o k . Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 177 - Grimms Kenntnisse der beiden Halbvokale waren nach der Beschäf- tigung mit Vuks Grammatik gefestigt; mit Recht zweifelte er Vuks Behauptung an, daß es in den alten slavischen Büchern nirgendwo das ъ gebe (Anh. II, S. 1 Vgl. J. Grimm, Deutsche Grammatik 2, 1878, S. 177; 3, 1890, S. 243 2 Vgl. Skok, a.a.O., unter na. 3 Vgl. S. 187 dieser Arbeit. 4 Vgl. Skok, a.a.O.,unter k b i und unter p c e l a . Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access b. Zur Lautlehre Sie beschränkt sich auf die Feststellung der Alternationen von к : b, с , с und g : z , z , с ; die letzte wird auch in dem umgekehrten Verhältnis als z : z , g ( k n e z , k n e z e v , k n e g i n j a ) , statt g š' z , z angegeben. Grimm hat bereits in der Anzeige bemängelt, daß Vuk nur "die Übergänge des Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access kehllauts in den Zischlaut blosz da angeben, wo sie sich im serbi- sehen ereignen, nicht ihre Ursache entwickeln" konnte (GKS IV, 225). Er hat sich selbst dieser Frage zugewandt. In seinen Aufzeichnungen klammert er Vuks Alternationen von k , g : b ( r e b i , l e b i ) aus und beschränkt sich auf die Ergebnisse der sog. ersten und zweiten Pa- latalisation, die er meistens Umlaut nennt. Er hat auch eine Defi- nition gegeben: "die regel hat zwei stufen und scheint so zu lau- ten: 1. wurzelhaftes k, g, ch, wandelt sich in tsch, zh, sch sobald ein organisches e der flexion daran stöszt? 2. wurzelhaftes k, g, ch wandelt sich in tz, z, s, sobald ein organisches i (oder ja, je) der flexion daran stöszt, woraus sich zugleich erklärt, warum die kehllaute kein jer hinter sich leiden, organisches e und i sagen wir deshalb, weil, nachdem sich allmählich die flexionsvocale ver- wirrten, unorganisches e und i durchaus nicht jene Wirkung äuszern." (GKS IV, 225). Grimms Definition der beiden Palatalisationen is t im wesentlicher richtig, aber unvollständig. Er hat vor allem die unterschiedliche Einwirkung des 1 , je nach seiner etymologischen Herkunft, nicht er- kannt. Deshalb meinte er, "der dualis fem. otschi, ouschi verletzt die regel", die sonst im PI. о с е в а , и в е з а eingehalten werde (GKS IV, 225) . Durch Vuks Auslassung des h befand sich Grimm vor der Schwierigkeit, die Ersatzlaute v , g , j in die Regel einzubeziehen? einen der Versuche enthält sein Stichwort: טi n s u n d 8 (Anh. II, S. 234 ). 1 Auch in seiner "Deutschen Grammatik" weist Grimm mehrmals auf die beiden Palatalisationen im Slavischen hin: 2., 1878, S. 368?, 3., 1890, S. 482. Vera Boji 9783954793044 1 Auch in seiner "Deutschen Grammatik" weist Grimm mehrmals auf die beiden Palatalisationen im Slavischen hin: 2., 1878, S. 368?, 3., 1890, S. 482. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM i f 1 In Grimms Materialien sind sonst keine Angaben über die Akzente enthalten. Offenbar besaß er keine umfangreichen Kenntnisse die- ses Problems. b. Zur Lautlehre Grimm s te llt die Palatalisation nicht nur bei der Flexion, son- dern auch bei der Wortbildung fest {d u š a , d i z a t i , e n i j e z a n ) . Er be- merktauch richtig, daß im Serbischen im Nom. PI. fern, und Akk. PI. mask, das e "unroganisch" ist und keine Palatalisation bewirkt, daß aber auch vor dem "organischen" i im Nom. PI. der Adjektive die Pa- latalisation abgeschafft is t1 (GKS IV, 226). Grimms Ergebnisse sind bemerkenswert, trotz der Unvollständigkeit seiner Definition. Die weiteren Notizen von Grimm beziehen sich auf den Übergang des I in o , den er als einen an das Italienische erinnernden Hang zur Vokalisierung bezeichnet (GKS IV, 204). Die Gegenüberstellung Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access von vas und va s deutet auf sein Interesse für die distinktive Funk- tion des Akzents hin1. c. Zur Deklination der Substantive Es sind zunächst die Notizen zu den einzelnen Kasus festzustel- len. Die meisten beziehen sich auf die einsilbigen Maskulina, bei denen Grimm offenbar diejenigen mit dem sog. beweglichen a und die mit Stammerweiterung im Plural festzustellen suchte. Die Endung г im Gen. PI. ( l j u d i ) , die im Serbischen, von wenigen Ausnahmen ab- gesehen, nur bei den Feminina vorkommt, bezeichnet er als weiblich (Anh. II, S.235 ). Im Falle von l j u d i handelt es sich aber um einen alten mask. г-Stamm. Die stammerweiternden Neutra, zu deren Darstellung in Vuks Gram- matik er einen Beitrag geleistet hat, ziehen weiterhin seine Auf- merksamkeit an; er vergleicht sie in der Pluralbildung mit den ahd. Tiernamen auf-гг. Er zieht einen Vergleich zwischen der Entwicklung der Kasusendungen im Serbischen gegenüber den übrigen slavischen Sprachen. Bemerkenswert is t die Vielseitigkeit der von ihm benutzten Quellen zu diesen Fragen (Anh. II, S.235ff.). Grimm hat aus den Volksliedern viele Belege für die Kongruenz mit den Zahlen z w e i - v i e r gesammelt (Anh. II, S.245f.). Er hatte gleich nach der Beendigung der Arbeit an der Grammatik an Vuk ge- schrieben, daß ihm die Erklärung der Substantivendung nach diesen Zahlen als Genitiv Sg. nicht richtig erscheint (Prepiska II, 44). Er hat auch in der Anzeige der Grammatik Vuks Deutung angefochten: "Die s. 49. 50 entwicklete meinung, dasz zu den cardinalien der zwei-, drei- und vierzahl das männliche und neutrale subst. in den gen. sing, gesetzt werde, hält schwerlich nähere prüfung aus. es wäre eine seltsame construction, die weder durch die übrigen slavi- sehen dialecte, noch durch andere sprachen bestätigt wird, aller- dings haben die subst, brda, rasta etc. den schein des gen. sing, der aber doch verschwindet, sobald man wahrnimmt, dasz auch adjecti- va und participia mit derselben endung a dazu gesetzt werden, z. b. 1 In Grimms Materialien sind sonst keine Angaben über die Akzente enthalten. Offenbar besaß er keine umfangreichen Kenntnisse die- ses Problems. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access I, 94 dva se draga и selu gledala; I, 196 dva tzvijeta u bostanu rasla; II, 10 grad gradila t r i brata rodjena und so überall; hier kann unmöglich draga, rodjena, tzvijeta, rasla, gledala, gradila ein gen. sing. sein, was sind sie aber denn? c. Zur Deklination der Substantive nominative, wie sie die construction verlangt und entweder die übrig gebliebene dual- formen (gegen das, was s. 25 und XXII. der Gramm, behauptet wird) die auch auf die drei und vierzahl erstreckt wurden; oder plura- lische neutralformen, die hier nach einer analogie der altdeutschen spräche angewandt werden." (GKS IV, 227). Grimms Aufzeichnungen zeigen, daß er dieses Problem weiter ver- folgt hat; sie enthalten auch die Feststellung, daß es sich hier um "spuren des dual" handelt (Anh. II, S.245 ). Die Notizen zeigen, daß Grimms Aufmerksamkeit besonders den For- men mit einem kombinatorischen Lautwandel und den besonderen syn- taktischen Konstruktionen galt. Diese Tatsache deutet darauf hin, daß die unkomplizierten Formen für seine Sprachbeherrschung kein Problem darstellten. Er verfolgt z.B. auf dem Gebiet der Syntax den Gebrauch des partitiven Genitivs {dok j e m o je na ramenu g l a v e ) und des sog. dativus ethicus {Űe a i m e n i) (Anh. II, S.246 ). d. Zum Adjektiv Grimms Eintragungen enthalten zunächst einige Ergänzungen zu Vuks Darstellung der Adjektive (Anh. II, S.236ffJ• Er hat bereits in seiner Anzeige "die angabe des dem nom. gleichen acc. sing. masc unbelebter dinge" vermißt (GKS IV, 227). In den Notizen gibt er einige Belege dazu, die er dem Akk. Fern, gegenüberstellt. Offen- bar hat ihn die serb. Form 8 a v ß 8 v a , 8 v e , die durch Umstellung der Konsonantengruppe v8~ entstanden ist, besonders interessiert, wes- halb er ihre Entsprechungen in fast allen slavischen Sprachen zu- sammengetragen hat. Vuk hat in der Grammatik nur die Form 8 a v , im Wörterbuch jedoch auch v a 8 , ohne zusätzliche Erklärung angegeben. Grimm verzeichnet außerdem die als Adverbia dienenden neutr. Ad- jektivformen, über die Vuk in seiner Grammatik keine Auskunft ge- geben hat. Der größte Teil von Grimms Notizen zum Adjektiv beziehen sich rößte Teil von Grimms Notizen zum Adjektiv beziehen sich Der größte Teil von Grimms Notizen zum Adjektiv beziehen sich Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access auf seine, in der Vorrede aufgestellte Theorie von der starken und schwachen Adjektivform im Slavischen. Er hat an seiner Deutung, die Bopp angegriffen hat, festgehalten und sie auch in die 1״Deutsche Grammatik" aufgenommen; dennoch zeigen seine Ausführungen dort eine gewisse Modifikation: in der Darstellung des deutschen starken Ad- jektivs lehnt er Bopps Annahme der Agglutination eines Pronomens ab, läßt jedoch eine solche Erklärung für das slavische Adjektiv zu. Er betont ausdrücklich, daß die starke (unbestimmte) Form älter is t, und verweist auf seine Vorrede zu Vuks Grammatik. Für das slavi- sehe Adjektiv s te llt er zwei syntaktische Regeln auf: "1. das a ttri- butive adj. schwach: siepi vol, 2. das prädicative stark: vol je slep,..", die aber nicht auf alle slavische Sprachen anwendbar sind. In bezug auf die in seiner Vorrede zu Vuks Grammatik aufgestellte Deklination der beiden Adjektivformen meint er, daß "sie noch kei- neswegs... weggeräumt" sei1. Vermutlich steht in diesem Zusammen- hang seine Notiz z i v t i b r a t a c , wo er die prädikative, unbestimmte Form und den poss. Dat. t i wohl als Vokative mißverstanden hat. Sei- ne Materialien enthalten eine Sammlung von Belegen für die "starke" Adjektivform in der deutschen und den verschiedenen slavischen Spra- chen. 1 Vgl. Deutsche Grammatik 4, 1890, S. 676f.; vgl. auch 1, 1893, S. 751. - Grimms Materialien zum poss. Adj. erscheinen ebenfalls in seiner Grammatik; er vergleicht das germ. Ableitungsmorphem - 8 k - mit dem slav. - 8 k i , s te llt aber für die Maskulina in der Semantik die Typologie auf: - 8 k - : - o v / - e v (p a u l i s c h , p a u l i n i a c h : p a v l o v ) Vgl. ebd., 2, 1878, S. 360. g , , , 2 Alle diese Angaben hat er in die "Deutsche Grammatik" 3, 1889, S. 225 aufgenommen. Vera Boji - 9783954793044 e. Zu den Zahlwörtern, Pronomina und Partikeln In seinen Eintragungen verfolgt er die Anhängung der Parti- kein an die Pronomina auch in anderen Sprachen; für das Serbische wie für das Gotische s te llt er sie in den obliquen Kasus fest (m e n i - k a r , t e b i k a r ) ^ . Sein Sammeln von diesen Angaben steht vermutlich im Zusammenhang mit seiner Bemühung, die Entstehung der Flexion aus den Partikeln und der Wortarten durch Reduzierung der Begriffe (vgl. 2 - p u t ) zu erklären . Die Aufzeichnungen zum Relativpronomen š t o zeigen ebenfalls eine Ergänzung zu Vuks Ausführungen, indem Grimm nach Heranziehung ver- schiedener Quellen die Regel aufstellt, daß bei einem lebendigen Subjekt das pers. Pronomen wiederholt wird. Grimms Verbindung des serb. s i n o è und russ. a e g o d n ja mit dem aksl. s i j is t aufschlußreich in bezug auf den Umfang seiner slavistischen Kenntnisse und sein Vermögen, die historisch-vergleichende Methode auch auf diesem Ge- biet anzuwenden (Anh.II, S.239f.). Zu den Partikeln hat Grimm auch einige Ergänzungen gesammelt. Bei den meisten von ihnen handelt es sich um Angaben aus Vuks Wör- terbuch, die in das entsprechende Grammatikkapitel nicht eingegan- gen sind. Grimms Zuordnung des konditionalen Hilfsverbs b i zu den Partikeln geht ebenfalls auf die Angabe in Vuks Wörterbuch ("Parti- kel, um den Optativ anzuzeigen") zurück. Nur sadm a, d o v l e , d o n le und a ko Bogda sind im Wörterbuch nicht vorhanden. Interessant sind Grimms Anmerkungen zur serb. Präposition u, in deren syntaktischer Differenzierung (Gen. oder Akk./Lok.) er die aksl. Präpositionen u {o u ) und ѵъ richtig erkannt hat (Anh. II, S. 24 e. Zu den Zahlwörtern, Pronomina und Partikeln Beim Zahlwort hat sich Grimm besonders für die Multiplikativa interessiert. Er verfolgt ihre Bildungsweise in den slavischen Spra- chen und s te llt fest, daß die Bildung mit p u t eine Eigentümlichkeit der serbischen Sprache darstellt, während das aksl. k r a t , dem er die l i t . Entsprechung hinzufügt, in allen übrigen slav. Sprachen nachweisbar is t; das russ. und poln. r a z ist wiederum in die 2 altpreuß. Sprache eingegangen (Anh. II, S. 239 ). Bei den Pronomina enthalten Grimms Notizen einige Ergänzungen zu Vuks Ausführungen. Bereits in seiner Anzeige wies er darauf hin. 1 Vgl. Deutsche Grammatik 4, 1890, S. 676f.; vgl. auch 1, 1893, S. 751. - Grimms Materialien zum poss. Adj. erscheinen ebenfalls in seiner Grammatik; er vergleicht das germ. Ableitungsmorphem - 8 k - mit dem slav. - 8 k i , s te llt aber für die Maskulina in der Semantik die Typologie auf: - 8 k - : - o v / - e v (p a u l i s c h , p a u l i n i a c h : p a v l o v ) . Vgl. ebd., 2, 1878, S. 360. g , , , 2 Alle diese Angaben hat er in die "Deutsche Grammatik" 3, 1889, S. 225 aufgenommen. Vera Boji - 9783954793044 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access daß in Vuks Grammatik der Akk. fern, j u nicht erwähnt wird und daß außer der Form t i z i in den Liedern auch n j o j z i vorkommt (GKS IV, 227) . In seinen Eintragungen verfolgt er die Anhängung der Parti- kein an die Pronomina auch in anderen Sprachen; für das Serbische wie für das Gotische s te llt er sie in den obliquen Kasus fest (m e n i - k a r , t e b i k a r ) ^ . Sein Sammeln von diesen Angaben steht vermutlich im Zusammenhang mit seiner Bemühung, die Entstehung der Flexion aus den Partikeln und der Wortarten durch Reduzierung der Begriffe (vgl. 2 - p u t ) zu erklären . daß in Vuks Grammatik der Akk. fern, j u nicht erwähnt wird und daß außer der Form t i z i in den Liedern auch n j o j z i vorkommt (GKS IV, 227) . g 2 Dieses Problem hat er ausführlich in der Schrift "Uber den ur- sprung der spräche" von 1852 behandelt (GKS I, 225 - 298). 1 S. diese Angaben in der "Deutschen Grammatik" 2, 1878, S. 11. f. Zu den Verben In Grimms Materialien ist den Verben der größte Raum gewidmet. Seine Aufzeichnungen beziehen sich hauptsächlich auf zwei Fragen- komplexe: den Verbalaspekt und die Klassifikation der Verben. 1 S. diese Angaben in der "Deutschen Grammatik" 2, 1878, S. 11. 2 Dieses Problem hat er ausführlich in der Schrift "Uber den ur- sprung der spräche" von 1852 behandelt (GKS I, 225 - 298). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM 1 S. diese Angaben in der "Deutschen Grammatik" 2, 1878, S. 11. 2 Dieses Problem hat er ausführlich in der Schrift "Uber den ur- sprung der spräche" von 1852 behandelt (GKS I, 225 - 298). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Grimm hat bereits in der Vorrede den slavischen Verbalaspekt behandelt und nach seinen Spuren in der deutschen Sprache gesucht. Die Notizen in seinem Handexemplar zeigen, daß er dieses Problem und seine Behandlung in der damaligen Wissenschaft mit großer Auf- merksamkeit verfolgt hat (Anh. II, S.240f.). Er verweist auf Thiersch, der den griech. Aorist als den Ausdruck der Vergangenheit "ohne Dauer" definiert hat; dem gegenüber drücke das Imperfekt die Vergangenheit als "dauernd" aus. Grimm lehnt den im "Allgemeinen Anzeiger der Deutschen" erschie- nenen Artikel ab, in dem versucht wird, das Vorhandensein des Ver- balaspekts in der deutschen Sprache mit morphologischen Kriterien nachzuweisen. Er s te llt fest, daß die deutsche Sprache den semanti- sehen Unterschied zwischen pf. und ipf. Verben nur durch besondere syntaktische Konstruktionen umschreiben kann ( e r i s t g e h e n d - e r g e h t f o r t ) . Er geht auch in der "Deutschen Grammatik" auf diese Fra- ge ein: "Wenn der begriff des verbums in das part. präs. und das verb, subst. aufgelöst wird, so entspringt ein regerer ausdruck der continuität, des nicht aufhörens der handlung. 1 Vgl. Deutsche Grammatik 4, 1898, S. 5. 2 Vgl. ebd., 2, 1878, S. 853. g 2 Vgl. ebd., 2, 1878, S. 853. 1 Vgl. Deutsche Grammatik 4, 1898, S. 5. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. Deutsche Grammatik 4, 1898, S. 5. 2 Vgl ebd 2 1878 S 853 f. Zu den Verben man könnte die wir- kung dieser Umschreibung der in allen slavischen sprachen eigen- thümlich entfalteten form des verbum actionis imperfectae verglei- chen.” 1 Er gre ift auch die in der Vorrede gestellte Frage über den Einfluß der Präpositionen auf die mit ihnen zusammengesetzten Verben im Deutschen auf und meint, daß einige von ihnen "ohne zum wesen der conjugation erforderlich zu sein, einen gewissen einflusz auf die praeterita ausüben", weshalb sie mit dem Verb im Präsens nicht üblich seien ( v e r s t a r b : s t i r b t ) . Diese Erscheinung vergleicht er mit dem Einfluß der Präpositionen auf die slav. Verben, betont aber den Unterschied: sie "dienen, die perfective bedeutung zu erheben; sie gehen auch (wie unser be- er- ver-) durch alle modos, nicht durch alle tempora, präsens (im wahren, unfuturischen sinn), imper- fect und part, praes, versagen sich ihnen.״ Für die deutsche Spra- che s te llt er fest, daß eine solche Differenzierung im Imperfekt nicht existiert (Anh. I I , S. 240 ). In bezug auf die Klassifikation der Verben is t Grimms Anmerkung interessant, mit der er Vuks Behauptung, daß die Unterscheidung Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access zwischen den trans, und intrans* Verben unwichtig sei, nur auf die Formenlehre einschränkt (Anh. II, S. 240 ). "Moguònost da se za neke glagole veže akuzativ objekta cini najistaknutiji sintaksički k ri- te rij grupisanja naših glagola, posle onih koje daje glagolski vid .” Grimm hat viel Material zur Konjugation zusammengetragen. In der Vorrede hatte er Vuks Einteilung der Verben in die Konjugationsklas- sen als vereinfachend k ritis ie rt. Vuk hat die 1. Sg. Präs. -am , -em , - i m zum Kriterium für seine Einteilung genommen; er hat zwar darauf hingewiesen, daß die Kenntnis des In fin itiv s wichtig sei, konnte aber die beiden Kriterien in seiner Darstellung der Konjugation nich׳ verbinden. Die Konjugation gehört zweifellos zu den schwierigsten Kapiteln in der Morphologie. DobrovskJ z.B. hat den Infinitivstamm zur Grundlage seiner Einteilung in Konjugationsklassen gewählt. In Grimms Materialien is t auffallend, daß seine Angaben neben der 1. Sg. Präs. auch den In fin itiv enthalten. 1 Vgl. P. Ivic, О Vukovom Rjecniku.., S. 61. 2 Grimms Kenntnisse der slavischen Hilfsverben erscheinen in der ”Deutschen Grammatik” , wo er die Bildung der Vergangenheitstempora im Deutschen und Slavischen typologisch betrachtet und feststellt, daß die slav. Sprachen das b i t i ”ohne rücksicht auf intransitiven oder transitiven sinn” verwenden. "Sie können es, weil sie sich dazu eines wahrhaft activen part. prät. bedienen, das von dem pas- siven das passiv umschreibenden... völlig absteht, z. b. ... serb. pleo sam...” - Vgl. Deutsche Grammatik 4, 1898, S. 194. 3 Vgl. Skok, a.a.O., unter l a c a n und p i t a t i l . Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access p g 3 Vgl. Skok, a.a.O., unter l a c a n und p i t a t i l . Vera Boji Downloaded from PubFactory at 01/10/2 1 Vgl. P. Ivic, О Vukovom Rjecniku.., S. 61. f. Zu den Verben (Er hat bereits in de: deutschen Fassung von Vuks Grammatik die fehlenden Infinitive er- gänzt.) Er s te llt Gruppen von Verben zusammen, die einen Konsonan- tenwandel im In fin itiv (g re b e m : g r e p s t i ; c rp e m : e r p e t i ) , oder im Präsens (vueem : v u ò i usw., le z e m : l e g a t i usw.) aufweisen (Anh. II, S.243 ). Offenbar hat Grimm eine differenziertere Einteilung als diejenige von Vuk angestrebt, die in vielen Untergruppen alle Be- Sonderheiten berücksichtigen sollte. Das zeigt u.a. sein Verzeichnis der Verben mit ,,eingeschobenem vocal” (b e re m : b r a t i ; p e re m : p r a t i ; s a l j e m : s i a t i ) , der unregelmäßigen Verben { i ö i mit Zusammensetzun- 2 gen) und "auxuliaria" (Anh. I I , S. 241 ). Er versucht zugleich, die historische Entwicklung der einzelnen Formen zu erklären (kunem, и < l ; die sog. Vereinfachung der Gruppe t l : r a s l a ) . (Anh. II, S. 243). Er zieht dabei Angaben aus verschiedenen slavischen Sprachen heran. Es is t bewundernswert, daß er in den serb. Adjektiven lakom 3 und p i t o m die Reste des Part. Präs. Pass, erkannt hat (Anh. II, S. 2 ־ 185 ־ Aus Grimms Materialien zu den Verben is t besonders deutlich zu sehen, daß er sich eine erweiterte serbische Grammatik als eine hi- storisch-vergleichende Grammatik der slavischen Sprachen vorgestellt hat, so wie seine "Deutsche Grammatik" eine historisch-vergleichende Grammatik der germanischen Sprachen geworden is t. Der Verwirklichung einer solchen Konzeption standen zur damaligen Zeit viele Hindernis- se im Wege. Dobrovsk^s altslavische Grammatik enthielt bekanntlich viele Mängel1, die Vorarbeiten für die einzelnen slavischen Sprachen und die Quellenforschung, auf der sie beruhen müßten, befanden sich in den Anfängen. Grimm selbst konnte eine solche Konzeption nicht verwirklichen. Die Arbeit an der "Deutschen Grammatik" und dem Wör- terbuch hat keinen Raum für die dazu erfordaliche Vertiefung in die 2 Slavistik gelassen . Erst F. f. Zu den Verben Miklosich hat Grimms Forderung nach der historisch-vergleichenden Grammatik der slavischen Sprachen er- fü llt, allerdings nicht in einem Werk, sondern durch Ausarbeitung einer altkirchenslavischen (1848 - 1850) und einer vergleichenden slavischen (1852 - 1883) Grammatik. 1 Vgl. Jagic, Is to rija .., a.a.O., S. 125f. 2.Die 1838 übernommene Vorbereitung des riesigen "Deutschen Wörter- buchs" hat Grimm auch an der Ausarbeitung des geplanten 5. T. der "Deutschen Grammatik" gehindert. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. Jagic, Is to rija .., a.a.O., S. 125f. g. Der Zeitraum von Grimms Beschäftigung mit der serbischen Sprache Grimms Eintragungen in seinem Handexemplar stammen offensicht- lieh nicht alle aus derselben Zeit. Ihre Datierung könnte eine Aus- kunft über den Zeitraum seiner slavistischen Studien geben. Als An- haltspunkte für die Datierung können die von Grimm benutzten Quellen, sein Briefwechsel und die graphischen Eigenschaften seiner Handschrift dienen. Die Aufnahme des slavischen Sprachmaterials in Grimms Werke bietet weitere Beweise über die Dauer und den Umfang seiner slavistischen Studien. Die meisten Quellen, aus denen Grimm seine Materialien schöpft, sind entweder vor oder kurz nach der "Kleinen serbischen Grammatik" erschienen. Eine Ausnahme stellen Murkos slovenische Grammatik von Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1832 und Brlics "illyrische" von 18331dar. Die inhaltliche Geschlos- senheit der Notizen, in denen diese beiden Quellen, neben den älte- ren, genannt werden, deutet darauf hin, daß Grimm oft gleichzeitig in verschiedenen Schriften nach den ihn interessierenden Angaben gesucht hat (vgl. z.B. die Notizen zu o c i , u e i , zu den M ultiplikati- 2 va , dem Pronomen e t o und z.T. zu den Verben; Anh. IX, S. 236,239, 241f.). Griiwn verweist außerdem auf Bopps Grammatik von 1835 (Anh. II S.233,237 ). Die jüngste unter den genannten Quellen is t der erste Band der Wiener Liederausgabe von Vuk aus dem Jahre 1841 (Anh. I I , S. 235,244 ). Diese Tatsachen lassen die Schlußfolgerung zu, daß ein Teil von Grimms Materialien aus der Zeit nach 1832 stammt. Ein Teil seiner Aufzeichnungen ist dagegen offenbar älter. Das g ilt vor allem für die meisten Notizen über Vuk und die Sammlung von Zitaten aus der Leipziger Liederausgabe von 1823/24, die vermut lieh in der Zeit kurz nach Erscheinen der "Kleinen serbischen Gram- matik" entstanden sind. In diesen Notizen zeigt auch Grimms Hand- schrift noch einen Einfluß der sog. deutschen Schrift3, deren Ab- Schaffung und Ersetzung durch die lateinische er 1822 in seiner Grammatik verkündet hat. Das serbische Sprachmaterial schreibt er in der angepaßten lateinischen Schrift ( a ch = 8 Л t e c h = c , t z = с , eh = z) , gebraucht zuweilen die einzelnen kyrillischen Zeichen von Vuk (für Ô und d) ; die späteren Aufzeichnungen, besonders diejeni- gen zu den Verben, sind zum größten Teil in kyrillischer Schrift geschrieben4. Es läßt sich zusammenfassend sagen, daß Grimms Materialien in seinem Handexemplar in der Zeit bis zum Beginn der 40er Jahre ge- sammelt wurden. 1 Brlics Grammatik is t in einer erweiterten Aufl. 1842 und 1854 er- schienen. Grimms Verweise beziehen sich auf die erste Auflage. 2 Zu den Multiplikativa te ilte ihm auf seine Anfrage Kopitar einige Angaben zu - k r a t am 22. April 1837 mit. - Vgl. Vasmer, S. 164. 3 Vgl. bes. Grimms Exzerpt aus dem "Literarischen Conversations- blatt" von 1824 im "Anhang" der Faksimileausgabe von 1974. 4 Zur Veranschaulichung dieser Tatsache wird im Anhang II Grimms Schreibweise wiedergegeben. g. Der Zeitraum von Grimms Beschäftigung mit der serbischen Sprache Ein Teil is t vermutlich in der ersten Hälfte der 20er Jahre, der übrige nach 1832, vermutlich nach Bopps Grammatik von 1835 und während der Ausarbeitung des vierten Teils der "Deut- sehen Grammatik" entstanden. Der Verweis auf die "Allgemeine Zei- tung" von 1839/40 deutet ebenfalls darauf hin. 1 Brlics Grammatik is t in einer erweiterten Aufl. 1842 und 1854 er- schienen. Grimms Verweise beziehen sich auf die erste Auflage. 2 Zu den Multiplikativa te ilte ihm auf seine Anfrage Kopitar einige Angaben zu - k r a t am 22. April 1837 mit. - Vgl. Vasmer, S. 164. 3 Vgl. bes. Grimms Exzerpt aus dem "Literarischen Conversations- blatt" von 1824 im "Anhang" der Faksimileausgabe von 1974. 4 Zur Veranschaulichung dieser Tatsache wird im Anhang II Grimms Schreibweise wiedergegeben. V B ji 9783954793044 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Grimms Sammeln des serbischen Sprachmaterials stand zu Beginn zweifellos im Zusammenhang mit seiner Idee von einer erweiterten serbischen Grammatik. Das zeigen deutlich seine Notizen zur Palata- lisation und zur Deutung der Kasusendung nach den Zahlen, mit denen er Vuks Ausführungen korrigieren wollte. Da er zur gleichen Zeit an seiner eigenen Grammatik arbeitete, nahm er bei der Sprachver- gleichung auch das erarbeitete slavische Sprachmaterial auf. Die Einbeziehung der slavischen Sprachen is t mit der Zeit zu einem fe- sten Bestandteil seiner vergleichenden Methode geworden. Der Beweggrund für Grimms Beschäftigung mit der serbischen Spra- che war in den späteren Jahren u.E. nicht mehr die Erweiterung der serbischen Grammatik, sondern seine vergleichende Forschung auf dem Gebiet der deutschen Sprache. Das bestätigt die Tatsache, daß in Grimms Werken viele Angaben aus der serbischen Sprache Vorkommen, die in seinem Handexemplar der Grammatik nicht enthalten sind1. Grimm hat der serbischen Sprache und der Slavistik im allgemei- nen bis zum Ende seines Lebens Interesse entgegengebracht. In seiner deutschen Sprachgeschichte von 1848 befinden sich ebenfalls Anga- ben aus der serbischen Sprache und Verweise auf die Arbeiten von Miklosich und Šafafīk2, was beweist, daß er die slavistische Lite- ratur weiterhin verfolgt hat. Aus seinem Briefwechsel geht hervor, daß er auch Vuks Wörterbuch von 1852 benutzt hat (Prepiska II, 64). Grimm hat schließlich auch in sein "Deutsches Wörterbuch", an dem er bis zum Ende des Lebens gearbeitet hat, die slavischen Sprachen einbezogen. Seine Angaben im Wörterbuch zeigen in bezug auf die Materialien im Handexemplar eine Erweiterung der slavistischen Kenntnisse, z.B. 1 Vgl. z. B. die ausführliche Behandlung der serbischen und slavi- sehen Komposita in der "Deutschen Grammatik" 2, 1878, S. 940, 947f 2 Vgl. Geschichte der deutschen Sprache, 4. Aufl. 1880, z.B. S. X: Bericht über Safarîks Deutung der Herkunft der Slaven; S. 9: über Miklosichs Etymologie von z l a t o ; S. 14: Über serbische Frauenna- men; S. 39: Apharäsis in r a l o < o r a l o , o r a t i ģ, S. 92: p o s e s t r i t i 8е л p o b r a t i t i 8 e , Erklärung der Sitte, Verweise auf Volkslieder; S. 100: Verweis auf Vuks Schrift "Montenegro und Montenegriner" von 1837; usw. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Vgl. auch unter: a n , a u g e , b i b e r , b i e g e n , b i e r usw. Eine voll- ständige Auswertung des slavistischen Materials bei Grimm würde das Thema einer gesonderten Studie ausfüllen. 2 Vgl. S. Trops, Jakob Grimm als Übersetzer serbokroatischer Volks- lieder. In: Euphorion 16. Erg.H., 1923, S. 106 - 120. 3 Vgl. J. Milovic, Übertragungen slavischer Volkslieder aus Goethes Briefnachlaß, 1939, S. XV. 4 Vgl. S. 121 dieser Arbeit. Vera Boji 9783954793044 g. Der Zeitraum von Grimms Beschäftigung mit der serbischen Sprache \ na r b e i t x der wurzel... a r b ... entspringt dadurch wichtiger aufschlusz, den uns aber die sl. spräche zumal eröffnet. Wie nun die Slaven überhaupt die deutsche dem vocal folgende l i - quida ihm vorausschicken... zeigen sie rab", poln. böhm. r o b und r a b o t a , poln. böhm. r o b o t a , welche jenem a r b j a und a r b a i p e ... ge- паи entsprechen."1 паи entsprechen."1 Es is t deutlich zu sehen, daß aus Grimms Arbeit an der serbi- sehen Grammatik seine Beschäftigung mit der Slavistik erwachsen is t. Seine Materialien im Handexemplar enthalten bereits einen solchen Ansatz. h. Der Umfang von Grimms Kenntnissen der serbischen Sprache Uber Grimms Kenntnisse der serbischen Sprache wurden im Zusam- menhang mit den in seinen "Kleineren Schriften" abgedruckten und ihm zugeschriebenen Übersetzungen der serbischen Volkslieder ent- gegengesetzte Urteile ausgesprochen. Tropš vertrat die Meinung, daß 2 Grimm ohne fremde Hilfe nicht fehlerfrei übersetzen konnte ; die- se Behauptung wurde von Milovic mit scharfen Worten abgelehnt3. Grimms Kenntnisse der serbischen Sprache müssen in ihrer Ent- Wicklung gesehen werden. Er hat während seiner Teilnahme am Wiener Kongreß mit Kopitars Hilfe angefangen Serbisch zu lernen. Nachdem Vuks Wörterbuch erschienen war, konnte er beim Lesen der Lieder "das meiste herausbringen" (Brief an Kopitar vom 10. Okt. 1823; Prepiska I, 718). "Ich verstehe alles mit Hilfe des Wörterbuchs, bis auf Einzelnes." (Brief an Vuk vom 24. April 1824, Prepiska II, 45) . Es ist feststellbar, daß Grimms Kenntnisse der grammatischen Struktur der serbischen Sprache vor der intensiven Beschäftigung mit ihr nicht gefestigt waren. Er konnte die serbischen Formen leicht mißverstehen, z.B. die abhängigen Kasus nach Präpositionen { i z T r s i b a - aus T r s o h i t s c h a ; GKS IV, 197; o s im m a t e r e - a u ß e r ... 4 d i e M ü t t e r ; Anh. I, S.210 ), oder die Aoriste und Imperative . Diese Lücken waren nach seiner Beschäftigung mit Vuks Grammatik überwiegend beseitigt. Unter seinen handschriftlichen Aufzeichnun- gen befindet sich z.B. die Feststellung, daß in Vuks Wörterbuch to p irrtümlich als Femininum bezeichnet is t (Anhang II, S.235 ). Das mißverstandene m a t e r e ist ebenfalls korrigiert. Seine Ergänzun- gen der Grammatik von Vuk und das gesammelte Material in seinem Handexemplar zeigen deutlich, daß er während der Beschäftigung mit Vuks Grammatik die Struktur der serbischen Sprache eingehend kennen- gelernt hat. Mit diesen Kenntnissen konnte er wissenschaftlich ar- beiten. to p irrtümlich als Femininum bezeichnet is t (Anhang II, S.235 ). Das mißverstandene m a t e r e ist ebenfalls korrigiert. Seine Ergänzun- gen der Grammatik von Vuk und das gesammelte Material in seinem Handexemplar zeigen deutlich, daß er während der Beschäftigung mit Vuks Grammatik die Struktur der serbischen Sprache eingehend kennen- gelernt hat. Mit diesen Kenntnissen konnte er wissenschaftlich ar- beiten. Grimms Kenntnisse des Wortschatzes und der besonderen Redewen- dungen in der serbischen Sprache sind aber weiterhin unvollkommen geblieben. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access h. Der Umfang von Grimms Kenntnissen der serbischen Sprache Er konnte gelungene Übersetzungen leisten, war aber, be- sonders in bezug auf die Idiomatik, vor Fehlübersetzungen nicht abgesichert. In seiner Rezension der Liederübersetzung von Talvj z.B. meint er richtig, daß serb. t i j o , p o t i j o nicht mit f l ü s t e r n d übersetzt werden soll, da es bloß als Gegensatz zu g l a a o v i t o ge- braucht wird, "die schönen zeilen tscharna goro, puna t i si lada; srtze moje, puno t i si jada! hätten wir lieber gegeben: schwarz gebirge voll du bist des schattens; herze meine, voll du bist des leides! wenn schon der reim verloren geht, als durch: schwarzer wald, dein schatten is t erquickend! armes herz, dein weh ist schwer und drückend! (2, 61) denn es braucht hier nicht gesagt zu werden, was sich von selbst versteht, dasz der schatten labe, noch dasz das leid belaste und der aus tiefer brust hervorgestiegene ausruf wird damit nur geschwächt." (GKS IV, 420f.). In beiden Fällen zeigt Grimm ein Einfühlen in die Sprache des Originals, seine über- setzung der angeführten Zeilen ist u. E. derjenigen von Talvj vor- zuziehen. Auf der anderen Seite enthalten die von Trops festge- stellten Übersetzungsfehler von Grimm ein Mißverständnis der Be- deutung einzelner Worte und besonderer Ausdrücke. Grimms Kenntnisse der serbischen Sprache waren auf die Hilfe der Lexika angewiesen. Unter seinen handschriftlichen Eintragungen befindet sich eine Notiz, die auf ein häufiges Nachschlagen in Vuks Wörterbuch beim Lesen hinweist: Zur Präposition 8a verzeich- net Grimm einen Beleg: 8a ѵ г в о к а d e l a und gibt eine zunächst über- raschende Übersetzung: vom hohen b e r g e , ( d e l a /. d i j e l a ) (Anh. II, S. 245 ). Seine Quelle is t das in Vuks Danica abgedruckte Volks- lied "Srbi и Donavertu ,״in dem der Vers: ״Sa visoka dela Šelem- berga" vorkommt1.Vuks Wörterbuch verweist bei d e o , d e l a auf d i j e l , und dort lesen wir unter 2) uz d i j e l , b e r g a n , (mit Angabe des ON D u g i d i j e l ) . Aus diesen Angaben hat Grimm seine Übersetzung zusammengestellt. 1 Vgl. Danica. Zabavnik za godinu 1826, S. 112. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Zu dieser Wende haben wahrscheinlich sowohl W. Schlegels scharfe Kritik der spekulativen Etymologien als auch W.v.Humboldts Sprachauffassung, Savignys historische Schule und Grimms eigene Einsicht in die Unergiebigkeit der Methode von A. Kanne beige ־ tragen. h. Der Umfang von Grimms Kenntnissen der serbischen Sprache Es zeigt sich deutlich, daß er beim Lesen, oft auch bei gängigen Wörtern, in Vuks Wörterbuch nachschlagen mußte. Wir haben darüber hinaus in der Analyse der Grammatiküberset- zung festgestellt, daß die aus dem Kontext isolierten Wörter und Wortverbindungen für Grimm eine besondere Schwierigkeit darstell- ten und daß er in solchen Fällen weitgehend von den Angaben im Wörterbuch abhängig war. In bezug auf Grimms Kenntnisse der serbischen Sprache läßt sich zusammenfassend sagen, daß er nach der Beschäftigung mit Vuks Grairanatik eine gute Einsicht in die grammatische Struktur besessen hat. Diese Kenntnisse boten eine Grundlage für die wissenschaftli- che Arbeit auf diesem Gebiet, so daß er auch zu den selbständigen Ergebnissen koninen konnte, die wir in der Analyse der deutschen Fassung der Grammatik und seiner handschriftlichen Materialien ge- zeigt haben. Grimms Kenntnisse des Wortschatzes und der Ausdrucks- eigentümlichkeiten und somit seine praktische Beherrschung der serbischen Sprache waren dagegen unvollkommen. D . SCHLUSSBETRACHTUNG Jacob Grimms Sprachauffassung zeigt eine ideelle Entwicklung, die wir am Beispiel seiner Ansichten über den Ursprung und den Entwicklungsgang der Sprache verfolgt haben. Die Arbeit an der "Deutschen Grammatik" brachte eine Wende in seinen Ansichten und leitete eine Entwicklung ein, deren eingehende theoretische Be- gründung wir in seinen Arbeiten der späten Phase finden. Grimms Ansichten sind in der frühen Phase durch die romanti- sehe Sprachauffassung beeinflußt, namentlich durch die sog. Offen ־ barungstheorie, die die Vorstellung von der Vollendung der Spra ־ che im goldenen Zeitalter und ihrem allmählichen Niedergang ent- hält. Wir haben Grimms mythologische Sprachbetrachtung wegen ihres spekulativen Charakters "vorwissenschaftlich" genannt. Mit der "Deutschen Grammatik" wandte Grimm sich bewußt der empi rischen Forschung zu, die zugleich die Überwindung romantischer Spekulationen bedeutete. Der romantische Einfluß is t in der Einlei ־ tung zur Grammatik von 1819 in Grimms Ansicht von der "Urvollendung der Sprache noch spürbar. Grimms Begriff der Sprachvollendung unterscheidet sich aber von demjenigen der Romantik. Er versteht darunter den Reichtum der Sprache an Flexionsformen und an Begriffen für sinnliche Vor- Stellungen, während die Romantik alle Formen als Fesseln betrach- tet. Da die Sprachentwicklung über die Reduktion von Flexionsfor- men und sinnlichen Elementen zu einer immer höheren Abstraktions ־ stufe verläuft, erscheint sie für Grimm als ein Niedergang der ur ־ sprünglichen Vollendung. Grimms Ansichten aus dieser Zeit enthalten eine spezifische Komponente: die in seinem Sinne vollendete Sprache eigne sich be- sonders für die Poesie. Die Sprachentwicklung bedeutet daher auch einen allmählichen Verlust der poetischen Kraft der Sprache. 1 Zu dieser Wende haben wahrscheinlich sowohl W. Schlegels scharfe Kritik der spekulativen Etymologien als auch W.v.Humboldts Sprachauffassung, Savignys historische Schule und Grimms eigene Einsicht in die Unergiebigkeit der Methode von A. Kanne beige ־ tragen. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Grimms theoretische Äußerungen der späten Phase zeigen einen veränderten Begriff der Sprachvollendung. Mit dem natürlichen, menschlichen Ursprung der Sprache als Ausgangspunkt entfiel die Vorstellung von ihrer Urvollendung. Grimm sieht nun die Entwick- lung der Sprache, deren Ursprung er aus der Denkfähigkeit und dem Kommunikationsbedürfnis der Menschen erklärt, als ständige Vervoll- kommnung im Einklang mit dem Fortschritt des menschlichen Geistes* Er behält seine Vorliebe für die komplizierten Flexionen der älte- ren Sprachzustände bei; sie hat jedoch nichts Romantisches mehr an sich, vielmehr is t sie für die meisten Sprachforscher des 19. Jhs. charakteristisch. 1 "man hat in der etymologie bisher vorzugsweise die äuszeren be- standtheile der Wörter... gepflegt, den ursprung, fortschritt und Übergang der begriffe... vernachlässigend, die Vorstellungen mehr von den wortgesetzen abhängig gemacht, als worte und gedanken in einander sich verschlingen lassen.” (GKS I, S. 123). Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access D . SCHLUSSBETRACHTUNG Wir haben an Grimms Beurteilung des Englischen festgestellt, daß er die poetische Kraft der Sprache nicht mehr in Abhängigkeit von dem Formenreichtum sieht. Er betrachtet die Ent- Wicklung der Sprache, ähnlich wie W. von Humboldt, als den "fort- schritt und Zuwachs ihrer inneren kraft". In Grimms Darstellung der Entwicklung der sprachlichen Formen zeigt sich seine Auffassung der Sprache als der formenden geistigen Kraft, die in der mittels Sprache sich vollziehenden Begriffsbil- dung und Denkentfaltung wirkt. Seine Deutung der Entstehung und des Zerfalls von Flexionen im Zusammenhang mit der Denkentfaltung zeigt, daß er, ähnlich wie Humboldt, die formende Kraft der Spra- che nicht nur auf ihre Entstehungszeit, sondern auch auf die ge- samte in die Zukunft reichende Entwicklung der Sprache bezieht. Aus Grimms Ansichten von dem engsten Zusammenhang von Sprache und menschlichem Geist resultiert der Unterschied zwischen seiner Auffassung des sprachlichen Organismus und derjenigen der natur- wissenschaftlichen Schule und der Junggrammatiker. Da Grimm in der materiellen Form des jeweiligen Sprachorganismus Reste des Gewese- nen und Keime des Werdenden sieht, unterscheidet sich seine Auffas- sung des sprachlichen Organismus von der naturwissenschaftlichen Auffassung der Ausnahmslosigkeit der Lautgesetze bei den Junggram- matikern. Grimms Forderung an die vergleichende Sprachwissenschaft, in ihr Forschungsfeld nicht nur die Lautgestalt der Wörter, sondern auch die Wortinhalte einzubeziehen1, steht im Einklang mit seiner ־ 193 - Auffassung des geistigen Charakters der Sprache. Auffassung des geistigen Charakters der Sprache. Eine wichtige Komponente in Grimms Sprachauffassung is t seine Verbindung der Sprache mit der Geschichte ihres Trägers. Sie zeigt sich bereits in dem ersten Band der "Deutschen Grammatik" und wird in der Akademieabhandlung von 1852 ausführlich theoretisch begrün- det. Die Sprache erscheint bei Grimm als Widerspiegelung der Ge- schichte und als die Geschichte selbst. Die Sprachforschung erhält die Aufgabe der Erhellung von Vorgeschichte. Diese Aufgabenstellung bestimmt u.a. auch Grimms Zuwendung zur historischen Sprachfor- schung und sein Interesse für die hinter den Wörtern stehenden Sachen. Dieser Ansatz von Grimm hat in der späteren Sprachpaläonto- logie seine Fortsetzung gefunden. Der Zusammenhang zwischen Sprache und ihrem Träger bedeutet bei Grimm auch die Betrachtung der sprachlichen Gegebenheiten und Ver- änderungen im Hinblick auf gesellschaftliche Faktoren: blühende Entwicklung oder Stagnation und Verwilderung von Sprachen sind ge- sellschaftlich bedingt. Andererseits kann die Sprache, da sie eine gesellschaftliche Funktion hat, durch anachronistische Regelungen zum Hemmnis der kulturellen Entwicklung ihres Trägers werden. Die- se Ansichten hat er, wie wir gesehen haben, u. a. besonders deut- lieh im Zusammenhang mit der sprachlichen Situation bei den Ser- ben hervorgehoben. In Grimms Sprachauffassung is t der Ansatz ei- ner soziologischen Sprachbetrachtung vorhanden, der für seine Zeit neu war und auch lange danach ohne Fortsetzung geblieben ist. Wir haben Grimms Sprachauffassung weiter im Zusammenhang mit den Problemen der Schriftsprache betrachtet, um eine Ausgangsba- sis für den Vergleich seiner und Vuk Karadzics Ansichen zu schaffen. In Grimms Ansichten über die Entstehung der Schriftsprache stehen die kulturellen Faktoren im Vordergrund. Eine kulturtragende Mund- art entwickelt sich über eine Art synthetischen Dialekts zur Schriftsprache durch ihre Pflege in der Literatur. Obwohl Grimm auch andere gesellschaftliche Voraussetzungen für die Entstehung der Schriftsprache gesehen hat, zeigt er eine etwas unrealisti- sehe Einschätzung der Macht der Dichter, die offensichtlich von der romantischen Dichterauffassung beeinflußt is t. Grimm hat die Schriftsprache als Mittel der geistigen und kultu- rellen Entwicklung verstanden. Er war gegen den Dualismus der Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM Grimm hat die Schriftsprache als Mittel der geistigen und kultu- rellen Entwicklung verstanden. Er war gegen den Dualismus der Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Schriftsprache als einer besonderen Literatursprache und einer ge- sprochenen Variante. Die Schriftsprache, als jedem erwerbbare Norm, wird durch die lebendige Pflege in der Sprachgemeinschaft und die "sprachgewalt" hervorragender Dichter1 weiterentwickelt. Die B il- dung der Sprache wirkt auf die Volksbildung zurück. 1 Er nannte Goethe einen solchen Dichter. -Vgl. "Rede auf Schiller" von 1859, GKS I, S. 390. 2 A. Schleicher war, entgegen der Behauptung von Beneä, also nicht der erste, wenn er auch umfangreichere slavistische Arbeiten als Grimm geliefert hat. - Vgl. Benes, a.a.O., S. 121. Auffassung des geistigen Charakters der Sprache. Die im Grün- de richtigen Vorstellungen von Grimm gehen allerdings von einem auch heute nicht verwirklichten Bildungsideal aus. Grimm hat das Verhältnis zwischen der Schriftsprache und den Mundarten im wesentlichen richtig gesehen: Die Schriftsprache als allgemeinverbindliche Norm muß gegenüber den Mundarten abgegrenzt werden. Die ehemals gleichwertigen Mundarten betrachtet er im Ver- gleich mit der kulturtragenden, zum Zeichen der Bildung geworde- nen Schriftsprache als sozial "gesunken". Grimms Irrtümliche Gleichsetzung der Sprache mit der Nation is t zeitbedingt. Seine Bemühung, über die Sprache zur Einigung der deutschen Nation beizutragen, entspringt der Auffassung von der aktiven Rolle des Wissenschaftlers in der Geschichte des Volkes. Jacob Grimm war der erste deutsche Sprachforscher, der sich der Slavistik gewidmet und dabei eine slavische Sprache, nämlich 2 die serbische, auch praktisch recht gut beherrscht hat . Es ent- sprach seiner Natur und seinem wissenschaftlichen Weitblick, sich der vernachlässigten Sprachen und Forschungsgebiete anzunehmen, zu denen damals, als die Indogermanistik im Mittelpunkt des Inter- esses stand, auch die deutsche Sprache gehörte. Seine historische Erforschung der deutschen Sprache hat zu Entdeckungen geführt, die bewußt gemacht haben, daß der großen Synthese eingehende Ein- zelanalysen vorausgehen müssen, d. h. vor der Rekonstruktion der uridg. Sprache die ältesten Formen der Einzelsprachen erschlossen werden müssen. Der von ihm gewiesene Weg wurde von den Junggramma- tikern fortgesetzt. Grimms Interesse für die Slavistik is t aus seiner Z Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access beit mit Vuk Karadzic erwachsen. Wir haben festgestellt, daß er sich bis zum Ende seines Lebens mit ihr beschäftigt hat. Seine ausführlichsten Arbeiten auf dem Gebiet der serbischen Sprache sind seine Überarbeitung von Vuks Grammatik und seine Vor- rede. Wir haben festgestellt, daß die deutsche Fassung der serbi- sehen Grammatik durch Grimms Beitrag an wissenschaftlicher Quali- tät gewonnen hat: die Lücken wurden gefüllt und die überflüssigen Exkurse beseitigt, so daß die "Kleine serbische Grammatik" im Ver- gleich mit dem Original eine größere Vollständigkeit in der Darstel- lung des grammatischen Stoffes und eine übersichtlichere, gelunge- nere Komposition aufweist. Grimm hat bereits in der "Vorrede" eine bemerkenswerte slavi- stische Leistung mit der etymologischen Erklärung der slavischen reduzierten Vokale erreicht. Er hat darüber hinaus einige Etymolo- gien geliefert, die Miklosich unter Berufung auf ihn in sein alt- slavisches Wörterbuch übernommen hat. 1 Dazu kommen seine,nicht zu unserem Thema gehörenden Erfahrungen aus der Beschäftigung mit der damals noeti lebendigen serbischen Volksdichtung, an der er nach eigenen Worten das Wesen des Epos studieren wollte. Seine Deutung des kollektiven Charakters der Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Auffassung des geistigen Charakters der Sprache. Auch Grimms Korrektur von Vuks Deutung der Substantivendung nach Zahlen und seine Definitio- nen der ersten und zweiten Palatalisation reichen über die Serbi- stik hinaus. Es entsprach seiner vergleichenden Methode, daß er die Beschäftigung mit der serbischen Sprache in slavistische For- schung ausweitete. Seine Eintragungen im Handexemplar der "Kleinen serbischen Grammatik" veranschaulichen diesen Weg. Die Zusammenarbeit mit Vuk hat positive Ergebnisse für Grimms wissenschaftliche Arbeit gebracht. Sie sind in erster Linie darin zu sehen, daß Grimms vergleichende Methode durch Einbeziehung der slavischen Sprachen eine wesentliche Erweiterung erhalten hat. Die slavischen Sprachen gehören neben der griechischen und latei- nischen zu den am meisten herangezogenen Fremdsprachen außerhalb der von ihm weitgefaßten germanischen Sprachgruppe. Grimm hat aus seiner Zusammenarbeit mit Vuk auch andere Anre- gungen geschöpft. Sie haben in seinem Versuch der Orthographiere- form und seiner Konzeption des "Deutschen Wörterbuchs", in dem er die Teilung in eine "edle" und eine "pöbelhafte" Sprache aufgeho- ben hat, ihren Niederschlag gefunden1. 1 Dazu kommen seine,nicht zu unserem Thema gehörenden Erfahrungen aus der Beschäftigung mit der damals noeti lebendigen serbischen Volksdichtung, an der er nach eigenen Worten das Wesen des Epos studieren wollte. Seine Deutung des kollektiven Charakters der Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access via free access Vuk Karadžifcs Sprachauffassung haben wir im Zusammenhang mit der Entwicklung seiner Ansichten über die serbische Schriftspra- che betrachtet. In dieser Entwicklung is t vom Anfang bis zum En- de eine Konstante vorhanden, nämlich die Auffassung, daß die Schriftsprache auf der Grundlage der Volkssprache geschaffen wer- den soll. Im Laufe der Zeit ändern sich Vuks Begriff der Volks ־ spräche und seine Ansichten darüber, wie der Weg zur serbischen Schriftsprache verwirklicht werden kann. Diese Veränderungen in seinen Ansichten führen zu Korrekturen seiner Konzeption der Schriftsprache. Vuk versteht unter der Volkssprache bis in die Mitte der 30er Jahre die Dorf spräche• Er wählt sie zum Vorbild für die Schrift- spräche, weil sie die reine serbische Sprache darstelle, während die städtische Sprache durch fremde Einflüsse verdorben sei. In Vuks Wahl der Sprache des einfachen Volkes zum Vorbild spiegelt sich der Einfluß von Herders Sprachauffassung wider,namentlich die Forderung nach der Rückkehr zur Volkssprache, in der Herder das Ursprüngliche bewahrt sah. In Vuks Auffassung, daß nur ein- fache, ungebildete Menschen neue Wörter schaffen können, wirkt der Rousseauisch-Herderische Begriff des Naturmenschen. Aus die- ser Auffassung erwächst Vuks Ablehnung der Neubildung von Wör- tern durch die Gebildeten. Epik aus ihrer formelhaften Struktur steht den modernen, von A. Lord und R. Jakobson vertretenen Ansichten nahe. - Vgl. Grimms Rezensionen von Vuks Ausgaben der Volksdichtung in GKS IV, V, VIII, bes. IV, S. 197-205, und A. Lord, Der Sänger erzählt. Wie ein Epos entsteht, 1965; R. Jakobson, Die Folk ־ lore als eine besondere Form des Schaffens. In: ders., Se ־ lected Writings IV, 1966, S. 1-15. Auffassung des geistigen Charakters der Sprache. Auf Grund der Ansicht von der Rein- heit der Sprache des einfachen Volkes fordert Vuk von den Schrift- stellern die Treue zur Volkssprache. Vuk sieht in bezug auf die Sprache die Gebildeten und die Schriftsteller in einem Gegensatz zum Volk. Herders Auffassung der Sprache als Denkart und als Ausdruck des "Nationalcharakters" taucht in Vuks Ansicht auf, daß die serbischen Schriftsteller wegen ihrer fremdsprachigen Ausbildung nicht serbisch denken und sprechen können und daß sie ihre nationale Eigenart verloren ha ־ ben. Er nennt sie Sprachverderber. Epik aus ihrer formelhaften Struktur steht den modernen, von A. Lord und R. Jakobson vertretenen Ansichten nahe. - Vgl. Grimms Rezensionen von Vuks Ausgaben der Volksdichtung in GKS IV, V, VIII, bes. IV, S. 197-205, und A. Lord, Der Sänger erzählt. Wie ein Epos entsteht, 1965; R. Jakobson, Die Folk ־ lore als eine besondere Form des Schaffens. In: ders., Se ־ lected Writings IV, 1966, S. 1-15. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access In Vuks Argumentation gegen die Schriftsteller ist der Ein- fluß von A. Šiskovs Äußerungen über die russischen Schriftstel- 1er spürbar. Nach Vuks wie auch Siskovs Ansicht verderben die Schriftsteller die Sprache, weil sie, wegen ihrer fremden Denk- art und Unkenntnis der eigenen Sprache, Fremdwörter einführen oder neue Wörter nach fremdem Vorbild schaffen. Vuks Forderung nach der Sprache des einfachen Volkes als der reinen serbischen Sprache erscheint analog mit šiškovs Verteidigung des "korennyj russkij jazyk". Beide entwickeln aus diesen Grundsätzen einen engen Purismus, den die spätere Sprachentwicklung überwunden hat Vuks Ansichten über die Reinheit der Volkssprache prägen auch seine Haltung gegenüber den ksl. Elementen in der Schrift- spräche. Zu Beginn zeigt er eine gewisse Unentschlossenheit, die unter dem Einfluß der slavenoserbischen Sprachpraxis steht. Seine Forderung nach der Serbisierung der ksl. Elemente macht aber den Unterschied zwischen seinen und zeitgenössischen Vorstellungen vom *1mittleren S til״* deutlich. Vuks Auffassung der Beziehung zwischen der serbischen und der ksl. Sprache bedeutet von Beginn der 20er Jahre an eine scharfe Trennung der beiden Sprachbereiche, sowohl in Bezug auf die Pho- netik und Morphologie als auch die Lexik. Dieses Prinzip hat in den 20er Jahren positive Folgen für die Festigung der phone- tisch-morphologischen Struktur der sich bildenden serbischen Schriftsprache gehabt. Vuks Ablehnung der lexikalischen Entleh- nung für abstrakte Begriffe, für die die Volkssprache keine Be- Zeichnungen kennt, is t jedoch puristisch. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Auffassung des geistigen Charakters der Sprache. Vuks Haltung gegenüber den Entlehnungen und Lehnbildungen ist auch in der Endphase puristisch geblieben und hat negative Folgen für die junge Schriftsprache gehabt. Obwohl er in der ei- genen Praxis, der Bibelübersetzung, von den puristischen Prinzi- pien abgewichen is t, hat er keine theoretischen Konsequenzen daraus gezogen. Vuks Purismus in der Endphase ist ein Zeichen da- für, daß er die Kriterien der Funktionalität der Schriftsprache außer acht gelassen hat. Wir haben gesehen, daß der von ihm ab- gelehnte Wortschatz der damaligen Schriftsteller sich dennoch in der Schriftsprache durchgesetzt hat. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 198 - Vuks Bemühungen um die Kodifizierung der Volkssprache als Schriftsprache zeigen im Verlaufe der Zeit einen veränderten Be- g riff der Volkssprache. Zu Anfang setzte Vuk Volks- und Schrift- spräche gleich. Aus seiner ungenügenden Kenntnis der Dialektsi- tuation heraus nahm er an, daß die Volkssprache, bis auf die un- terschiedliche Vertretung des e , überall gleich sei. Die während der Arbeit am Wörterbuch erweiterten Kenntnisse der Dialekte ver- langten eine neue Bestimmung des Verhältnisses zwischen Volks- und Schriftsprache. Vuk suchte, offenbar unter Kopitars Einfluß, die Lösung in der Zulassung aller Dialekte. In seiner Argumenta- tion, daß bei allen Völkern, so bei den Griechen und den Deut- sehen, in allen Dialekten geschrieben wurde, bevor sich einer von ihnen als Schriftsprache durchsetzte, klingen Grimms Gedanken über die Entstehungsgeschichte der deutschen Schriftsprache an. Auch die bereits hier erscheinende, später oft wiederholte Auf- fassung, daß die Schriftsprache nicht durch Mischung der Dialekte gebildet werden soll, steht im Einklang mit Grimms Ansichten. Vuks Berufung auf die Evolution der deutschen Sprache vor der Schaffung der einheitlichen schriftsprachlichen Norm und sein gleichzeitiger Kodifizierungsversuch, den er mit «einer Gramma- tik und dem Wörterbuch von 1818 unternommen hat, führten bei ihm zu Widersprüchen zwischen Theorie und Praxis, die sich darin äußerten, daß er die von seiner Norm abweichenden mundartlichen Züge als Fehler behandelte. In der zweiten Hälfte der 30er Jahre hat Vuk zwei Korrekturen an seiner Norm vorgenommen (Einführung des h , Abschaffung der sog. ”neuesten Jotierung"). Sie spiegeln die neue Entwicklung in seiner Konzeption der Schriftsprache wider. Unter der Berufung auf die Dubrovniker Sprache und literarische Tradition gibt er bewußt das Prinzip der Vorbildlichkeit der Dorfsprache zugunsten der Stadtsprache auf. Auffassung des geistigen Charakters der Sprache. Diese Wende deutet auf Vuks Einsicht hin, daß die Anforderungen an die moderne Schriftsprache über den in der Folklore und in der dörflichen Lebensform abgesteckten Rahmen hinausgehen. Zu dieser Wende hat auch Jacob Grimm durch seine Be- tonung der Rolle der Literatur in der Entwicklung der Schriftspra- che beigetragen. Grimm hat seine Ansichten darüber auch in der Rezension von Vuks Liedern geäußert, nämlich daß ״sobald sich Ser- bien zur cuitur emporarbeitet", die Volkslieder zu kostbaren Re- liquien und ihre Sprache zur Geschichte gehören werden. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 199 - Vuk hat durch die Aufnahme von Elementen der Dubrovniker Spra- che in seine Norm einen synthetischen Dialekt geschaffen, der mit keinem der Volksdialekte völlig identisch war. Sein Begriff der Volkssprache bedeutet in der Endphase, daß die Schriftsprache aus Elementen der Volkssprache zusammengesetzt ist. Vuk hat für den synthetischen Dialekt das Prinzip der allge- meinen grammatischen Regelmäßigkeit aufgestellt. Wir haben gese- hen, daß er bei der Bestimmung der grammatischen Regelmäßigkeit der zur Wahl stehenden mundartlichen Züge die diachronisch-verglei- chende Methode heranzog, deren Ansätze wir bereits in seinen durch Grimm angeregten Arbeiten aus den 20er Jahren festgestellt haben. Vuks Konzeption des synthetischen Dialekts is t eine eingehen- de Beschäftigung mit den Dialekten vorausgegangen. Seine Kenntnis der Unterschiede zwischen den stokavischen Mundarten hat ihn zu der Einsicht geführt, daß eine einheitliche Schriftsprache nicht durch Pflege aller Dialekte geschaffen werden kann. Vuk hat in seine Dialektforschung auch kroatische Dialekte einbezogen. Wir haben festgestellt, daß er sich auch mit dem kajkavischen Dialekt beschäftigt und einen guten Überblick über dessen Eigentümlich- keiten besessen hat. Mit der Schaffung des synthetischen Dialekts hat Vuk, zusam- men mit den Illyrern, die Idee einer einheitlichen Schriftsprache für ein Volk der Serben und Kroaten verfolgt, die in der Sprach- Vereinbarung von Wien auch formuliert wurde. In dieser Idee wirkt die Herderische Gleichsetzung von Sprache und Volk nach, die wir auch bei Jacob Grimm festgestellt haben. Bei dem Versuch, einen Vergleich zwischen Grimm und Vuk zu ziehen, drängt sich zunächst der Eindruck ihrer Unterschiedlich- keit auf. Dieser Eindruck entsteht bereits in Anbetracht des un- terschiedlichen Umfangs ihres Werkes, in erster Linie der beiden Grammatiken und Wörterbücher. Der 42 Seiten umfassenden serbi- sehen Grammatik von Vuk steht die vierbändige, fast dreieinhalb- tausend Seiten enthaltende Grammatik von Grimm gegenüber. 1 Das von B. Danicic begonnene und von der JAZU gerade beendete Wörterbuch der kroatischen oder serbischen Sprache wurde offen- sichtlich nach dem Vorbild des Grimmischen Wörterbuchs konzi- p ie rt. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Auffassung des geistigen Charakters der Sprache. Auch Vuks Wörterbuch der serbischen Volkssprache kann dem von Grimm konzipierten und begonnenen sechzehnbändigen historischen Wörter- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 00051004 - 200 ־ buch der deutschen Schriftsprache nicht zur Seite gestellt wer- den^. Dem unterschiedlichen äußeren Umfang entspricht auch der unterschiedliche wissenschaftliche Gehalt ihres Werkes. Der ide- ellen V ielfalt von Grimm, der die Sprache als Phänomen des menschlichen Geistes und als konkrete Einzelsprache, sowohl ge- schichtlich als auch in ihrem gegenwärtigen Zustand betrachtet, steht Vuks Konzentration auf die Problematik der zu begründen- den serbischen Schriftsprache gegenüber. Grimms Herkunft aus ei- ner hochentwickelten Kultur mit reger geistiger Auseinanderset- zung bot einen völlig anderen Ausgangspunkt für wissenschaftli- che Tätigkeit, als ihn der Autodidakt Vuk im damaligen, überwie- gend bäuerlichen Serbien besaß, wo nicht die geistige Auseinander- setzung, sondern der Freiheitskampf gegen die Türken das Leben beherrschte. Grimm konnte auf eine entwickelte Wissenschaft zu- rückgreifen, an die in der Aufklärung und der Romantik aufgewor- fenen Fragen anknüpfen und sie weiterentwickeln. Vuk dagegen stand am Beginn einer Neuentwicklung, für die er selbst die er- sten Grundsteine zu legen hatte. Diese unterschiedlichen kulturellen Voraussetzungen spie- geln sich auch in dem Entwicklungsgang von Grimms und Vuks Sprach- auffassungen wider. In Grimms Entwicklung zeigen sich ideelle Ge- gensätze zwischen der frühen und späten Phase, die im Einklang mit den Gegensätzen der zeitgenössischen Geistesentwicklung in Deutschland stehen. Grimms Entwicklung verlief von einer mytholo- gisch ausgerichteten Romantik zu der von Herders Gedanken gepräg- ten, dem Volk zugewandten Spätromantik; bei ihm sind sowohl auf- klärerische als auch romantische Züge und eine gewisse Verbin- dung zur naturwissenschaftlichen Schule vorhanden. Vuks Ansich- ten sind in erster Linie von der Spätaufklärung geprägt, nament- lieh von den Ideen Herders, die von der Spätromantik wieder auf- gegriffen wurden. Seine Entwicklung zeigt keine großen ideellen Sprünge, sondern aus der Praxis gewonnene Korrekturen. Grimms und Vuks Ansichten über die Entwicklung der Schrift- spräche zeigen bis in die zweite Hälfte der 30er Jahre mehr Un- terschiede als Gemeinsamkeiten. Während bei Grimm die Wirkung 1 Das von B. Danicic begonnene und von der JAZU gerade beendete Wörterbuch der kroatischen oder serbischen Sprache wurde offen- sichtlich nach dem Vorbild des Grimmischen Wörterbuchs konzi- p ie rt. Auffassung des geistigen Charakters der Sprache. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access der kulturellen Faktoren, namentlich die Bedeutung der Schrift- tradition in den Vordergrund t r it t , macht Vuk die Sprache des einfachen Volkes zum Vorbild für die Schriftsprache. Grimm hebt die "Sprachgewalt" des dichterischen Genies und die schöpferische Kraft der Poesie bei der Sprachbildung hervor; in seiner Sprach- auffassung wirkt der romantische Poesiebegriff nach. Für Vuk is t das einfache Volk der Sprachschöpfer, in seiner Sprachauffassung spiegeln sich Herders Ansichten über die Volkssprache wider. Nach Vuks Korrektur seiner Sprachkonzeption in den 30er Jahren, d. h. nach seiner Verbindung von Dorf- und Stadtsprache und der Einbe- Ziehung der Dubrovniker Literatursprache erscheinen seine und Grimms Ansichten über die Schriftsprache als nah verwandt. Aus den genannten Unterschieden in Grimms und Vuks Sprachauf- fassungen ergibt sich auch ihre unterschiedliche Beurteilung der Rolle der Dichter bei der Sprachbildung. Der von der romantischen Poesieauffassung beeinflußten, etwas unrealistischen Überschätzung der Macht der Dichter bei Grimm steht Vuks Unterschätzung ihrer Rol- le in der Entwicklung der Schriftsprache gegenüber. Grimm steht als Zeitgenosse von Goethe und Schiller unter dem Eindruck einer blühenden Literatur, deren reiche Tradition durch Neuentdeckungen und Lachmanns, Beneckes und seine eigenen Ausgaben bestätigt wurde. Vuks Haltung gegenüber den Schriftstellern is t dagegen durch das von ihm bekämpfte slavenoserbische Sprachchaos von vornherein negativ geprägt. Seine Beurteilung der Dichter is t auch von dem durch die Spätaufklärung geschaffenen Bild des vom Volk entfrem- deten Schriftstellers beeinflußt. Grimm und Vuk sehen die Sprache als Kommunikationsmittel an. Der größte und wichtigste Unterschied in ihren Ansichten zeigt sich aber in der Beurteilung der Sprache als Medium der Kunst. Bei Grimm erscheinen, im Einklang mit seiner Auffassung der Spra- che als der Trägerin der geistigen Entwicklung, die beiden Funk- tionen der Sprache als gleichbedeutend und sich gegenseitig för- dernd. Bei Vuk steht die Sprache als Kommunikationsmittel im Vor- dergrund; er zeigt bei der Anwendung des Kriteriums der reinen Volkssprache auf die Literatur ein gewisses Nichtbegreifen der Funktion der Sprache als Kunst. Grimm und Vuk zeigen viele gemeinsame Ansichten. Die Gemein- samkeiten sind einerseits durch Herders Einfluß sowohl auf Grimm Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access als auch auf Vuk, andererseits durch die aus ihrer Zusammenar-- beit erwachsene gegenseitige Beeinflussung erklärbar. als auch auf Vuk, andererseits durch die aus ihrer Zusammenar-- beit erwachsene gegenseitige Beeinflussung erklärbar. 1 Grimm meinte: "Die spräche überhaupt in eine erhabne, edle, tra- uliche, niedrige und pöbelhafte zu unterscheiden taugt nicht... des ausdrucks pöbelhaft (plebejum) im sinne von bäurisch sollte man sich enthalten, seit das volk (populus) und das volksmäszi- ge als merkmal des freien erkannt worden is t." (GKS VIII,S.337f.) 1 Aus raumsparenden Gründen werden hier arabische Ziffern an Stelle der römischen eingeführt. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM Auffassung des geistigen Charakters der Sprache. Grimm und Vuk vertreten die Herderische Auffassung der Spra- che als Denkart des Volkes und die Ansicht über die Identität von Sprache und Volk. Bei beiden wirkt Herders Forderung nach Rück- kehr zur Volkssprache nach: Vuk wendet sie zunächst konsequent an, indem er Volks- und Schriftsprache gleichsetzt; bei Grimm findet sie ihren Niederschlag in der Einbeziehung der gesprochenen Volks- spräche im ״,Deutschen Wörterbuch". Grimm und Vuk streben einen volkssprachlichen Charakter der Schriftsprache an. In bezug auf die deutsche Sprache, in der be- reits eine schriftsprachliche Norm existierte, konnte das nur ei- ne Demokratisierung der Schriftsprache bedeuten, in dem Sinne, daß auf dem Gebiet der Lexik die Adelungische Teilung in eine ed- le und eine pöbelhafte Sprache überwunden wurde1. Der volkssprach- liehe Charakter der Schriftsprache bleibt nur auf dem Wege der allgemeinen Bildung erreichbar. Der Begriff des volkssprachlichen Charakters der Schriftspra- che hat durch Vuk entsprechend der Sprachsituation bei den Serben eine unterschiedliche Anwendung erfahren. Da die Serben keine allgemeingültige schriftsprachliche Norm besaßen, konnte Vuk bei der Begründung der Schriftsprache die Herderische Vorstellung von der Sprache des einfachen Volkes und der Volkslieder im we- sentlichen verwirklichen. Grimms und Vuks methodische Verfahren weisen, trotz der vor- herrschenden Unterschiedlichkeit, Berührungspunkte auf, in denen sich der gegenseitige Einfluß zeigt. Gemeint sind die synchroni- sehen Ansätze bei Grimm, insbesondere sein Versuch der Orthogra- phiereform, und die Heranziehung der Sprachgeschichte und Sprach- Vergleichung bei Vuk. Beide, Grimm und Vuk, haben darüber hinaus die Auffassung von der aktiven Rolle des Wissenschaftlers in der Geschichte des Volkes vertreten und auch in die Praxis umgesetzt. 1 Grimm meinte: "Die spräche überhaupt in eine erhabne, edle, tra- uliche, niedrige und pöbelhafte zu unterscheiden taugt nicht... des ausdrucks pöbelhaft (plebejum) im sinne von bäurisch sollte man sich enthalten, seit das volk (populus) und das volksmäszi- ge als merkmal des freien erkannt worden is t." (GKS VIII,S.337f.) Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 203 - ANHANG I Verzeichnis der Abweichungen der deutschen Fassung (1824) von Vuks Original (1818) der serbischen Gramnnatik 1 Aus raumsparenden Gründen werden hier arabische Ziffern an Stelle der römischen eingeführt. Vera Boji - 9783954793044 ANHANG I Verzeichnis der Abweichungen der deutschen Fassung (1824) von Vuks Original (1818) der serbischen Gramnnatik ANHANG I Verzeichnis der Abweichungen der deutschen Fassung (1824) von Vuks Original (1818) der serbischen Gramnnatik NG I der deutschen Fassung (1824) serbischen Gramnnatik Deutsche Fassung Seite: [Hier und im weiteren Text sind al־ le im Original enthaltenen Defini- tionen und Angaben in lateinischer Sprache ausgelassen.] ... 28 einfache Laute.., die mit folgenden Buchstaben geschrieben und ausgesprochen werden, wie nach■ steht: Aussprache Gedrucktes grie-/latei- chi- /ni- sches/sches Alphabet. A а а wie das deutsche a Б б b wie das deutsche b В в V wie das deutsche w Г г g wie das deutsche g Д д d wie das deutsche d ъ Ь dj ungefähr wie dj (unga- risch ду) Е е e wie das deutsche e Жж x ungefähr wie gelindes sch (franz. j, ungar.zs) 3 3 z wie ein sanftes z (franz z) wie das deutsche i И и i J •כ •נwie das deutsche j К к к wie das deutsche к Л л 1 wie das deutsche 1 Jb л Íj ungefähr wie l j (ita l. gl, ungar, ly) Mм m wie das deutsche m H н n wie das deutsche n н>н> nj ungefähr wie nj (ita l. дп, ungar, ny) 0 о o wie das deutsche о П п P wie das deutsche p Р р r wie das deutsche r С с s wie ein scharfes s (ss) Т т t wie das deutsche t hier arabische Ziffern an Stelle Original: Deutsche Fassung [Anm.] Pa je to narod uzeo и podsmije i и svima pripovijet- каша, fle se kaže da je što ka ־ luder govorio, tako se izgova- raju (još kroz nos) ta slova. 30 [Anm.]... kao i vkus medu lju ־ dima što je jednom narodu blagoglasno, to drugome može b iti najprotivnije,.. [Anm.]... kao i vkus medu lju ־ dima što je jednom narodu blagoglasno, to drugome može b iti najprotivnije,.. 4 ... Griechen und Italiener würden daran die Zähne verbeißen. [Anm.] Dieß ist eine allgemeine und natürliche Regel. Die griechische und lateinische Sprache sind voll davon, z.В................bespokoj, nishoždu i proč. 31 5 In Briefen gemeinder Serben, die ni< von Grammatik gehört haben, las ich. Und wenn ein d oder t: p Ja sam Čatio u knjigama, što su pisali prosti Srblji, ko- j i nijesu čuli za etimologi- ju ... A kad dode d i i i t: a) pred c, č, dŽ, onda se svagda izbaci, n.p. ... patka, pače... a) vor c, č, dž zu stehen käme, dam wird es immer weggeworfen, z.B. pat- ka, die Ente, Diminut. pačiea... d u c z и ž i u g, n.p. ... nate- zati, nateŽem, nategnuti;.. 7 z - Ž und in g, z.В.. . .natezati, anziehen (frequentativum), natežem, ich ziehe an; nategnuti, anziehen (perfectivum);.. Deutsche Fassung Deutsche Fassung Seite: Seite: [Hier und im weiteren Text sind al־ le im Original enthaltenen Defini- tionen und Angaben in lateinischer Sprache ausgelassen.] ... 28 einfache Laute.., die mit folgenden Buchstaben geschrieben und ausgesprochen werden, wie nach■ steht: 28 prosti glasova. koji se najpametnije mogu zapisati evo ovim slovima a, b, • • Aussprache sches/sches Alphabet. A а а wie das deutsche a Б б b wie das deutsche b В в V wie das deutsche w Г г g wie das deutsche g Д д d wie das deutsche d ъ Ь dj ungefähr wie dj (unga- risch ду) Е е e wie das deutsche e Жж x ungefähr wie gelindes sch (franz. j, ungar.zs) 3 3 z wie ein sanftes z (franz. z) wie das deutsche i И и i J •כ •נwie das deutsche j К к к wie das deutsche к Л л 1 wie das deutsche 1 Jb л Íj ungefähr wie l j (ita l. gl, ungar, ly) Mм m wie das deutsche m H н n wie das deutsche n н>н> nj ungefähr wie nj (ita l. дп, ungar, ny) 0 о o wie das deutsche о П п P wie das deutsche p Р р r wie das deutsche r С с s wie ein scharfes s (ss) Т т t wie das deutsche t hier arabische Ziffern an Stelle Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Aus raumsparenden Gründen werden hier arabische Ziffern an Stelle der römischen eingeführt. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 204 - bh ch ungefähr wie t j (ungar. ty) nicht ganz tsch, son■ dern gelinder und wenige] zischend, etwa tch, genai wie das schwedische к vor e, i, y- У У и wie das deutsche и ц ц с wie das deutsche z (tz) ч ч CS wie das deutsche tsch и и fehlt wie ita l. ge ш ш sh wie das deutsche sch Slova se ova razdjeljuju na samoglasna i na poluglasna. Slova se ova razdjeljuju na samoglasna i na poluglasna. ... n. p. r-da, r-va-nje,.. 2 z. B. r 3־a, der Rost? r-ka, das Schnarchen; r-va-nje, das Ringen,.. 2 z. B. r 3־a, der Rost? r-ka, das Schnarchen; r-va-nje, das Ringen,.. 7 z - Ž und in g, z.В.. . .natezati, anziehen (frequentativum), natežem, ich ziehe an; nategnuti, anziehen (perfectivum);.. ־ Sveto pismo kaže, da niko ne može dvëma gospodinoma rabo ־ ta ti, a dekoji bi Ijudi celi, da im slova pokazuju i kako se rije č i izgovaraju, i šta zna- če, i od kuda su postalel Kvintilijan kaže, da su slova samo zato izmišljena, da se njima zapisuju rije č i onako kao što se izgovaraju; i to su znali,i sad znadu svi pra- vi gramatici i и jeziku vešti spisatelji i knjiŽevnici; sa ־ mo one nadriknjige (fušeri književni), koji su nešto na- u č ili, pa misle da več ništa пета što ne znadu, ne mogu da to razumiju i da priznadu. m - v, n.p. tama, tavnica... (no ovo je več etimologičeska premjena) i t.d. [ Anm.] ... zbornik... jetzt, da die Aussprache auszubessern ange- fangen wird, ist auch dieses ver- bessert, und sbornik gemacht! 8 к ־c, in с und in 6, z.B. ... peko, ich habe gebraten; junak... 32 Allein dieser Grundsatz is t ohne ־ hin nicht durchzuführen. oder sind alle diese hergebrach ־ ten und angenommenen Schreibungen zu verdammen? Ako stanemo metati po jedno jer (b) kod g, z, к, s, с onda nam treba načiniti još nekoliko srednjije i l i mršavije jerova; pa cemo tek na pošljetku vi- deti, da se opet ne može za- pisati kao što bi se celo. ־ Sveto pismo kaže, da niko ne može dvëma gospodinoma rabo ־ ta ti, a dekoji bi Ijudi celi, da im slova pokazuju i kako se rije č i izgovaraju, i šta zna- če, i od kuda su postalel Kvintilijan kaže, da su slova samo zato izmišljena, da se njima zapisuju rije č i onako kao što se izgovaraju; i to su znali,i sad znadu svi pra- vi gramatici i и jeziku vešti spisatelji i knjiŽevnici; sa ־ mo one nadriknjige (fušeri književni), koji su nešto na- u č ili, pa misle da več ništa пета što ne znadu, ne mogu da to razumiju i da priznadu. 9 m in v: z.B. tama, die Finsterniß, tavnica, das Gefängnis;., (aber dieß g ilt nicht allgemein, da Einige auch tamnica, u.s.w. sagen). m - v, n.p. tama, tavnica... (no ovo je več etimologičeska premjena) i t.d. m - v, n.p. tama, tavnica... (no ovo je več etimologičeska premjena) i t.d. s ־s: z.B. ... umjestiti, stellen, umjeitati (eben das, frequentativ) 10 n ־n j:.. 7 z - Ž und in g, z.В.. . .natezati, anziehen (frequentativum), natežem, ich ziehe an; nategnuti, anziehen (perfectivum);.. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 00051004 ־ 205 ־ [Anm.] ... zbornik* .., a sad od kako je jezik počeo poprav ־ lja ti, i to je popravljeno da bude sbornikb Í к - ć , с i и č, n.p. ... peko, peci, peče, peci; junak... 32 [Anm.]... Ajde mogio bi i to b it i. . . a ne oč, knež, ra- došč i žalošč... i od pišati... Ako stanemo metati po jedno jer (b) kod g, z, к, s, с onda nam treba načiniti još nekoliko srednjije i l i mršavije jerova; pa cemo tek na pošljetku vi- deti, da se opet ne može za- pisati kao što bi se celo. ־ Sveto pismo kaže, da niko ne može dvëma gospodinoma rabo ־ ta ti, a dekoji bi Ijudi celi, da im slova pokazuju i kako se rije č i izgovaraju, i šta zna- če, i od kuda su postalel Kvintilijan kaže, da su slova samo zato izmišljena, da se njima zapisuju rije č i onako kao što se izgovaraju; i to su znali,i sad znadu svi pra- vi gramatici i и jeziku vešti spisatelji i knjiŽevnici; sa ־ mo one nadriknjige (fušeri književni), koji su nešto na- u č ili, pa misle da več ništa пета što ne znadu, ne mogu da to razumiju i da priznadu. [Anm.] ... zbornik* .., a sad od kako je jezik počeo poprav ־ lja ti, i to je popravljeno da bude sbornikb Í к - ć , с i и č, n.p. ... peko, peci, peče, peci; junak... 32 [Anm.]... Ajde mogio bi i to b it i. . . a ne oč, knež, ra- došč i žalošč... i od pišati... Ako stanemo metati po jedno jer (b) kod g, z, к, s, с onda nam treba načiniti još nekoliko srednjije i l i mršavije jerova; pa cemo tek na pošljetku vi- deti, da se opet ne može za- pisati kao što bi se celo. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 7 z - Ž und in g, z.В.. . .natezati, anziehen (frequentativum), natežem, ich ziehe an; nategnuti, anziehen (perfectivum);.. (Aber dieß g ilt eben ־ falls nicht allgemein). 71 71 ... Ali buduči da u pisanju ovako daleko nijesu iš li ni Grci ni Latini, zato nijesam ni ja čeo (ni smjeo; a dokle su oni iš li, mislim da je slo- bodno i meni, i svakome dru- дот); a drugi ко и napredak ako ćedbude tako pisati, može slobodno: ni jedan mu pametan čovek ne može reći da пета pravo: zašto se tako govori; 71 71 ... Ali buduči da u pisanju ovako daleko nijesu iš li ni Grci ni Latini, zato nijesam ni ja čeo (ni smjeo; a dokle su oni iš li, mislim da je slo- bodno i meni, i svakome dru- дот); a drugi ко и napredak ako ćedbude tako pisati, može slobodno: ni jedan mu pametan čovek ne može reći da пета pravo: zašto se tako govori; 11 [Punkt 1) aus dem "Dodatak” ] ... Weil aber im Schreiben weder Grie- chen, noch Lateiner so weit gegan ־ gen sind, habe ich noch nicht so zu schreiben gewagt, glaube aber, daß es mir oder Ändern in Zukunft erlaubt sein wird, der allgemeinen Aussprache gemäßer zu schreiben; 11 [Punkt 1) aus dem "Dodatak” ] ... Weil aber im Schreiben weder Grie- chen, noch Lateiner so weit gegan ־ gen sind, habe ich noch nicht so zu schreiben gewagt, glaube aber, daß es mir oder Ändern in Zukunft erlaubt sein wird, der allgemeinen Aussprache gemäßer zu schreiben; Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ašnjimnalazi se ѵеб i u knjiga- ma űekojim (n.p. и Dositeovim, i u G. profesora Ignjatovica). [Anm.]... zašto su oni viűeli de Nijemci ovako krpe i nateZu и njiovom jeziku, pa bi i oni radi da uvedu и svoj. Ovo je öetvrta služba, koja se iste od slova, t . j . da pokazuju i razliku glasa и rijeCma! ... ono je. Sto se nalazi и Srbuljama... h ... n.p. ora, Via, lad, rom, rabar; manuti, dunuti, maovina, duovnik i t.d. ... suv, gluv, duvati... smej i t.d. šnjim kann man schon in einigen Büchern, z.B. bei Dositheus und Ignjatowitsch finden. šnjim kann man schon in einigen Büchern, z.B. bei Dositheus und Ignjatowitsch finden. 13 ...jenes je (welches in den Srbu- ljama zu finden is t)... [Anm.] Altes Kirchenbuch, das den Serbischen Dialekt näher is t, als die neuen Auflagen. h ... n.p. 7 z - Ž und in g, z.В.. . .natezati, anziehen (frequentativum), natežem, ich ziehe an; nategnuti, anziehen (perfectivum);.. ora, Via, lad, rom, rabar; manuti, dunuti, maovina, duovnik i t.d. ... suv, gluv, duvati... smej i t.d. 14 h ... ora (orëch־b) , die Nuß; Via (Vlach-b) , ein Walach; lad (chladi der Schatten; гот (chrom*,) , lahm; rabar (сЬгаЬагъ), tapfer; manuti (machnuti), schwingen; dunuti (duchnuti), blasen); maovina (ma- сhovi na) , das Moos; ma (machb), der Schwung; gra (grach*) , die Fc sole; duovnik (duchovnikb), der Beichtvater, u.s.w. ... suv (such-ь), trocken,gluv (gluchb), taub; kuvati (kuchati), kochen; duvati (duchati), blasen... smej (smëch־b) , das Lachen; u.s.w. 14 h ... ora (orëch־b) , die Nuß; Via (Vlach-b) , ein Walach; lad (chladi der Schatten; гот (chrom*,) , lahm; rabar (сЬгаЬагъ), tapfer; manuti (machnuti), schwingen; dunuti (duchnuti), blasen); maovina (ma- сhovi na) , das Moos; ma (machb), der Schwung; gra (grach*) , die Fc sole; duovnik (duchovnikb), der Beichtvater, u.s.w. ... suv (such-ь), trocken,gluv (gluchb), taub; kuvati (kuchati), kochen; duvati (duchati), blasen... smej (smëch־b) , das Lachen; u.s.w. [Anm.] ... fala, f a lit i, u fa titi, zafatiti, d o fa titi i t.d. [Anm.] ... fala (hvala), das Lob; f a lit i (hvaliti), loben; u fa titi (uhvatiti), fangen; zafatiti (za־ h va titi), schöpfen; dofatiti (do- h va titi), erwischen; u.s.w. Anm. Auch in solchen Wörtern zwischen Vokalen wird in einigen Gegenden (in Serbien und in Bos- nien) ganz ausgelassen, und ge- sprochen kuati, duati, u.s.w. Œ Nije slovo, nego je čitav slog (шт, i l i ot) i rije č. Od ęrije, dok nije bilo a rtije i stampe, morali su lju d i sve knjige pisati na pergamentu (uglaāenoj koži), koji je bio mlogi skuplji od najljepše a rti- ģe;zato su se starali da pišu sto se može krace; i tako su po- celi nadmetati t it le i skraciva- t i onakove rije č i, koje cesto do- laze, i koje svaki lasno može po- goditi, n.p. cKs, o*c, dvdb, b\, дь, znači hristos, otac, david, ® ist gar kein einzelner Buch- stab, sondern Abkürzung für шт oder ot, wie dergleichen Abkür- zungen (title ) für mehrere gang- bare und leicht zu errathende Wörter eingeführt worden sind, z.B. cfis, oc, dvdt», Łfw, g\, be- deutet Hristos, Christus; otac, Vater; David, David; Bog, Gott; Gospod, Herr, u.s.w. [Anm.] Vergl. Dobrowsky in s titt. p. 60. sqq. Einige Altslavische Grammatiker sagen,.. 19 In der Tat eine unbequeme Aus- hülfe, welche niemanden über die Unvollkommenheit des Altsla- vischen Alphabets täuschen wird. 22 [Punkt 4) aus dem "Dodatak:] ,.. z.B. mije (der Schlauch) und grije (die Sünde) haben im Nom. gleichen Accent und Laut, aber in den übrigen Endungen bekommt letzteres grija (Gen.), grljovi (Nom. Plur.), u.s.w. màslo (das Schmalz) und vèslo (das Ruder) haben im Nom. beinahe einen glei- chen Laut, aber veslo hat im Plur. wie auch Gen. Sing. gleiche Endung; aber maslo wird im Plur. nicht ganz so scharf, wie im Gen. Sing. accentuirt; ... 22 [Punkt 4) aus dem "Dodatak:] ,.. z.B. mije (der Schlauch) und grije (die Sünde) haben im Nom. gleichen Accent und Laut, aber in den übrigen Endungen bekommt letzteres grija (Gen.), grljovi (Nom. Plur.), u.s.w. màslo (das Schmalz) und vèslo (das Ruder) haben im Nom. beinahe einen glei- chen Laut, aber veslo hat im Plur. wie auch Gen. Sing. gleiche Endung; aber maslo wird im Plur. nicht ganz so scharf, wie im Gen. Sing. accentuirt; ... ... N.p. mîje i grije imaju и imen. jednak glas, a u ostalim padežima grije ima gríja, grîjo- vi i t.d. Màslo i vèslo imaju u imen. gotovo jednak glas, i veslo ima u mlož. broju kao i u rod. jed., a maslo se u mlož. broju ne izgovara sasvim tako oštro, kao i и rod. jed. ... 71 ־ 37 37 Von dem Geschlechte des Nomens. 7 z - Ž und in g, z.В.. . .natezati, anziehen (frequentativum), natežem, ich ziehe an; nategnuti, anziehen (perfectivum);.. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 207 - [Anm.] ... I samo d i t napinju se sauno novi književnici Sremač- ki i Bačvanski da čitaju kao a i 6 ... 35 Ovo je bio govor о Slavenskim slovima, kojije mi nemamo... nekolike rije č i о našim slo- vima... dž je i kod nas... ... koni, i l i konji; koński i l i konjski... Kod nas aekoji u č ite lji Slaven- skije gramatika kažu,.. a li je to slab izgovor... 36 nego je to, s oproštenjem, kr- pel, koji pokazuje nedostatak Slavenske Bukvice. [Anm.] ... I samo d i t napinju se sauno novi književnici Sremač- ki i Bačvanski da čitaju kao a i 6 ... 16 [Anm•] ... Neugelehrte Sirmier und Batscher mühen sich, ein db und tb, Я und с zu lesen... 18 Soviel von den Altslavischen Buchstaben, welche die Serben nicht haben,.. einige Worte über Serbische Buchstaben,.. dž dient den Serben... 18 Soviel von den Altslavischen Buchstaben, welche die Serben nicht haben,.. einige Worte über Serbische Buchstaben,.. dž dient den Serben... Einige Altslavische Grammatiker sagen,.. 18 Soviel von den Altslavischen Buchstaben, welche die Serben nicht haben,.. einige Worte über Serbische Buchstaben,.. dž dient den Serben... Einige Altslavische Grammatiker sagen,.. 35 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Von dem Geschlechte des Nomens. In der Serbischen Sprache gibt es drei Geschlechter, nemlich: männliches, weibliches, und neu ־ traies. Alle Substantive endigen sich entweder auf einen Consonanten, oder auf einen der drei folgen- den Vocale: auf a, oder auf e, oder auf o. Und bios nach diesen Ausgängen kann man das Geschlecht der Wörter bestimmen. I. Alle Wörter, die sich conso- nantisch endigen sind in der Re- gel Masculina, z.B. covek, der Mensch; kamen, der Stein; nož, das Messer; panj, der Baumstamm; mač, das Schwert; car, der Kai ־ ser; raj, das Paradies; kraj, das Ende; mis, die Maus, u.s.w. Ausgenommen sind nachstehende Feminina: Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 208 - 23 3 1) Positiv, ohne Rücksicht auf die Endung oder Bedeutung; z.B. avet, das Gespenst; ac (etwa: Närrchen); blago- v ij est, Marä-Verkündigung; blagodat, der Segen; vaš, die Laus, večer, der Abend; vlast, die Macht; vrlet, die Steile; golet, kahle Gebirgsgegend; duz, die Länge; zue, die Galle, zano- vije t, Geißklee; zapovijest, der Be- fehl; zvijer, das wilde Thier; zelen, das Grüne; zob, der Haber; kap, der Tropfen; kob, die Begegnung; k lije t, eine Kammer; kokos, die Henne; korist, der Nutzen; kost, das Bein; kostrijet, Ziegenwolle; krv, das Blut; krmelj, der Eiter in den Augen; laž, die Lüge, last, die Leichtigkeit; ljubav, die Liebe; mast, das Schmalz; moc, die Kraft; močar, die Nässe vom Regen; muar, Muharklee; napast, Unglück; na- rav, das Naturell; nemoc, die Krank- heit; noe, die Nacht; oblast, die Macht; pamet, der Verstand, das Ge- dächtniß; paprat, das Farnkraut, pec, der Ofen; plijesan, der Schimmel; pod- gor, Gegend unter dem Berge; pomoè, die Hülfe; postát, die Reihe bei der Ernte etc., pripovijest, das Sprich- wort; prnjad, faules Holz; prostriž, am Rocke, oder Mantel die Öffnung um die Hand durchzustecken; raž, der Rog- gen, riječ, das Wort; rukovet, die Handvoll; svast, die Schwägerin; svi- jest, die Besinnung; slast, die Süßig- keit, Wollust; smrt, der Tod; stvar, das Ding; strmen, die Steile; studen, die Kälte; cud, das Naturell; uš, die Laus; cijev, die Röhre; erven, die Röthe; caä, der Ruß; cast, das Gast- mahl; čeljust, der Kinnbacken; čest, die Ehre. 2) Alle Wörter die von Adjectiven auf ost gebildet werden, z.B. mladost, die Jugend; milost, die Gnade; mudrost, die Weisheit; radost, die Freude, u.s.וו 3) Alle Collectiva auf ad, welche junge Thiere bezeichnen; z.B. Von dem Geschlechte des Nomens. telad, die Kälber; jarad, junge Ziegen; pra- sad, die Ferkel; čeljad, die Frauens- leute im Hause; momcad,die Bursche, u. s.w. Hierher rechne man auch drvljad, Scheit-Holz; und burad, Fässer; eben so perut, das Gefieder. 24 24 II. Alle Wörter, die sich auf a endi- gen, sind in der Regel Feminina, z.B. voda, das Wasser; zemlja, die Erde; munja, der Blitz; zmija, die Schlange, u. s. w. Vera Boji - 9783954793044 s. w. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 209 - Ausgenommen sind Masculina: 1) Die sich im Altslavischen (also ursprünglich) auch ch endigen, z.B. ora (orechi), die Nuß? pra (prachi), der Staub; stra (strachi), die Furcht; gra (grachi), die Fasole. 2) Die Manns-Namen; z.B. Radoica, Mi- lija , Zarija, Pantelija, u.s.w. 3) Wörter, die männliche Ämter, Wür ־ den oder Verrichtungen bedeuten, z.B. sudija, der Richter; vladika, der Bi- schof; vojvoda, der Feldherr; sluga, der Diener, u.s.w. Doch letztere nur im Singularis, denn im Plur. sind sie aus Übergewicht der Form weiblich, z.B. naš sudija (unser Richter), naše sudije; naš vladika (unser Bischof), naše via- dike; moj sluga (mein Diener), moje slu- ge, u.s.w. Ja die Lieder dulden auch im Sing. das Femininum, z.B. Sługo moja, Oblačicu Rade - Da t i da Bog, Sibinjska vojvodo - I I I . Alle Wörter, die sich auf e und auf о endigen, sind in der Regel Neu- tra, z.B. polje, das Feld; spasenije, die Erretung; pisanje, das Schreiben; sunce, die Sonne; pile, das junge Huhn; pero, die Feder; zlato, das Gold; drvo, das Holz, u.s.w. Ausgenommen: Ausgenommen: 1) Masculina sind: a) Namen der Männer, z.B. Stanoje, Miloje, Dimitrije; Ranko, Branko, Raško, Boza, u.s.w. b) Wörter auf о, die sich im Altslavischen (also ursprünglich) auf 1 endigen; z.B. soko (sokoli), der Falk; kotao (koteli), der Kessel; svrdao (svrdeli), der Boh- rer, u.s.w. c) Wörter auf e, die sich im Altslavischen auf ech endigen, z.B. mije (mêchi), der Schlauch; grije (grechi), die Sünde, u.s.w. 1) Masculina sind: a) Namen der Männer, z.B. Stanoje, Miloje, Dimitrije; Ranko, Branko, Raško, Boza, u.s.w. b) Wörter auf о, die sich im Altslavischen (also ursprünglich) auf 1 endigen; z.B. soko (sokoli), der Falk; kotao (koteli), der Kessel; svrdao (svrdeli), der Boh- rer, u.s.w. c) Wörter auf e, die sich im Altslavischen auf ech endigen, z.B. mije (mêchi), der Schlauch; grije (grechi), die Sünde, u.s.w. Von dem Geschlechte des Nomens. "Sługo moja Oblaciću Rade - "Da t i da Bog, Sibinjska voi- vodo “ [Anm.] ... ja ne vjerujem da bi prost Srijemac kazao, n.p. О naši današnji Srpski knjiga; и Petrovi krāsni nepokošeni zeleni livada i t.d. 40 2) ... nesretnjico jedna!.. 3) ... Sara, Saro i t.d. 4) ... osim matere... stvari i stvarju; kokoši i kokoŠju; žuči i žučju i t.d. 41 Kod ovog sklonenija treba upamtiti: 1) Imena živi stva- r i, koja se svršuju na e i na- rašcuju na ta, ona и mlož. bro- ju postaju (sabiratelna) žen- 37 Srpska^imena suštestvitelna imaju č e tiri sklonenija. 37 Srpska^imena suštestvitelna imaju č e tiri sklonenija. 37 Srpska^imena suštestvitelna imaju č e tiri sklonenija. Die Serbischen Substantiva haben viererlei Declinationen; jede Deel nation unterscheidet zwei Numeros, Singularis und Pluralis, keinen Du lis , in jedem Numerus aber sieben Casus: Nominativ, Genitiv, Dativ, Accusativ, Vocativ, Instrumentalis und Localis. ... die im Gen. Sing. noch ein a hinzunehmen;.. [Anm.] ... gavranom... Man hört si auch unterm Volke... Die Serbischen Substantiva haben viererlei Declinationen; jede Deel nation unterscheidet zwei Numeros, Singularis und Pluralis, keinen Du lis , in jedem Numerus aber sieben Casus: Nominativ, Genitiv, Dativ, Accusativ, Vocativ, Instrumentalis und Localis. ... koja i и roditeinom imaju a; . . [Anm.] ... gavranom... može cuti i u govoru po varošima... 38 Kod ovog sklonenija treba upamtiti: 9) ...оси, осе, otaca... 39 4) Put ima u rod. jed. i putom i putem: a) n.p. ja saun za putom; sto ji pod pu- tom. b) n.p. ode putem. 27 Allgemeine Anmerkungen über diese Declination. 29 ...оси, (im Gen. Plur.) otaca... 29 ...оси, (im Gen. Plur.) otaca... 29 ...оси, (im Gen. Plur.) otaca... 30 Put (der Weg) hat im Instr. Sing. putom und putem, z.B. ja sam za putom, ich bin zur Reise bereit; sto ji pod putom, er is t unter Wegs ode putem, er is t fort seinen Weg. 30 Put (der Weg) hat im Instr. Sing. putom und putem, z.B. ja sam za putom, ich bin zur Reise bereit; sto ji pod putom, er is t unter Wegs ode putem, er is t fort seinen Weg. Po коте se sklanjaju sva ime- na ženskoga i (ostala) muško- да roda, koja se svršuju na à; п• p• +•* 31 Begreift alle Feminina auf a, samt den Masculinis, die im Gen. e be- kommen. [Anm.] A u pjesmama su ovakova imena i и jed. broju ponajvise ženskoga roda, n.p. 32 2) ... nesrecnico jedna! .. 33 3) ... Sara, Saro и.s.w. Die auf ca, zuweilen auf e. z.B. Milica, о Milice; Anica, о Anice! и.s.w. 35 4) ... mit Ausnahme von matere (di< Mütter) ...stvar, stvari und stvar־ ju; kokoŠ, kokoši und kokoŠju; žuč (die Galle), žuCi und žuCju, и.s.w. 5) Mati und kòi haben ein epenthe- tisches er, wie das Paradigma lehr־ 36 Anmerkungen. 1) Verschiedene Neutra dieser Deel haben ein epenthetisches et, en und es, welches im Gen., Dat., Instr., Loc. des Sing., so wie im ganzen Plur. hinzutritt. Nämlich Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 32 2) ... nesrecnico jedna! .. 33 3) ... Sara, Saro и.s.w. Die auf ca, zuweilen auf e. z.B. Milica, о Milice; Anica, о Anice! и.s.w. 35 4) ... mit Ausnahme von matere (di< Mütter) ...stvar, stvari und stvar־ ju; kokoŠ, kokoši und kokoŠju; žuč (die Galle), žuCi und žuCju, и.s.w. 5) Mati und kòi haben ein epenthe- tisches er, wie das Paradigma lehr־ 36 Anmerkungen. 1) Verschiedene Neutra dieser Deel haben ein epenthetisches et, en und es, welches im Gen., Dat., Instr., Loc. des Sing., so wie im ganzen Plur. hinzutritt. Nämlich Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 40 41 Kod ovog sklonenija treba upamtiti: 1) Imena živi stva- r i, koja se svršuju na e i na- rašcuju na ta, ona и mlož. Von dem Geschlechte des Nomens. 2) Feminina, die im Altslavischen (also ursprünglich) auf 1 ausgehen, z.B. misao (myslb), der Gedanke; so (solb), das Salz; ponikao, das Hervorgesprosse- ne; pogibao (pogibelb), der Untergang, u.s.w. - 25 Es giebt außerdem noch einige Mascu- lina auf и (solche, die im Altslavi- sehen auf ch ausgehen), z.b. kožu, der Pelz, repu, die Pestilenzwurz; du, der Geist, u.s.w. Einige Feminina haben den Plur. auf i, z.B. n iti, das Weber ״ trumm; prsi, die Brust; oči, die Augen; usi, die Ohren. Der Sing. gebricht ih- nen entweder, oder er ist anderes Ge- schlechts. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 210 - Die Serbischen Substan viererlei Declinationen; nation unterscheidet zwe Singularis und Pluralis, lis , in jedem Numerus abe Casus: Nominativ, Genitiv Accusativ, Vocativ, Instr und Localis. ... die im Gen. Sing. noc hinzunehmen;.. [Anm.] ... gavranom... Ma auch unterm Volke... 27 Allgemeine Anmerkungen üb Declination. 29 ...оси, (im Gen. Plur.) o 30 Put (der Weg) hat im Ins putom und putem, z.B. ja putom, ich bin zur Reise sto ji pod putom, er is t u ode putem, er is t fort se 31 Begreift alle Feminina au den Masculinis, die im G kommen. [vgl. S. 24,bei Grimm ] [Anm.] Daß die Lieder se Sing. weiblich nehmen, is merkt. 32 2) ... nesrecnico jedna! 33 3) ... Sara, Saro и.s.w. ca, zuweilen auf e. z.B. о Milice; Anica, о Anice 35 4) ... mit Ausnahme von m Mütter) ...stvar, stvari ju; kokoŠ, kokoši und kok žuč (die Galle), žuCi und и.s.w. 5) Mati und kòi haben ein tisches er, wie das Parad 36 Anmerkungen. 1) Verschiedene Neutra d haben ein epenthetisches und es, welches im Gen., Instr., Loc. des Sing., s 37 Srpska^imena suštestvitelna imaju č e tiri sklonenija. ... koja i и roditeinom imaju a; . . [Anm.] ... gavranom... može cuti i u govoru po varošima... 38 Kod ovog sklonenija treba upamtiti: 9) ...оси, осе, otaca... 39 4) Put ima u rod. jed. i putom i putem: a) n.p. ja saun za putom; sto ji pod pu- tom. b) n.p. ode putem. Po коте se sklanjaju sva ime- na ženskoga i (ostala) muško- да roda, koja se svršuju na à; п• p• +•* [Anm.] A u pjesmama su ovakova imena i и jed. broju ponajvise ženskoga roda, n.p. 27 Allgemeine Anmerkungen über diese Declination. 42 Prilagatelna imena mogu se, po n ji- ovon značenju, ra z d ije liti и t r i re- da: Prva pokazuju kakvocu imena su- štestvitelni; i ova su opet dvoja- ка: 1) koja pokazuju kakvo je sto, n.p. dobar covek, zelen lis t, mutna voda, šarena tica, brz konj i t.d. ! 2) koja pokazuju oda šta je što, п. p. gvozden kotao, drvena kuca, sre- brno puce, kožna aljina i t.d. 27 Allgemeine Anmerkungen über diese Declination. bro- ju postaju (sabiratelna) žen- skoga i l i muškoga roda, i Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 « 211 - a) et schieben alle Neutra auf e ein, welche junge Thiere bedeuten, als tele (Kalb); prase (Ferkel), čeljade (Dienstmagd), Gen. Čelja- deta, u.s.w. b) en alle Neutra auf me, als ime (Name), seme (Sa- me), vreme (Zeit), vime (Euter), u.s.w. c) es schalten nur wenige Neutra auf o, auch nicht, wie ei- nige andere Slavische Sprachen thun, bereits im Sing., sondern nur im Plur. ein, als: nebo, Plur nebesa, čudo (Wunder), Plur. ču- desa, daneben aber auch čuda. Un- üblich sind die Plur. kolesa, pe- resa, etc. 3 sklanjaju se po prvom i po tre- сет skloneniju, n.p. tele, tele- ta, telad, i l i teoci; prase, pra- sad i l i prasci; čeljade, čeljad; momee, momčad i t.d. A li imena ovakova mrtvi stvari (koja su ponajviše tuűa), imaju i mlož. broj, n.p. tane, taneta, и mio?, br. taneta; japundže, silembe, coše, cebe i t.d. 37 3 sklanjaju se po prvom i po tre- сет skloneniju, n.p. tele, tele- ta, telad, i l i teoci; prase, pra- sad i l i prasci; čeljade, čeljad; momee, momčad i t.d. A li imena ovakova mrtvi stvari (koja su ponajviše tuűa), imaju i mlož. broj, n.p. tane, taneta, и mio?, br. taneta; japundže, silembe, coše, cebe i t.d. 2) Die in voriger Anmerkung unter a gedachten Neutra mit epentheti- schem et bilden keinen Plur. Neu- trius (wie andere Slavische Spra- chen teleta, čeljadeta), sondern setzen entweder einen männlichen Plur. nach erster Deel., als: teo ci (Kälber), prasci (die Ferkel), oder den Sing. des weiblichen Col lectivums auf ad (s. dritte Deel, als telad, prasad; čeljad. Einige Namen lebloser Dinge (meistens fremde Wörter) auf e schieben gleichfalls et ein und behalten auch die neutrale Pluralform, z.В tane (die Kugel), Gen. taneta, Nom. PI. taneta; japundŽe (der Mantel), silembe (der Brustriemen am Reitpferde) coše (das Eck), cebe (die Kotze), u.s.w. 38 4) Pseto... pašcad (zenskoga roda).. 5) Pseto... pašČad (weiblich von pasce)... 5 ) Ramo ima и rod. ramena (kao rame). 5 ) Ramo ima и rod. ramena (kao rame). 6) Ramo (die Schulter) hat im Gen ramena (von rame, welches andere Mundarten im Nom. setzen). 6) Ramo (die Schulter) hat im Gen ramena (von rame, welches andere Mundarten im Nom. setzen). [Vgl. Anmerkungen zu den Adjectiv Declinationen S. 44f. bei Grimm] Druga pokazuju Čije je Sto; i ova su opet dvojaka: 1) koja pokazuju da :e što samo jednoga izvjesnog imer.a suštestvitelnog, n.p. popov. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 212 ־ kovačev, vojvodin i t.d. 2) koja pokazuju, da je što više imena suštestvitelnije, i l i jednoga ko- да mu drago, n.p. popovski, ko- vački, vojvodski; govedi, kozji, r ib lji, t ič ji, Öetinji i t.d. Treòa idu u red drugije, nego se od n ji razlikuju, što nijesu postala od imena stuštestvitelni, nego od drugije različni dijelo- va govorenja, kao n.p. 1) od na- rječija (osobito od mjesta i vre- mena), n.p. tamošnji, današnji, lanjski, tokoršnji i t.d. 2) od glagola, n.p. stajaca košulja, pietica igla, oraca zemlja, ku- povni gunj, muzovna krava, te- kuca voda, i ostala ovakova pri- Častija. 3) od imena b ro jite lj- nije, n.p. prvi, drugi, treåi i t.d. kovačev, vojvodin i t.d. 2) koja pokazuju, da je što više imena suštestvitelnije, i l i jednoga ko- да mu drago, n.p. popovski, ko- vački, vojvodski; govedi, kozji, r ib lji, t ič ji, Öetinji i t.d. Der Serbe unterscheidet gleich dem Altslaven zwei Declinationen, deren einer oder der ändern die ein־ zelnen Adjectiva heimfallen; und un* abhängig davon eine doppelte Bie- gungsform, welcher in der Regel je- des Adj., gehöre es zu der ersten oder zweiten Declination, zugleich fähig is t. In der Benennung dieser Flexionen weichen die Grammatiker von einander ab. Die Form, welche sie voranzusetzen pflegen, heißt bald die unbestimmte, bald die ab- stracte, bald die abgeschnittene, verkürzte (terminatio indefinita, abstracta, apocopata); die entgegen־ gesetzte zweite Form aber bald die bestimmte, bald die unabgeschnitte- ne oder ganze (term, definita, con- 9 creta, non apocopata sive integra). Die beiden ersten Namen sind von de: Bedeutung her genommen, der letzte bezieht sich auf das Formelle selbs* Ohne hier über die Schicklichkeit dieser Benennungen zu entscheiden, wollen wir der letzten, welche die älteste zu sein scheint, gebührend folgen. Ganz (cijel) heißt jedes Adjectivum, zu dessen Wurzel oder Ableitung im Nom. Sing. Masc. der Voc. i t r it t ; abgeschnitten (usje- čen, eingeschnitten, von usjeći, einschneiden), dasjenige, welchem dieser Vocal mangelt, gleichsam ab- gehauen ist. Das abgeschnittene en- digt daher gewöhnlich auf Consonan- Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM О dvojakom svršivanju prilaga- telni imena. [Vgl. Anmerkungen zu den Adjectiv Declinationen S. 44f. bei Grimm] Prilagatelna imena prvoga re- da svršuju se и muškome rodu jedinstvenog broja dvojako, t . j . na poluglasno slovo, i i i na i: zašto mi govorimo, i sluáamo Űe se govori, n.p. dobar i dobri, zelen i zeleni, drven i drveni, dvogub i dvogubi i t.d. Ovo su prvo nazvali Slavenski gramati- ci prilagatelnim usječenim (so usjecenijem), a drugo cijelim (bez usjeCenija). Može b iti, da bi bilo pametnije reci, da je ovo drugo prilagatelno na- raslo od prvoga, nego da je prvo usjeceno: zašto prvo sto- j i u prvome svom obliku (kao n.p. muž и imenitelnom pade- žu) i pokazuje kakvocu neiz- vjesne stvari (na pitanje ka- kav?), n.p. zee je plašljiv; pošten Čovek ne umije lagati; meni je mio erven pojas; zia- tan je prsten skupij i nego srebrn; Gojko se fcini (da je) pametan i t.d. A ovo je drugo na<5injeno (sa dodatkom i) od prvoga, i kakvocom opredjelju- je stvar (na pitanje koji?) kao Njemački Ölen der, die das), n.p erveni pojas iz je li miSi; izgu- bio sam srebrni prsten; prodao sam moj neobrani vinograd i t.d. Ovo dvojako svrŠivanje nasi 39 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 213 - prilagatelni imena samo se nalazi u muškome rodu (je- dinstvenog broją), a u žen- skome i u srednjem rodu sa- то se po glasu razlikuje, n.p . bolja je aljina sirò- ka, nego űska; kamo ona sîrokâ aljina? Tu je i ûskâ i sîrokâ; ovo je prase debèlo; posto ces mi dati tvoje debelÒ prase? i t.d. prilagatelni imena samo se nalazi u muškome rodu (je- dinstvenog broją), a u žen- skome i u srednjem rodu sa- то se po glasu razlikuje, n.p . bolja je aljina sirò- ka, nego űska; kamo ona sîrokâ aljina? Tu je i ûskâ i sîrokâ; ovo je prase debèlo; posto ces mi dati tvoje debelÒ prase? i t.d. ten, z.B. dobar (gut), zelen (grün), drven (hölzern); oder auf о statt 1, als cijo (ganz), zreo (re if), truo (faul), veseo (lustig). Ganz lauten dieselben Wörter: dobri, zeleni, cije- l i , zreli, tru li, veseli. Femininum und Neutrum lauten in der ganzen Form buchstäblich wie in der abgeschnitte- nen, doch eine verschiedene Accentua- tion unterscheidet beide. Der Unter- schied zwischen ganz und abgeschnit- ten auf die Bedeutung angewandt, so pflegt letzteres das Unbestimmte aus- zudrücken, z.B. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Obadva se stepena sklanjaju po 47 drugom skloneniju prilagatelni imena. [Anm.]... Po ovom bi trebalo ka ־ zati težji, težja... a li se ne govori. [Vgl. Anmerkungen zu den Adjectiv Declinationen S. 44f. bei Grimm] zec je plašijiv , der Hase is t furchtsam, pošten Čovek ne umije lagati, ein redlicher Mann kann nicht lügen; meni je mio crven pojas, mir is t der rothe Gürtel lieb; zlatan je prsten skuplji nego srebrn, ein goldner Ring is t theuerer, als ein silberner; Gojko se cini (da je) pame- tan, der Gojko s te llt sich vernünftig; u.s.w. Das ganze hingegen das Bestimm- te, z.B. crveni pojas iz je li misi, die Mäuse haben den rothen Gürtel gefres- sen; izgubio sam srebrni prsten, ich habe den silbernen Ring verloren; pro- dao sam moj neobrani vinograd, ich habe meinen ungelesenen (unbeernteten) Weingarten verkauft, u.s.w. Hier auch einige Beispiele des verschiedenen Accents der Adj. Fem. und Neutr., um denselben Unterschied auszudrücken: bolja je aljina siròka, nego űska, besser ist ein weites, als ein enges Kleid; kamo ona sîrokâ aljina, wo ist das weite Kleid? Tu je i ùskâ i sîrokâ, hier ist das enge und das wei- te, ovo je prase debêlo, dieses Ferkel ist fett; posto ceš mi dati ono tvoje dêbelô prase, wie theuer gibst du mir dein fettes Ferkel? u.s.w. 40 [Anm.](... kod svetog Lazara) na crvenoj s v ili vezeno zlatom (kazu da je vezla carica Milica na éivot knezu Lazaru)... [Vgl. Anmerkungen S. 45 bei Grimm] [Vgl. Anmerkungen S. 45 bei Grimm] [Vgl. Anmerkungen S. 45 bei Grimm] D îvljî, mâlî, vèlikî, ne usi- jecaju se nikako. Dîvljî treba- lo bi da bude dîvalj, dîvalj, dîvlja, dîvlje; mâlî, mài ( i l i máo), mâla, malo; a vèlikî, vélik, velîka, velîko; a li se ne govori ni jedno, nego osta- ju tako: d îv ljî, dîvljâ, dîvlje; 43 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 214 - Prilagatelna imena imaju t r i 41 roda i dva sklonenija. Alle Serbischen Adjectiva zerfallen wie oben bemerkt worden ist, in zwei Deciinationen. 4 4 Anmerkungen zu den Adjectiv- Dec1inationen. Anmerkungen zu den Adjectiv- Dec1inationen. 1) Einige Adjektiva sind nur der ab- geschnittenen, nicht der ganzen Form fähig, namentlich: a) alle Possessi- ven auf ov, ev, als: popov (des Gei- stlichen), kovačev (des Schmidts) ; b) Possessiva auf in, als: vojvodin (des Wojwoden). ( j ) 2) Andere sind nur der ganzen Flexior fähig, nicht der abgeschnittenen, nämlich: a) Ableitungen von Substan- tiven mittels der Endungen i, l j i , n ji, z.B. goveäi (Rinds-), kozji (Ziegen-), r ib lji (Fisch-), t ie ji (Vogel-), detinji (Kinds ; )־b) Ablei- tungen auf ski, als: popovski (prie- sterlich), kovački (schmidtisch), vojvodski (wojwodisch); c) Ableitun- gen von Adverbiis, zumal loci et temporis, als: tamosnji (dortig), da- nasnji (heutig), lanjski (vorjährig), tokorsnji (seyn sollend), u.s.w.; d) Ableitungen von Verbis, z.B. sta- jaca kosulja (sonntägliches Hemd), pietica igla (Stricknadel), oraca zemlja (Ackerland), kupovni gunj (gekaufter Mantel), muzovna krava 4 5 (Melkkuh), tekuca voda (fließendes Wasser), und andere Participia mehr. Vergl. unten die Note zum Particip erster Conjugation; e) sämmtliche Comparative; f) sämmtliche Ordinal- zahlen; g) einzelne Adjectiva, na- mentlich: d îv ljî (wild), vèlikî (groß). Denn die abgeschnittenen For- men dîvalj, dîvlja, dîvlje; mal (ode! mao), mala, màio; vélik, velìka, ve- līko sind unerhört. Man kann zwar füi mal und vélik sagen: malen und go- lem, aber das sind andere Wörter, und z.B. in der Redensart jesi l i veliki narastao? (bist du schon groß geworden?) wird kein Serbe weder velik noch golem gebrauchen. 4 5 Obadva se stepena sklanjaju po 47 drugom skloneniju prilagatelni imena. [Anm.]... Po ovom bi trebalo ka ־ zati težji, težja... a li se ne govori. Beide Stufen gehen nach der zweiten Declination ganzer Adjectiva, 47 [Anm.]... Die ursprünglichen, voll- ständigen Formen težji, težja... sine allmählich aus dem Gebrauch gekommen. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 215 - [Anm.] Mlogi spisatelji i knji- ževnici nasi (a osobito po Sri- jemu i po Bačkoj) pisu jeda- najst, dvanajst i t.d. a li od naroda n ijesam mogao cuti da tako govori 48 48 [Anm.] Viele (besonders in Sirmien und Batscher-Comitat) schreiben auch jedanajst, dvanajst, u.s.w.; aber ich habe niemanden so reden hören. [ Anm.] Niemand halte trie s t... für gemein und verdorben... Anmerkungen zu den Adjectiv- Dec1inationen. .odnositelno mjestoimenije ito , imaju svi sadašnji Slaven- ski narodi, a и Slavenskom je- ziku пета да! 53 Po značenju mogu se glagoli ra z d ije liti и t r i reda: 1) neki glagoli pokazuju da ко radi sto izvjesno izvan sebe, što svagda mora stajati и vin. padežu, n.p. ženskoga и imen. mioz., tako isto i nekoliko kad znači manje od pet, n.p... Mjestoimenija su petoroguba: I. Koja pokazuju t r i lica: ja, t i, on; к ovima òemo űzeti и sklanjanju i II. Povratno sebe, koja se evo ovako skia- njaju: ... 50 ... nego se govori (osobito ti) kao za neko čudo, n.p. l i jepa t i je ... ... ne opominjem se vise za mi • • • 51 I II . Koja što prisvojavaju ... IV. Kojima se što pokazuje... ... ôvâj, ôvâ, òvô... 52 V. Kojima se što p ita ... VI. Koja se odnose na stvar... I. c îji,c îj (cî) R.cîjega (čijēga),čijeg (cîjêg), D.cîjemu (cijêmu),čijem (cîjêm ), V.čijega (čijega),cîjeg,cijêg ( č ij) , Z.- T.Čijim (cím) S.čījemu (cijêmu),čījem (cîjêm) I . č iji, cîj (cî) R .čijije (c îje ),c îjî, D.čijima, čijim, V.cîjê, Z.- T.čijima, cîjim S.cîjima, cîjim 54 . . .odnositelno mjestoimenije ito , imaju svi sadašnji Slaven- ski narodi, a и Slavenskom je- ziku пета да! 53 Po značenju mogu se glagoli ra z d ije liti и t r i reda: 1) neki aber adjectivisch construirt zu werden, als... ženskoga и imen. mioz., tako isto i nekoliko kad znači manje od pet, n.p... j werden, als... 54 Es gibt sechserlei Pronomina: I. Die drei persönlichen und II. Das Reciprocum (welches wieder Nom. noch Voc. haben kann) werden folgendergestalt fle c tir t:... 56 ...sondern werden blos (beson«ders ti) gleichsam als interjectio ad- mirantis eingeschlatet, z.B. l i - jepa t i je ... 57 ... die Beispiele von mi sind sei- tener... 58 I I I . Possessiva... IV. Demonstrativa... 59 ... ovâkî, ovákS, ovâkô... onákí, onâkâ, onákd... ta ki... v.Interrogativa... VI. Relativa... Nom. cîj ( č iji) , wessen. Gen. čijega (êga), eg (êg), Dat. čijemu (êmu), em (êm), Acc. čijega (êga), eg (êg), Voc. - Instr.cîjm (cîjim), Loс. cíjemu (êmu), em (êm) Nom. cîj ( c îji) , Gen. cîj ( îji) , îje ( ì jije ) , Dat. cîjma (ìjima), îjm (ìjim ), Acc. cîjê, Voc. ־ Instr. Anmerkungen zu den Adjectiv- Dec1inationen. Imena su brojiteljna petoro- guba: Es sind fünferlei Zahlwörter zu betrachten: Cardinalia, Distribu- tiva, Ordinalia, Multiplicativa und Adverbalia. 1) Osnovna, koja sami broj рока- zuju, i l i kojima se odgovara na pitanje koliko? n.p. jedan... 1) Cadinalia, jedan... 1) Cadinalia, jedan... 49 47 4 Jedan, jedna... po prvom sklo- neniju prilagatelni imena (kao cijelo prilagatelno)... Jedan, jedna... nach der ersten abgeschnittenen Declination... Jedan, jedna... nach der ersten abgeschnittenen Declination... [Anm.] Da ne reče kakav jeziko- popravitelj da je ovo (trie st...) podio i pokvareno... [ Anm.] Niemand halte trie s t... für gemein und verdorben... für 51 Jedan, jedna, jedno pokazuje ime suštestvitelno (и sva t r i roda) и imen. jed., n.p. jedan čovek, jedna гепа, jedno dijete; dva, t r i ce tiri, pokazuju imena sušt. muškoga i srednjeg roda и rod. jed., a ženskoga u imen. mlož., n.p. dva čoveka, t r i ka- mena, č e tiri brda, dvije vòde, t r i gòre, ce tiri zène i t.d. a pet, sest, sedam i t.d. рока- zuju imena sušt. (и sva t r i ro- da) и rod. mlož., n.p. pet ljûdi, pet Žena, sedam sêla, osam vesâla i t.d. 48 51 - [Anm.] I оѵЯе se do deset u skia- njanju pretvori osnovno ime, u muškom rodu, u sušt., n.p. pet, petorice; šest, šestorice i t.d. Kao dvoje sklanja se oboje i obadvoje? kao troje svetroje, a ostala sva kao četvoro. 52 3) Ordinalia, z.B. ... Von stotine und iljade... Nie können Ordinalia abgeschnit- ten fle ctie rt werden. 53 ... petom и.s.w. und dies sind die wahren einfachen Ausdrücke, indem jedan put aus zwei Wörtern zusammenfließen. Unter diesen pflegt nekoliko, wenn es weniger als fünf anzeigt, wie dva, t r i, ce tiri, das männliche und neutrale Substantiv im Gen. Sing. zu erfordern, mit Femininis Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 52 3) Ordinalia, z.B. ... 49 53 Unter diesen pflegt nekoliko, wenn es weniger als fünf anzeigt, wie dva, t r i, ce tiri, das männliche und neutrale Substantiv im Gen. Sing. zu erfordern, mit Femininis Izmeäu övije nužno je nesto и- ęamtiti kod nekoliko: kakogoä sto dva, t r i, ce tiri pokazuju imena sušt. muškoga i srednjeg roda и rod. Anmerkungen zu den Adjectiv- Dec1inationen. mlož.[sic!], a Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 216 - aber adjectivisch construirt zu werden, als... 54 Es gibt sechserlei Pronomina: I. Die drei persönlichen und II. Das Reciprocum (welches wieder Nom. noch Voc. haben kann) werden folgendergestalt fle c tir t:... 56 ...sondern werden blos (beson«ders ti) gleichsam als interjectio ad- mirantis eingeschlatet, z.B. l i - jepa t i je ... 57 ... die Beispiele von mi sind sei- tener... 58 I I I . Possessiva... IV. Demonstrativa... 59 ... ovâkî, ovákS, ovâkô... onákí, onâkâ, onákd... ta ki... v.Interrogativa... VI. Relativa... Nom. cîj ( č iji) , wessen. Gen. čijega (êga), eg (êg), Dat. čijemu (êmu), em (êm), Acc. čijega (êga), eg (êg), Voc. - Instr.cîjm (cîjim), Loс. cíjemu (êmu), em (êm) Nom. cîj ( c îji) , Gen. cîj ( îji) , îje ( ì jije ) , Dat. cîjma (ìjima), îjm (ìjim ), Acc. cîjê, Voc. ־ Instr. cîjma (îjima),îjm (îjim) Loc. cîjma (îjima), îjm (îjim) ... oče naš, koji je s i... 63 Anm. Die altslavische Grammatik gebraucht ihr interrogatives čto noch relativ, wohl aber kennen alj heutigen slavischen Sprachen sol- chen Gebrauch: russisch čto, böh- misch poln. co, krainisch kar (fül kajr) und ki. Die bekannte Eintheilung der Ve] ba in Activa, Passiva und Deponen■ tia ist in der Serbischen Sprache, wie in sämmtlichen slavischen, müßig, d.h. ohne Einfluß auf die ženskoga и imen. mioz., tako isto i nekoliko kad znači manje od pet, n.p... Mjestoimenija su petoroguba: I. Koja pokazuju t r i lica: ja, t i, on; к ovima òemo űzeti и sklanjanju i II. Povratno sebe, koja se evo ovako skia- njaju: ... 50 ... nego se govori (osobito ti) kao za neko čudo, n.p. l i jepa t i je ... ... ne opominjem se vise za mi • • • 51 I II . Koja što prisvojavaju ... IV. Kojima se što pokazuje... ... ôvâj, ôvâ, òvô... 52 V. Kojima se što p ita ... VI. Koja se odnose na stvar... I. c îji,c îj (cî) R.cîjega (čijēga),čijeg (cîjêg), D.cîjemu (cijêmu),čijem (cîjêm ), V.čijega (čijega),cîjeg,cijêg ( č ij) , Z.- T.Čijim (cím) S.čījemu (cijêmu),čījem (cîjêm) I . č iji, cîj (cî) R .čijije (c îje ),c îjî, D.čijima, čijim, V.cîjê, Z.- T.čijima, cîjim S.cîjima, cîjim 54 . . Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Anmerkungen zu den Adjectiv- Dec1inationen. bolovati, spavati, ic i, sjediti; gori drvo, tece voda, uzdisati i t.d. I ovi se glagoli zovu srednji (verba neutra). Flexionslehre. Es gibt nur eine active Conjugation, und die Formen des la t. Passivum und Deponens müssen umschrieben werden. Fast ebenso unfruchtbar scheint und hier die Unterscheidung in Activa (Tran- sitiva) und ihnen entgegengesetzte Neutra (Intransitiva). Sie hat es blos mit der Bedeutung zu thun, nicht mit der Form, und ohnehin Unbestimmtes genug. Es gibt zwar Neutra, die keine Activa werden können, aber es gibt sehr viele Verba, denen man bald einen transi- tiven Sinn unterlegt, bald einen intransitiven, z.B. pjevati pjes- mu, ein Lied singen; igrati konja, das Pferd springen lassen; kopati vinograd, den Weinberg hacken; orati njivu, das Feld ackern; vi- kati ljude, die Leute rufen; doch wenn es heißt z.B. bolje je pjeva- ti,nego plakati, besser ist sin- gen, als weinen; mator se konj ne uči igrati, ein altes Pferd wird nicht springen gelehrt; nit* umi- jem o ra t', ni kopati, ich kann we- der ackern, noch hacken; ječam trče, a rakija vice, die Gerste läuft, der Branntwein schreit; so sind alle diese Verba auch Neutra. Den activen Verbis, welche unse- re auf einen dritten einwirkende Handlung bedeuten, pflegt man so- dann diejenigen entgegenzusetzen, deren Handlung auf uns selbst zurückwirkt : S 4 pisati knjigu, sjeci drva, kopa- t i vinograd, jesti ljeb, nositi vodu i t.d. I ovi se glagoli zo- vu djejstvitelni (poslujuci?). 2) neki glagoli pokazuju da se posao vraca na onoga, koji да radi, n.p. brijemse, kaješse, nadatise, nakanjivatise i t.d. I ovi se glagoli zovu povraca- telni, koji и pravom smislu nije- su drugo nista, nego glagoli djej- stvitelni: zašto i njiov posao stoji svagda u vin. padezu (samo sto nije izvan onoga, koji ga ra- di), n.p. b rijā ti se (sebe), ve- selitise, kao b rija ti да, veseliti te i t.d. 3) neki glagoli pokazu- ju samo stanje lica i l i stvari, i l i posao, koji ne ide ni na što, što bi stajało u vin. padežu, ne- go je u onome koji да radi, n.p. bolovati, spavati, ic i, sjediti; gori drvo, tece voda, uzdisati i t.d. I ovi se glagoli zovu srednji (verba neutra). [Anm. ] Naši Slavenski gramatici ovakove glagole zovu stradatelni- та, а пеке otložitelnima. Anmerkungen zu den Adjectiv- Dec1inationen. cîjma (îjima),îjm (îjim) Loc. cîjma (îjima), îjm (îjim) ... oče naš, koji je s i... 63 Anm. Die altslavische Grammatik gebraucht ihr interrogatives čto noch relativ, wohl aber kennen alj heutigen slavischen Sprachen sol- chen Gebrauch: russisch čto, böh- misch poln. co, krainisch kar (fül kajr) und ki. Die bekannte Eintheilung der Ve] ba in Activa, Passiva und Deponen■ tia ist in der Serbischen Sprache, wie in sämmtlichen slavischen, müßig, d.h. ohne Einfluß auf die , 54 Es gibt sechserlei Pronomina: I. Die drei persönlichen und II. Das Reciprocum (welches wieder Nom. noch Voc. haben kann) werden folgendergestalt fle c tir t:... Mjestoimenija su petoroguba: I. Koja pokazuju t r i lica: ja, t i, on; к ovima òemo űzeti и sklanjanju i II. Povratno sebe, koja se evo ovako skia- njaju: ... Loс. cíjemu (êmu), em (êm) 63 54 53 Po značenju mogu se glagoli ra z d ije liti и t r i reda: 1) neki glagoli pokazuju da ко radi sto izvjesno izvan sebe, što svagda mora stajati и vin. padežu, n.p. 53 Po značenju mogu se glagoli ra z d ije liti и t r i reda: 1) neki glagoli pokazuju da ко radi sto izvjesno izvan sebe, što svagda mora stajati и vin. padežu, n.p. 53 Die bekannte Eintheilung der Ve] ba in Activa, Passiva und Deponen■ tia ist in der Serbischen Sprache, wie in sämmtlichen slavischen, müßig, d.h. ohne Einfluß auf die Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 217 - pisati knjigu, sjeci drva, kopa- t i vinograd, jesti ljeb, nositi vodu i t.d. I ovi se glagoli zo- vu djejstvitelni (poslujuci?). 2) neki glagoli pokazuju da se posao vraca na onoga, koji да radi, n.p. brijemse, kaješse, nadatise, nakanjivatise i t.d. I ovi se glagoli zovu povraca- telni, koji и pravom smislu nije- su drugo nista, nego glagoli djej- stvitelni: zašto i njiov posao stoji svagda u vin. padezu (samo sto nije izvan onoga, koji ga ra- di), n.p. b rijā ti se (sebe), ve- selitise, kao b rija ti да, veseliti te i t.d. 3) neki glagoli pokazu- ju samo stanje lica i l i stvari, i l i posao, koji ne ide ni na što, što bi stajało u vin. padežu, ne- go je u onome koji да radi, n.p. Anmerkungen zu den Adjectiv- Dec1inationen. [Anm.]Ovo je uzeto iz Latinske gramatike, zašto Latini imaju glagole djejstvitelne i strada- telne, a ovi n iti su djestvitel- 64 ni ni stradatelni, nego izmedu nji srednģi (kao i srednji rod izmēdu muškoga i ženskoga). j ( ) [Anm. ] Naši Slavenski gramatici ovakove glagole zovu stradatelni- та, а пеке otložitelnima. [Anm.]Ovo je uzeto iz Latinske gramatike, zašto Latini imaju glagole djejstvitelne i strada- telne, a ovi n iti su djestvitel- 64 ni ni stradatelni, nego izmedu nji srednģi (kao i srednji rod izmēdu muškoga i ženskoga). 64 Primječ. 1) Ovo razdjelenie gla- gola gotovo ne vrijedi ništa: za- sto svaki djejstvitelni glagol то- že b iti srednji (ali pravi srednji ne može b iti djejstvitelni) , n.p. pjevati pjesmu, igrati konja, ко- pati vinograd, orati njivu, vika- t i ljude, to su djejstvitelni gla- goli; a li kad se rece (i razumje- va) n.p. bolje je pjevati nego plakati; mator se konj ne uči igra- ti; nit* umijem orat1 ni kopati; jecam tree a rakija vice, onda su ovo sve srednji glagoli. Reciproca. Sie entspringen im Serbischen durch den Anhang des Pronomens se für alle drei Per- sonen, z.B. brijemse (ich rasire mich); kajeŠse, du bereuest; na- datise, hoffen; nakanjivatise, sich anschicken, und unzählige andere, deren Formen nicht als eine besondere Flexionsart be- trachtet werden dürfen. j g g 2) Naši Slavenski gramatici na- zivaju još пеке glagole stradatei- піла, а пеке otložitelnima (depo- nentia): no takovi glagola n iti ima и Slavenskome ni u Srpskome (kao ni и Njemačkom) jeziku, nego su to oni sami (gramatici) uzeli i preveli iz Grckoga i iz Latin- skoga jezika: zašto su m islili, da 00051004 00051004 - 218 - svaki jezik ono mora imati, što ima Greki i Latinski; a što Sla- venski jezik ima, a Greki i La- tinski пета, onom nijesu b ili ni mukaet ( i i i ako su što i na- ļpomenuli, a oni su o tom tako sto rekii, kao što govori čovek 0 stvari, koju ni sam ne razu- mije), kao n.p. dvojako znače- nje prilagatelni imena (cijelo 1 usjeceno), kod lieni mjesto- imenija тпё 1 mi, tebè i t i, sebe i si, mene i mja i t.d. I Bog zna sta se jos и Slaven- skom jeziku nije tako izostavi- lo i izgubllo! n.p. odnositelno mjestoimenije što imaju svi da- našnji Slavenski narodi, a u Slavenskom jeziku пета да! Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Anmerkungen zu den Adjectiv- Dec1inationen. Da su да imali i stari Slaveni, о tom ne treba siami jat i, nego su да izostavili prevodnici; zašto su m is lili da je ono sve pokva- reno, govedarski i svinjarski, Što пета u Latinskome i и Grč- kom jeziku. ־Da se vratimo opet к stradatelnim i otložitel- nim glagolima, n.p. Latinin ka- ze jednom rije c i amor, laudor, rogor i t.d. a mi u našem jeziku moramo za svaku tu riječ űzeti dvije, t . j . ljube me, vale me, mole me i t.d. A Nijemac mora uzeti t r i rije č i, t . j . ich werde geliebt, (bivam ljubljen), ich werde gelobt (bivam vaijen) i t. d. Koje mislim da ce svi prizna- t i, da ne ide u sprezanje gla- gola i sklanjanje imena zove se samo ono, kad se jedna riječ pre- mjenjuje sama и sebi, n.p. Iju- bim, ljubiš, ljubi, ljubimo, ljubite, ljube, ljublja, lju b i- t i i t.d. kao kod imena Bog, Bo- да. Води, Boze, Bogom, Bogovi i t.d. A truždajusja ni je glagol otložitelni, kao ni bijusja stra- datelni, nego su oba djejstvitel- ni glagoli. Svi naši glagoli (djejstvite ln i i srednji) mogu se opet razdije- l i t i 1) na glagole savršitelne Ungleich wichtiger und aus der Eigenthümlichkeit slavischer Zun- ge hervorgehend ist die Einthei- lung der Verba in Perfectiva und Imperfectiva. Svi naši glagoli (djejstvite ln i i srednji) mogu se opet razdije- l i t i 1) na glagole savršitelne (verba perfectiva... 2) na gla- gole nesavršitelne (verba im- perfectiva)... Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 219 - I ovo je najvažnije razdelenije naši glagola: 1) zato. Sto su ovi glagoli razliöni izmēdu se- be, kako и znaöenju, tako i и sprezanju... Diese Eintheilung greift nun wirk- lieh in das Förmliche der Conjuga- tion ein,.. [ Anm.] Da die Grammatik nur wenige Beispiele beider Arten anführen kann, so is t im Wörterbuche bei je- dem Verbo durch den Beisatz pf. o- der impf, gelehrt, wohin es ge- höre. [ Anm.] Da die Grammatik nur wenige Beispiele beider Arten anführen kann, so is t im Wörterbuche bei je- dem Verbo durch den Beisatz pf. o- der impf, gelehrt, wohin es ge- höre. Anmerkungen zu den Adjectiv- Dec1inationen. 53 [Anm.] Vec ako bi ко uzeo (za ׳ otloXitelni glagol) öuti, koji ima znaöenje glagola savrSitel- noga, a spreZe se kao glagol ne- savrSitelni, t . j . ima polupro- SavSe vrijeme, priöastije (i djejepriöastije) sadaSnjeg vreme- na i suStestvitelno od priöasti- ja stradatelnoga: Cûjâ, Cûjâse, eùjâsmo, ôûjâste, ôùjâu; ôùjû- ci, Cuvënje (n.p. po üuvenju). 53 [Anm.] Vec ako bi ко uzeo (za ׳ otloXitelni glagol) öuti, koji ima znaöenje glagola savrSitel- noga, a spreZe se kao glagol ne- savrSitelni, t . j . ima polupro- SavSe vrijeme, priöastije (i djejepriöastije) sadaSnjeg vreme- na i suStestvitelno od priöasti- ja stradatelnoga: Cûjâ, Cûjâse, eùjâsmo, ôûjâste, ôùjâu; ôùjû- ci, Cuvënje (n.p. po üuvenju). 5 ׳ 5 [Anm.] Ausnahme macht Öuti (hören) , welches die Bedeutung eines Per- fectivums hat, aber die Flexio- nen eines Imperfectivums: ôùjâ, ôûjâSe, ôûjâsmo, Öűjáste, ôûjâu, Gûjüci, öuvenje, z.B. po Cuve- nju (nach Hörensagen). 54 ... posratise, posranje (ne bi s njim otiSao ni na posranje)... 54 ... posratise, posranje (ne bi s njim otiSao ni na posranje)... 54 55 Sto je gofl sklonenije kod imena i kod mjestoimenija, to je spre- zanje kod glagola; samo se po tom (u ovom smislu) razlikuju i- mena od glagola. Sto se imena i mjestoimenija premjenjuju samo po padežima i po broju, i naj- viSe mogu imati oko deset pre- mjena; a glagoli se premjenju- ju po licima, po broju, po vre- menima, i po naklonenijama, i mogu imati preko trideset pre- mjena (a Latinski glagoli ima- ju još viSe premjena, a Gröki joS viôe od Latinskije). 55 Sto je gofl sklonenije kod imena i kod mjestoimenija, to je spre- zanje kod glagola; samo se po tom (u ovom smislu) razlikuju i- mena od glagola. Sto se imena i mjestoimenija premjenjuju samo po padežima i po broju, i naj- viSe mogu imati oko deset pre- mjena; a glagoli se premjenju- ju po licima, po broju, po vre- menima, i po naklonenijama, i mogu imati preko trideset pre- mjena (a Latinski glagoli ima- ju još viSe premjena, a Gröki joS viôe od Latinskije). 67 Die Serbische Sprache besitzt außer dem Präsens und Futurum ein doppeltes Präteritum, wovon wir das erste: Imperfectum, das zweite Präteritum (im eigentli- chen Sinne) nennen wollen. Das Imperfectum is t nur den Imperfec- tiv is eigen, das eigentliche Präteritum hingegen allen Verbis. 6 Vremena po pameti пета viSe, nego samo t r i, t . j . Anmerkungen zu den Adjectiv- Dec1inationen. sadaSnje (koje je sad), proSavSe (Sto je proSlo) i buduce (Sto ce doci и napredak); a glagoli naSi imaju t r i prosta vremena osim buducega, t . j. sadaSnje i dva proSavSa (1-vo poluproSavSe, koje imaju samo nesavräitelni glagoli... a 2-go skoroproSavSe, koje ima- ju svi glagoli... Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 220 - mu govorio, b ili su mu dali i t.d. no ovo ne ide u sprezanje glagola, zašto glagol пета ni- kakve nove premjene, nego mu se doda pomagatelni glagol bio, kao i ostale rije c i sto se sla ־ žu u govoru. mu govorio, b ili su mu dali i t.d. no ovo ne ide u sprezanje glagola, zašto glagol пета ni- kakve nove premjene, nego mu se doda pomagatelni glagol bio, kao i ostale rije c i sto se sla ־ žu u govoru. [Anm.] Dobrowsky in den in s tit 1- sl. vet. nennt es p. 522. 527 prae teritum iterativum und nimmt es nicht in die Paradigmen auf. Das Futurum... könnte man in so fern aus der eigentlichen Flexions lehre ausschließen. Da inzwischen das Hülfswort häufig dem voraus- gehenden In fin itiv accrescirt, so verdient das Futurum noch mehr als jenes dritte Präteritum in die Conjugationen aufgenommen zu wer- den • 68 69 U Slavenskim gramatikama imaju c e tiri naklonenija (izjavitel ־ no, povelitelno, soslagatelno i neopredjelenő); a li glagoli ni naši ni Slavenski nemāju viSe od tr i, t . j. 1-vo izjavi- telno, и коте kakvo lice Sto radi, i l i pokazuje da je Sto ra- diio, i l i da ce raditi, n.p. pi- 5em,pisao sam, pisaèu i t.d. 2-go povelitelno, и коте se sto zapovijeda (nagovara, moli, i l i nudi) drugome коте da radi, n.p. pisi, pisite; ustani, donesi, daj, reci i t.d. 3- t e neopre- djeleno, и коте glagol stoji pust bez ikakva izvjesnoga posla, lica i broja (kao n.p. suštestvi- telno ime и imenit. padežu), n.p. pisati, raditi, govoriti, ic i, doci, cuti i t.d. - A 4-to sosia- gatelno uzeli su gramatici iz Grcke i iz Latinske gramatike, zašto Latinin kaže jednom rije - ci, n.p. habuerim, attuleris, scissem; a Srbljin, i svaki dru- gi Slavenin mora uzeti dvije raz- liene rije c i mjesto te jedne La- tinske, t . j. Anmerkungen zu den Adjectiv- Dec1inationen. Napomenuto je malo, da bijusja, pitaju- sja, tvorjusja, nijesu glagoli stradatelni, kao ni bojusja otložitelni; a ко je razumjeo što smo malo prije rekli о sprezanju glagola, kazace i sam, da pitani byli езть ne ide и sprezanje glagola, kao ni ašče by esmb byli pitam i bio sam karan (istina da se govori: bio sam karan, biceš bijen, b ili su doČekani; 70 a li u sadašnjem vremenu niko nece reci: bivam karan, bivaš moljen, nego karaju me, mole me, biju да, traže te i t.d. A i u prošavšem i u buducem vre- menu ne govori se kod sviju gla- gola sa stradatelnim pričasti- jem, n.p. bicu traŽen, bio sam moljen, nego tražice me, m olili su me, zvali su да i t.d .). Bios für das Participium besitzt die Serbische Sprache eine Passiv- form• form• Es stehen ihr aber außer diesem passiven Participium drei active Participia zu, nämlich eins für das Präsens, welches sich auf èi endigt und vom Volk im Reden wie ein la- teinisches Gerundium gebraucht wird, z.B. ode igrajuci, er ging spielend fort; pjeva kopajuci, er singt gra- bend; jede stojeci, er ißt stehend; im Schreiben könnte man solche For- men für wahrhafte Participia (d.h. zugleich Adjectiva) nehmen. z.B. îgrajûèî (der Spielende), igrajÛèe (das Spielende), auch jetzt das Volk einige wirklich adjectivisch, als tèkûcâ voda (fließendes Wasser), stàjacâ košulja (Feiertagshemd), spávacê alj ine (Schlafkleider), piè- ticâ iqla (Stricknadel). Das Parti- cipium Präteriti Activi is t doppelt und in den Paradigmen durch X. und II. unterschieden. Das erste geht aus auf vši, und bedeutet ungefähr auch das latéin. Gerundium, zumal bei Perfectivis, z.B. došavši on (ona, ono) u kucu, reče mu, nachdem er (sie, es) ins Haus gekommen war, sagte er ihm; poigravši malo, sje- de, nachdem er ein wenig getanzt hatte, setzte er sich; rekavŠi to, uze да za ruku, nachdem er dies ge- sagt hatte, nahm er ihn bei der Hand; poradivši dace Bog, nach ge- thaner Arbeit wird Gott geben. Über pošav statt pošavši, vergl. die No- te zum Paradigma erster Conjugation. Auch dies Particip stehet selten adjectivisch, doch hört man bivši knez, der gewesene Fürst; bivšemu knezu, dem gewesenen Fürsten; biv- ši vojvoda, der gewesene Anführer, u.s.w. - Das andere Participium Prät. Act. wird mit dem Consonanten 1 gebildet, der im Masc. nach der Regel in о Übertritt, z.B. bio (ge- wesen), bila, bilo, u.s.w. Anmerkungen zu den Adjectiv- Dec1inationen. da sam imao, ako do- neseš, da sam bio znao i t.d. A to svak može videti da nije spre- zanje glagola. Sprezanje ( i i i bo- lje reci sklonenije) glagola zo- ve se samo ono, (kao što je i ma- lo prije kazano), kad se glagol sam po sebi premjenjuje (kao ime- na i mjestoimenija kad se skia- njaju) n.p. trpim, trpiš, trpi. 69 Die altslavischen Grammatiken stel len zwar vier Modus auf, den Indi- cativ, Conjunctiv, Imperativ und In fin itiv . Allein der Conjunctiv beruht weder im Altslavischen, noch weniger im Serbischen auf ei- ner eigentümlichen Flexion, son- dern muß durch Umschreibungen aus- gedrückt werden; z.B. da sam imao, wenn ich gehabt hätte; ako done- sem, wenn ich bringen sollte; da sam bio znao, wenn ich gewußt hät- te; и.s.w. Misverstandne Analogie der lateinischen Sprache hat die- selben Grammatiker gleich jenem Conjunctiv auch ein Passivum der altslavischen Sprache aufzudrän- gen verleitet. Ein solches Passi- vum findet sich denn auch im Serbischen nirgends. Man sagt zwar umschreibend: bio sam karan, ich bin gescholten worden; biceš bi- jen, du wirst geschlagen werden; b ili su dočekani, sie sind gut empfangen worden; aber im Präsens wird doch niemand sagen: bivam karan, bivam moljen; sondern ka- raju me, sie schelten mich; mole me, sie bitten mich; biju да, sie schlagen ihn; traže te, sie suchen dich, u.s.w. Auch im Präterito und Futuro wird nicht bei allen Verbis mit dem Particip Pass, ge- sagt, z.B. bicu tražen, bio sam moljen, vielmehr traziòe me, er wird mich suchen; m olili su ite, sie haben mich gebeten; zvali su да, sie haben ihn geruft, u.s.w. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access via free access » 221 - trpimo, trpite, trpe, trplja, trpljaše, trpljasmo, trpljaste, trpljau, trpiste, trpiŠe, trpljeo, trpeci i t.d. A da sam bio znao, i aŠče by esmb pi- ta li, to su čitavi razgovori. Tako se и našim Slavenskim gra- matikama nalazi i stradatelni zálog, a glagoli да nemāju ni naŠi ni Slavenski. Anmerkungen zu den Adjectiv- Dec1inationen. Dieses Particip stehet zwar immer adjecti- visch und flexibel, aber nur prädi- cirend, d.h. mit dem Hülfswort jesam verbunden, um das zusammenge- setzte Prät. Ind. hervorzubringen; für sich allein steht es nie. Der Serbe kann es sowol dem Hülfswort nachsetzen und dann bleibt es ge- 70 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 • 222 - trennt, z.B. jesam bio, bila, ibi- lo; jest bio; als vorsetzen und dann verwachsen beide: biosam, bi- lasam, bilosam; bioje, bilaje, bi ־ loje; das Hülfswort darf nicht weg■ bleiben, in der ersten und zweiten Person wohl nie, in der drittem zu־ weilen. ־Das Participium Passivi endigt meistenstheils auf n, zu ־ weilen aber auch auf t, worüber di< achte Anmerkung zur zweiten Conju- gátion nachzusehen. [Anm.] Kad se ovo pravilo uvede i и Slavensku gramatiku (kao sto je i и jeziku) onda ne ce po do ־ jakoinjim pravilima ("izmenja- jušče и i l i ju na achi") morati b iti: zovachi, vjazachi, іісасЬг, strazdachi, zenachi, perachi, alcach* i t.d. n iti ce se mora ־ t i kazati "izemljutsja çlyvu plyehi, poju pechi, chosču cho ־ techi n iti ce od voskresaju moei b iti voskresnuchi; od derzaju, derznuchi; od kupuju kupiehi; od sretaju, sretoch*, od padaju padoehi; od pominaju, pomjanuehi i t.d. Vaija da su ovakove po- grjeske u Slavenskim gramatika ־ ma naòerale G. Dobrovskoga, da kaže и Slavinu (recenzirajuci Slavensku gramatiku G. Mrazovi- ca: Mit einem Worte, es muß in den Slavenischen Sprachlehren, die Russische nicht ausgenommen, ein ganz anderes System einge- führt werden (jednom rije č i, то- ra se и Slavenske gramatike, i и samu Rosijsku, sasvim drugi red uvesti). 7 57 Die eigentlich flectirten Tempora (nämlich: Präsens, Imperfectum und einfaches Präteritum, da das zusammengesetzte Präteritum und Fu turum mit Auxiliarien erzeugt wer ־ den) haben für die drei Personen folgende Kennzeichnung. 1) Erste Person Präsentis Sing. m, Plur. то; im Sing. des Imperf. und Prät. bloßer Vocal, ohne Con- sonans, doch lehrt die altslavi- sehe Grammatik, daß ein ch wegge- fallen is t, welches der Serbe hier so wenig, als im Gen. Plur. des Nomens ausspricht, wenn er es auch noch schriebe. Der Plur. Imperf. und Prät. hat immer smo. 2) Zweite Person Präs. Sing. S; Imperf. Sing. še; Prät. Sing. bio ־ ßer Vocal. Präs. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Anmerkungen zu den Adjectiv- Dec1inationen. Plur. te; Imperf. 71 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 71 Die eigentlich flectirten Tempora (nämlich: Präsens, Imperfectum und einfaches Präteritum, da das zusammengesetzte Präteritum und Fu turum mit Auxiliarien erzeugt wer ־ den) haben für die drei Personen folgende Kennzeichnung. 71 71 1) Erste Person Präsentis Sing. m, Plur. то; im Sing. des Imperf. und Prät. bloßer Vocal, ohne Con- sonans, doch lehrt die altslavi- sehe Grammatik, daß ein ch wegge- fallen is t, welches der Serbe hier so wenig, als im Gen. Plur. des Nomens ausspricht, wenn er es auch noch schriebe. Der Plur. Imperf. und Prät hat immer smo 2) Zweite Person Präs. Sing. S; Imperf. Sing. še; Prät. Sing. bio ־ ßer Vocal. Präs. Plur. te; Imperf. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 223 - und Prät• Plur. ste. 3) Dritte Person Präs. Sing. und Plur. Vocal, doch in beiden walte- te vor Alters t, das allmählich abgefallen ist; Imp. Sing. še, Plur. Vocal. Umgekehrt Prät. Sing. Vocal, Plur. še. Das Verhältniß der Vocale läßt sich im Allgemei- nen nicht angeben. Sowohl im Imp. als Prät. fallen die zweite und dritte Pers. Sing. stets zusammen. Im Prät. unterscheidet sich der Vocal der ersten Pers. Sing. (wenn er nicht individuell abweicht, wie zuweilen in zweiter Conj.) durch seine Unbetonung von dem betonten Vocal der zweiten und dritten Per- son. Auch allein durch den Ton unterscheidbar sind die ersten Personen des Sing. Imp. und Prät., wenn ihr Vocal a lautet (wie in der ersten Conj. immer, in der zweiten und dritten zuweilen); nämlich das a des Imperativs hat den dritten Accent, das des Prät. gar keinen welches man auch so ausdrücken kann: die erste Pers. des Imperf. lautet, in diesem Fal- le, immer wie die zweite und d rit- te des Präteritum. [Anm. = 2) Punkt aus "Dodatak"] [Anm. = 2) Punkt aus "Dodatak"] 4 ... tèdo, tè ... tède, tè...tède, tè... tèdosmo, tèsmo... tèdoste, tèste... tēdoše» teše. 4 5 II. ceo (ktèo, tèo, tio, ô tio ), cèla, èèlo. [ Hinzufügung des "II. Part. Prät." bei allen Verben, so: bio, bila, bilo, u.s.w.] [Vgl. S. 69f. bei Grimm] 5 II. ceo (ktèo, tèo, tio, ô tio ), cèla, èèlo. , [ Hinzufügung des "II. Part. Anmerkungen zu den Adjectiv- Dec1inationen. Kad bude bolja zgoda i više mjesta i vremena za pisanje Srpske gra- matike, onda ce se moci i oni još bolje ra z d ije liti i и re- dove postaviti; a za sad, kao pored rječnika, evo ovako da i maio pregledamo: ... [Vuks Beispiele sind in Tabellen geordnet, fehlende Infinitive er- gänzt.] 6 5 b) iva ti (ovo su gotovo sve ne- savršitelni, i l i da rečemo je- dan put učaščatelni (učestni, učesni?), verba frequentati- va... [Anm.] Meni se cini da пета upravo nikakve razlike izmeflu glagola nesavršitelnije (verba imperfectiva) i učaščatelnije (verba frequentativa); zašto je svaki učaščatelni glagol i nesavršitelni, a svaki nesa- vršitelni može b iti i učašča- te ln i. b) Einige auf ivati (es sind bei- nahe lauter Imperfectiva). 71 3) ... jeda, jedaše i t.d .; tako i od grebem, greba, gre- baše; a od zebem može b iti da bi ko rekao i zebija, zebijaše i t.d. К ovima ide i kunem, kunija, kunijaše i t.d. 9 68 2 ) . . . • • • 5) Istina da po opštemu pravi- lu kod sviju nesvršitelni glago- la postaje suštestvitelno na nje od stradatelnog pričastija kad se promijeni n na nje, n.p. kopan, kopanje; igran, igranje; pisan, pisanje, pečen, pečenje; mjeren, mjerenje; suflen, sude- nje; Ijubljen, Ijubljenje; slavljen, slavljenje i t.d. Ali se opet (osobito u Resavskome i u Sremačkom narječiju) kod kod glagola ovoga sprezanja, osobito koji se svršuju na lim i nim, govori još i druk- cije ( t.j. ne promjenjuje se 1 na 1j , ni n na n j, n.p. 9 68 2 ) . . . • • • 5) Istina da po opštemu pravi- lu kod sviju nesvršitelni glago- la postaje suštestvitelno na nje od stradatelnog pričastija kad se promijeni n na nje, n.p. kopan, kopanje; igran, igranje; pisan, pisanje, pečen, pečenje; mjeren, mjerenje; suflen, sude- nje; Ijubljen, Ijubljenje; slavljen, slavljenje i t.d. Ali se opet (osobito u Resavskome i u Sremačkom narječiju) kod kod glagola ovoga sprezanja, osobito koji se svršuju na lim i nim, govori još i druk- cije ( t.j. ne promjenjuje se 1 na 1j , ni n na n j, n.p. Anmerkungen zu den Adjectiv- Dec1inationen. Prät." bei allen Verben, so: bio, bila, bilo, u.s.w.] 60 [Anm.] Istina da narod ovakove rije č i и govoru svagda uzima kao gerundia, n.p. ode igrajuci, pje- va kopajuòi, kopa pjevaju&i, je- de stojeci i t.d .; a li bi se и pisanju mogle űzeti i za prava pričastija (kao prilagatelna imena), n.p. igrajÛôî, igrajÙ- câ, igrajûcê; zašto je narod dekoje uzeo i obratio u prilaga- telna imena, n.p. tèkûcâ voda, stàjacâ kosulja, spávacê aljine, plèticâ igla i t.d. [Anm.] Tako se i ova pričastija prošavšeg vremena upotrebljava- ju и govoru kao gerundia, i to 60 [Anm.] Istina da narod ovakove rije č i и govoru svagda uzima kao gerundia, n.p. ode igrajuci, pje- va kopajuòi, kopa pjevaju&i, je- de stojeci i t.d .; a li bi se и pisanju mogle űzeti i za prava pričastija (kao prilagatelna imena), n.p. igrajÛôî, igrajÙ- câ, igrajûcê; zašto je narod dekoje uzeo i obratio u prilaga- telna imena, n.p. tèkûcâ voda, stàjacâ kosulja, spávacê aljine, plèticâ igla i t.d. [Anm.] Tako se i ova pričastija prošavšeg vremena upotrebljava [Vgl. S. 69f. bei Grimm] [Vgl. S. 69f. bei Grimm] 00051004 00051004 • 224 - ponajviše kod svršitelni glago- la, n.p. došavši (on, ona, ono) u kucu reče mu; poigravŠi malo sjede; rekavši to uze да za ru- ku; poradivši dace Вод i t.d. ponajviše kod svršitelni glago- la, n.p. došavši (on, ona, ono) u kucu reče mu; poigravŠi malo sjede; rekavši to uze да za ru- ku; poradivši dace Вод i t.d. 64 86 Mit der Zeit wird es der Serbische! Grammatik schon gelingen, in die Gründe dieser Abweichungen tiefer einzudringen und sie alsdann zweck mäßiger zu ordnen. Vorläufig be- gnüge man sich mit folgenden A.n- merkungen: 4 ... a li prema glagolima Grčkim i Latinskim (i sami drugi de- koji Slavenski naroda) sve je ovo smilje i bosilje. Kad bude bolja zgoda i više mjesta i vremena za pisanje Srpske gra- matike, onda ce se moci i oni još bolje ra z d ije liti i и re- dove postaviti; a za sad, kao pored rječnika, evo ovako da i maio pregledamo: ... 4 ... a li prema glagolima Grčkim i Latinskim (i sami drugi de- koji Slavenski naroda) sve je ovo smilje i bosilje. 97 2) ... Dasselbe g ilt auch von den Ver- balien. Der Ressawer und Sirmier wird das 1 und n vor dem enje nicht leicht in nj und l j verwan- dein, sondern sagen: molim, то- lenje (das Bitten), balim, bale- nje (das Rotzen), bunim, bunjenje (das Aufschwingen), branim, brane- nje (das Wehren). Andere Mundart- ten ziehen moljenje, baljenje, bunjenje vor, wie sie vrcenje (das Bohren), trpljenje (das Er- tragen, srdenje (das Zürnen) vor- ziehen dem Ressawischen vrtenje, trpenje, srdenje. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 00051004 ־ 225 - molìin, molen je; bálim, balenje bunim, bunenje; branimc brane- nje i t.d. nego mi se cini da je ono prvo običnije. Tako isto može b iti da bi ko rekao (и Resavskome i и Sremačkom na- rječiju) i vrtenje, trpenje, srdenje i t.d. prem da je svu- da običnije vrcenje, trpljenje srdenje i t.d. Oväe je za sad pored rječnika dosta gramatike. О narječija- ma, o predlozima, i o meždu- metijaxna nije trijebe govori- t i, zašto su ovűe и rječniku. 69 Alphabeti Serborum ... molìin, molen je; bálim, balenje; bunim, bunenje; branimc brane- nje i t.d. nego mi se cini da je ono prvo običnije. Tako isto može b iti da bi ko rekao (и Resavskome i и Sremačkom na- rječiju) i vrtenje, trpenje, srdenje i t.d. prem da je svu- da običnije vrcenje, trpljenje, srdenje i t.d. molìin, molen je; bálim, balenje; bunim, bunenje; branimc brane- nje i t.d. nego mi se cini da je ono prvo običnije. Tako isto može b iti da bi ko rekao (и Resavskome i и Sremačkom na- rječiju) i vrtenje, trpenje, srdenje i t.d. prem da je svu- da običnije vrcenje, trpljenje, srdenje i t.d. Oväe je za sad pored rječnika 98 dosta gramatike. О narječija- ma, o predlozima, i o meždu- metijaxna nije trijebe govori- t i, zašto su ovűe и rječniku. 69 Alphabeti Serborum ... 69 Alphabeti Serborum ... 69 69 Alphabeti Serborum ... Von den Partikeln. 4) Den Accusativ und Localis regieren: na, auf; o, von, an, zu; govori о njemu er redet von ihm; visi о klinu, hängt 100 am Nagel; udario о kamen, stieß sich an den Stein; о božicu, zu Weihnachten; po, um, durch, nach, in, deswegen, als: otisao po äevojku, er ging um das Mäd- chen fort; poslao po devojci, er schick te durch das Mädchen; udario да po gla- vi, er stieß ihm nach dem Kopf; u, in (wenn и bei heißt, erfordert es den Gen als: и boga je svaŠta dosta, bei Gott is t alles genug). Die Präpositionen na und u, wenn sie auf die Frage: wo? die Ruhe eines Gegenstandes anzeigen, haben den Localis bei sich, stoji na brdu, stehet auf dem Berg; poginuo na Kosovu, ging auf dem Amselfeld unter; bio sam и crkvi, ich war in der Kirche. Deuten sie aber auf die Frage wohin? Bewegung an, so folgt ihnen der Acc., z.B. o ti- šao na brdo, er ging auf den Berg; ide na Kosovo, er geht auf das Amselfeld; idem и crkvu, ich gehe in die Kirche. 5) Den Acc. und Instr. regieren (so wie na und и den Acc. und Loc.) folgende: pod, unter; ode pod kucu, er geht un- ter das Haus; stoji pod kucom, er steht unter dem Haus; nad, nada, über: nad vodu, über das Wasser; nad vodom, über dem Wasser; nada se, über es; nada mnom, über mir; meflu, zwischen, unter; to je medu nama, das ist zwischen uns; daj medu nas, theile unter uns; pred, preda, vor; stoji pred kucom, steht vor dem Haus; iznesi pred kucu, trägt vor das Haus; metni preda me, setzt vor mich; stoji preda mnom, steht vor mir; auch pred smrt, vor dem Tod; pred bo- zie, vor Weihnachten; za, hinter, nach, um, bei: sjedi za kucom, steht hinterm Haus; odnesi za kucu, trägt hinters Haus; ide za njim, geht nach ihm; ne brini se za njega, kümmert sich nicht um ihn; za novee svaita dosta, um Geld alles genug; uzeti za ruku, bei der Hand nehmen. Zuweilen steht bei za der Genitiv: za života, während des Lebens, Vera Boji 9783954793044 2) Den Dativ regiert nur eine Präposi ־ tion: k, ka, zu, к meni, zu mir, ka Kru sevcu, zu Kruschewatz. Von den Partikeln. 99 Von den Partikeln. I. Präpositionen. 1) Den Genitiv regieren: bez, brez, ohne; blizu, nahe; viSe, oberhalb; do, bis, außer; iz, aus; iza (iz za), von hinten, von jenseits; z.B. iza brda, vom Berge her; iz van, außer; z.B. izvan pameti, außer Verstand; izmeäu, zwischen; iznad, von oben herab; ispod (iz pod), von unten; ispred, vornen, weg; kod, bei, z.B. kod mene, bei mir; kod kuce, zu Hause; kraj, am Ende, z.B. kraj sela, zu Ende des Dorfs; kraj mora, am Ufer des Meers; kraj mene, bei mir, neben mir; nakraj, am En- de, unten, z.B. sjedi nakraj sela, er wohnt unten im Dorfe, pokraj, neben, längs; ukraj, am Ende; mjesto, statt; nakom, hinter, z.B. nakom sebe spornen ostaviti, Andenken hin- terlassen; nasred, mitten in, z.B. nasred sela, polja, mitten im Dor- fe, im Feld; niže, unten, unter- halb; od, von; oko, okolo, um, her- um, z.B. oko kuce, um das Haus; oko boŽica, um Weihnachten; osim, außer; pored, neben; poslije, nach, nachher; posred, mitten durch, z. B. udario да posred glave, er stieß ihn mitten durch den Kopf; ode posred sela, er ging mitten durchs Dorf; preko, über; prije, vor (mit Bezug auf die Zeit); pro- tiv , gegen; radi, zaradi, poradi, wegen, um willen; svr (savr), von oben, z.B. sko&io svr kuce, er sprang oben vom Hause herab; savr glave do zelene trave, oben vom Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM i f glave do zelene trave, oben vom Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 226 - Kopf bis zum grünen Grase, s onu stra- nu, jenseits. Kopf bis zum grünen Grase, s onu stra- nu, jenseits. 2) Den Dativ regiert nur eine Präposi ־ tion: k, ka, zu, к meni, zu mir, ka Kru sevcu, zu Kruschewatz. 3) Den Accusativ regieren: kroz, durch; mimo, vorbei; niz, hinab; niz brdo, vo- du, den Berg, das Wasser hinab; uz, hin auf, uz brdo, vodu. Von den Partikeln. 3) Den Accusativ regieren: kroz, durch; mimo, vorbei; niz, hinab; niz brdo, vo- du, den Berg, das Wasser hinab; uz, hin auf, uz brdo, vodu. 100 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 227 - za mladosti, während der Jfugend, u.s.w« 6) Gen. und Instr. regierem: s, sa, mit, von, durch: s bratom, mi: dem Bruder; s brda, von dem Berge; s Jomja, von dem Pferde; sa zemlje, von dir* Erde; propao 1 s brata, er ist durch dei Bruder zu Grund gegangen; sa svoje p>ameti, durch seinen Verstand. 101 II. Adverbia. 1) Des Orts: cimo, hierher; blizu, nahe; gore, oben; daleko, weit; dovde, dovle, bis hierher; dokle, so wei.t als; dolje, unten; donde, donle, bis dtorthin; dotle, bis dahin; de, wo; degod, wo immer; de mu drago, wo immer, es say ,־wo es w ill; igde, wo immer; iz daleki, von weitem; iznutra, inwendig; izokola:, rund herum, von der Seite; izrebar, iz:rebra, von der Seite; izprijeka, voi der Seite; ku- da, wo; lijevo, links; na&aleko, weit; naprijed, voraus; nide, nigde, nirgends; ovamo, hierher, auf dieser Seite; ovde, hier; ovuda, hierher; odav׳de, odavle, von hier; odakle, von wo; odatle, von da; odsvakle, odsvakud, vo>n überall; od- ovud, von hier, von dieser Seite; ozgo, oben; ozdo, unten; okolo, herum; onamo, dorthin; onde, dort; onuda, dort vorbei; ostrag, von hinten; otkud (od kud), von wo; spolja, von außen; svagde, svade, überall; sprijed, vorne; tamo, dort; tu, dort, da; tuda, dorthin; unutra, drinnen, hinein; cak, weithin, bis an. 2) Der Zeit: va vijek, immer, ewig; ve- čeras, večeraske, diesen Abend; davno, lange; danas, danaske, heute; dockan, docne, spät; zimi, Winters; zimus, zi- muske, diesen Winter; zorom, frühe, bei Tagesanbruch; igda, ikad, irgendwann, je; istom, erst; jako, jetzt; jesenas, jesenaske, diesen Herbst; jutros, jutro- ske, heute früh; juče, gestern; kad, wann als; katkad, dann und wann; lani, vori- ges Jahr; lje ti, Sommers; Ijetos, lje - toske, heuer; najzad, najposlije, zuletzt nigda, nikad, niemals; nocas, nocaske, heint; noću, nocom, bei Nacht, Nachts; ovoga puta, gleich, auf der Stelle, jetzt odma, gleich; onda, ondaj, dann; onomad- ne, dieser Tage; poslije, später; potom, (po tom), hernach; prekjuče, vorgestern; preklani, vor zwei Jahren; preksinoc, vor 102 gestern Abend; prije, prede, vor, ehe; proljetos,verwichenen Frühling; rano, frühmorgens; sad, jetzt; sinoc, gestern Abend; sjutra, sutra, morgen; sjutradan. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM 102 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access den Tag darauf; skoro, unlängst; tada, dann; u d ilj. Immer, beständig. 3) Der Quantität: više, mehr; dovoljno, hinlänglich, nach Herzenslust; dosta, genug; izobila, überflüssig; istom, nur, nichts als; još, jošt, jošte, noch; ko- liko, wie viel, so viel; kolikogod, so- viel immer; malko, malo, malčice, ein wenig; manje, weniger; mlogo, viel; mrvi čku, mrvke, mrvčice, ein klein wenig, Krümchen, Bißchen; nekoliko, einiges, et was; nikoliko, gar nichts; ovoliko, so- viel; onoliko, soviel; toliko, soviel. II. Adverbia. 4) Der Qualität: bolje, besser; gotovo, beinahe; teško, schwer; slabo, schwach; carski, kaiserlich. [Anm.] Beinahe je- des Adjectiv läßt sich auf diese Weise in ein Adv. verwandeln. 5) Fragende Adverbia: zar, etwa? zašto, warum? je li (je l i ) , gelt? jer, warum? kamo, wo ist? kuda, wohin? n ije li (nije l i ) , nicht? pošto, wie theuer? 6) Bejahende, verneinende: da, ja; da kako, allerdings; doista, zaista, gewiß; ja, ja; jamacno, sicher; Ije, sicher; nije, nein. 5) Fragende Adverbia: zar, etwa? zašto, warum? je li (je l i ) , gelt? jer, warum? kamo, wo ist? kuda, wohin? n ije li (nije l i ) , nicht? pošto, wie theuer? ) p 6) Bejahende, verneinende: da, ja; da kako, allerdings; doista, zaista, gewiß; ja, ja; jamacno, sicher; Ije, sicher; nije, nein. I I I . Conjunctionen. I I I . Conjunctionen. a, aber, und; a kamo l i , viel weniger; ako, wenn; al, a li, aber, oder; ama, aber; baš, gerade, auch, sogar; bud, wen auch, sei es; buduci, weil, indem, da aber, weil es so ist; van da, außer, wenn; vec, sondern; vec ako, außer wenn; da, daß (da ja imam, daß ich hätte) wenn (da ja znam, wenn ich wüßte); dakle also, daher; eie, einmal; zašto, denn; i, und; i l i , oder, entweder; ja, ja li, entweder, oder; jer, denn; kako goä, so wie; l i , ob? nego, sondern; ni, weder, noch; n iti, weder, noch; no, aber; opet, abermals, wieder; pa, und, hernach, dann; pak, und, hernach, aber; prenda, wiewohl; takofler, auch, to, so. 103 IV. Interjectionen. A! ahi aa! aha! ah! ach! avaj! oweh! weh aj! hei! aja (ajja)! nein, auf keine Art! ao! (wundernd), ato (a to)! sieh da blago! wohl (mit dem Dat.) bl. meni! wohl mir! bog me! bei Gott! bre! (befeh- lend) gle! scheu, sieh! davori! he! wart! de! wohlan! dede, deder! ma cne, ma che! dede da vidim! laß sehen! dela! (befehlend) žiža! (sich verbrennend) ej ! hei! evo! sieh da (vor mir)! eno! sieh da (vor ihm) ! eto! sieh da (vor dir) ! 103 IV. Interjectionen. A! ahi aa! aha! ah! ach! avaj! oweh! weh aj! hei! aja (ajja)! nein, auf keine Art! ao! (wundernd), ato (a to)! sieh da blago! wohl (mit dem Dat.) bl. meni! wohl mir! bog me! bei Gott! bre! (befeh- lend) gle! scheu, sieh! davori! he! wart! de! wohlan! dede, deder! ma cne, ma che! dede da vidim! laß sehen! dela! (befehlend) žiža! (sich verbrennend) ej ! hei! evo! sieh da (vor mir)! eno! sieh da (vor ihm) ! eto! sieh da (vor dir) ! Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access - 229 « i i ! (wundernd) jao, jaoji! weh! jatka! der Arme! jado! weh! kuku men.i ! weh mir! kuku lele! weh! lele meni! weh !.׳Iju, Iju! (2u Wiegenkindern) more! (s. das Wörterbuch) na, naj! da! nemoj ! nicht doch! nu! nude! nuder (betheuernd) nuto! sieh! о! (rufend) oj ! (auf Rufen antwortend , in den Liedern ausrufend) op, opa! op,cu!p! opa,cupa! (tan- zend) oo! oho! pi! pfui! pik! (s. das Wör- terbuch) pomagaj! Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access I I I . Conjunctionen. zu Hülfe! pst! pst! puc! plumpf! ta! (ne cesi)gewiß! teško! weh (mit dem Dat. der Person: teško meni, tebi, njemu, nama! weh mir, d ir, ihm, uns) ba ću! la ri fari! и! uh! uk! (frierend, schau- dernd). ) Es gibt sodann viele Interjectionen für Thiere, zumal Hausthiere, sie zu locken, zu scheuchen, gehend und ste!hend zu machen. LockwÖrter: b iri, b iri! (zu welschen Hüh- nern) v it, v it! (zu Tauben) gic, gic! (zu Schweinen) 6a, da! (zu Pferden) ma, ma! (zu Kühen) mac, mac! (zu Katzen) cuk, cuk! (zu Hühnern) Scheuchwörter: oš, oša! (zu Hun- den) pis, pis! (zu Katzen) us, uš! (zu Schweinen) и j , uj! (zu Krähen, hui, hui) voc! (zum Ochsen) cuki! (zum Hunde) sibe! (zu jungen Hunden)sikel (zu Ferkeln) Soll der Ochse angetrieben werden, so heißt es: ca! soll er zurückgehen: stu! den Ochsen oder das Pferd zum Fortgehen zu bringen: de! Das Kalb von der Kuh zu trennen: ljok! Lämmer von den Müttern: k it, k it! k it, lue! Ziegen zu treiben: kec, keca! Schafe zu treiben: is, isa! die Ziege zum Melken ste- hen zu machen: t ir , t i r i ! 104 Merkwürdig sind einige, •die noch auf heid- nische Zeit zurückweisen und fast nur in den Liedern gebraucht wexden, vgl. lado! le l jo! kolaloml in dem Wörterbuch. In den Liedern finden sich auch zusammengesetzte, oft halbe und ganze Zeil«en füllende Inter- jectionen, ja der Refraim mag als eine erweiterte Interjection !betrachtet werden. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 00051004 - 230 - ANHANG II Zusammenstellung der Notizen aus Grimms Handexemplar mit Quellenangaben1 ANHANG II Zusammenstellung der Notizen aus Grimms Handexemplar mit Quellenangaben1 mit Quellenangaben1 [über Vuk:] [118] Wuks Gramm[atik] rec[ensiert] Wiener Lit[eratur]z[eitung] 1815, n46 ־ Volkslieder I - - n- 74 II - 1816 März [111] Literarisches] Con[versations]blatt n- 98. 28 April 1824 .3 Wuk Stefanowitsch (W. Böttiger) und Kruse? hatte anno 1819 übersetz[un]g des N[euen] T[estaments] ins Serbische verfaßt, die Petersburger Bibelgesfellschaft] wollte sie 1820. drucken lassen, der Druck fand zu Wien Hindernisse, dann wurde die Ubers[etzung] nach Petersburg geschickt u[nd] einer starken Revision unterworfen, wodurch sie, da man viel alte Kirchenausdr[iicke] einschiebt, an Brauchbarkeit fürs Volk verlieren dürfte. Wuk hat eine Wienerin geheiratet, die er während s[einer] Reise nach Sachsen in Temiswar ließ. W[uk] nachdem er studiert war Mauthinspector in einem Di- strict Serbiens; während Czarny Georgs Oberherrschaft Secre- tar des Senats zu Belgrad. Seit der Besitznahme durch die Türken privatisierte er theils in Serbien, theils in Oester- reich, reiste durch Polen nach Rußland, wo er Petersb[urg], Moskau, Kiew besuchte. - Jetzt kehrte er nach Kragujewatz, Miloschs Wohnsitze zurück. [Zum Titel- blatt:] belobt im litteraturblatt zum morgenblatt 1824 . nr. 69. 27. aug[ust] von X. über Wuk allg[emeine] zeitung 1839 n^ 257° und widerlegt - 1840 n- 165 p. 117. 118 die Übersetzung der gr[ammtik] ist von Tirol [118] Wuks Gramm[atik] rec[ensiert] Wiener Lit[eratur]z[eitung] 1815, n46 ־ Volkslieder I - - n- 74 II - 1816 März [111] Literarisches] Con[versations]blatt n- 98. 28 April 1824 .3 Wuk Stefanowitsch (W. Böttiger) und Kruse? [118] Wuks Gramm[atik] rec[ensiert] Wiener Lit[eratur]z[eitung] 1815 n46־ , Volkslieder I - - n- 74 II - 1816 März [111] Literarisches] Con[versations]blatt n- 98. 28 April 1824 .3 Wuk Stefanowitsch (W. Böttiger) und Kruse? Literarisches] Con[versations]blatt n- 98. 28 April 18 Wuk Stefanowitsch (W. Böttiger) und Kruse? hatte anno 1819 übersetz[un]g des N[euen] T[estaments] ins Serbische verfaßt, die Petersburger Bibelgesfellschaft] wollte sie 1820. drucken lassen, der Druck fand zu Wien Hindernisse, dann wurde die Ubers[etzung] nach Petersburg geschickt u[nd] einer starken Revision unterworfen, wodurch sie, da man viel alte Kirchenausdr[iicke] einschiebt, an Brauchbarkeit fürs Volk verlieren dürfte. [Zum Titel- blatt:] belobt im litteraturblatt zum morgenblatt 1824 . nr. 69. 27. aug[ust] von X. über Wuk allg[emeine] zeitung 1839 n^ 257° und widerlegt - 1840 n- 165 p. 117. 118 die Übersetzung der gr[ammtik] ist von Tirol 1 Grimms Notizen werden nicht chronologisch (zum Problem der Datie- rung s. 1 Grimms Notizen werden nicht chronologisch (zum Problem der Datie- rung s. oben S.185ff,), sondern nach Themen geordnet, um einen bes seren Überblick Über seine Interessengebiete und das gesammelte Ma terial zu ermöglichen. Die oft zufällige Anordnung im Handexemplar is t in der Faksimileausgabe von Mojasevic/Rehder wiedergegeben; di dort in einem nicht durchnumerierten Anhang abgedruckten losen Zet tel Grimms sind mit meiner Seitenzählung in Klammern angegeben; ebenfalls durch Klammąrn sind meine Ergänzungen der Stichworte Grimms gekennzeichnet. Grimms Unterstreichungen werden beibehalten [ Zu Siedlungs- und Kulturgeschichte: ] Leipzig 1724. 7 K. H. Ritter von Lang, Regesta sive rerum Boicarum autographa ad anum usque 1300. Bd. 1-4, München 1822-28. 8 G. Hugo, Rechtshistoriker und Prof. der Rechte in Göttingen. Seine "Geschichte des römischen Rechts", 1790, ist in mehreren erw. Aufl erschienen (11. Aufl. 1832), vgl. ADB Bd. 13. 9 J. A. Kanne, De vocabulorum enantiosemia sive observat, de confu- sione de linguis Babilonica specimen Ium. Nürnberg 1819. 10 J. Dobrovsky, Institutiones lingvae slavicae.., Wien 1822 11 Lindenblatts Jahrbücher oder die Chronik von Johannes von Posilge, hg. J. Voigt und F. W. Schubert, Königsberg 1823. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 1 Ottokars österreichische Reimchronik, hg. H. Pez 1745 (= Scripto- res rerum austriacarum veteres ac genuini, Bd. 3). 2 Biterolf und Dietleib. Deutsche Gedichte des Mittelalters, hg. von der Hagen und Büsching, Bd. I, 1820. lf und Dietleib. Deutsche Gedichte des Mittelalters, hg. r Hagen und Büsching, Bd. I, 1820. 3 Wirnt von Gravenberg, Wigalois, hg. G. F. Benecke, 1819; Vers 9894 4 B. Sastrow (1520-1603), Bürgermeister von Stralsund und Histori- ker. Seine "Selbstbiographie" enthält wichtige Angaben zur Ge- schichte von Pommern. - Vgl. Brockhaus/Efron Bd. 28 . 4 B. Sastrow (1520-1603), Bürgermeister von Stralsund und Histori- ker. Seine "Selbstbiographie" enthält wichtige Angaben zur Ge- schichte von Pommern. - Vgl. Brockhaus/Efron Bd. 28 . 5 J. Wolf, Politische Geschichte des Eichsfeldes I- II, 1792-93. 6 J. F. Schannat, Corpus traditionum Fuldensium, ordine chronolo- gico digestum. Leipzig 1724. schichte von Pommern. - Vgl. Brockhaus/Efron Bd. 28 . 5 J. Wolf, Politische Geschichte des Eichsfeldes I- II, 1792-93. 6 J. F. Schannat, Corpus traditionum Fuldensium, ordine chronolo- g 5 J. Wolf, Politische Geschichte des Eichsfeldes I- II, 1792-93. 6 J F S h t C t diti F ld i di h l 6 J. F. Schannat, Corpus traditionum Fuldensium, ordine chronolo- gico digestum. Leipzig 1724. 7 K. H. Ritter von Lang, Regesta sive rerum Boicarum autographa ad anum usque 1300. Bd. 1-4, München 1822-28. 8 G. Hugo, Rechtshistoriker und Prof. der Rechte in Göttingen. Seine "Geschichte des römischen Rechts", 1790, ist in mehreren erw. Aufl. erschienen (11. Aufl. 1832), vgl. ADB Bd. 13. ( ) g 9 J. A. Kanne, De vocabulorum enantiosemia sive observat, de confu- sione de linguis Babilonica specimen Ium. Nürnberg 1819. 10 J. [ Zu Siedlungs- und Kulturgeschichte: ] [ Zu Siedlungs- und Kulturgeschichte: ] [118] Valben, Syrfen, zakel u[nd] walack Ottoc[ar] 691- * Turinge u[nd] Surben (Sorben) Bit[erolf] 52- ^ Sarvie Wigal[ois]3 I spätere zurückdrängung slavischer spräche von der ostseeküste. letzter rest im Lüneburgischen. in altenborg wurde die slavische spräche gewaltsam ausge- ro tte t. in Hinterpommerp noch im 16. jh. wendisch gesprochen. Sastrow 2, 5Ф8 weicht sie in Lausitz? und in Schlesien? Wenden im eichsfeld с Wolfs geschichte des Ei[chsfelds] I. 33-39 ^ in regione Slavorum ad moin, trad. fuld. 2, 126 conf. notam ibd. 2 , 24. in Solavis I I Lang regesta^ II, 46 (1210) Heinrich Supan ,gehört zur familia eccles[iae] ratisbonensis Ibid. II, 114 (1220) Alberti comitis in Tirol literae de divisione prolis cujusdam inter pueros supanos et mona- sterium Burense. плакати (plorare) goth. flekan (piangere) О IV Hugo rechtsgfeschichte] ed. 1. § 312. der bauernjunge Uprauda. ״ Kanne enantiosema (Norim. 1819) p. 28. legt Uprauda dem Justinus "Justiniani imp. patri"bei. a ltsl. вгсток (oriens) Instit[utiones] p. 33^° [Im Text sind die Namen: Bulgaren, Serben, Croatien, Dalma- tien unterstrichen.] VI Übergewicht der griech. kirche in Litthauen. Lindenblatt p. 206 Ottoc[ar] 691- * [118] Valben, Syrfen, zakel u[nd] walack Ottoc[ar] 691- * Turinge u[nd] Surben (Sorben) Bit[erolf] 52- ^ Sarvie Wigal[ois]3 I spätere zurückdrängung slavischer spräche von der ostseeküste. letzter rest im Lüneburgischen. I g in altenborg wurde die slavische spräche gewaltsam ausge- ro tte t. I I Lang regesta^ II, 46 (1210) Heinrich Supan ,gehört zur familia eccles[iae] ratisbonensis плакати (plorare) goth. flekan (piangere) О IV О Hugo rechtsgfeschichte] ed. 1. § 312. der bauernjunge Uprauda. ״ Uprauda. Kanne enantiosema (Norim. 1819) p. 28. legt Uprauda dem Justinus "Justiniani imp. patri"bei. a ltsl. вгсток (oriens) Instit[utiones] p. 33^° [Im Text sind die Namen: Bulgaren, Serben, Croatien, Dalma- tien unterstrichen.] VI Übergewicht der griech. kirche in Litthauen. Lindenblatt p. 206 1 Ottokars österreichische Reimchronik, hg. H. Pez 1745 (= Scripto- res rerum austriacarum veteres ac genuini, Bd. 3). 2 Biterolf und Dietleib. Deutsche Gedichte des Mittelalters, hg. von der Hagen und Büsching, Bd. I, 1820. 3 Wirnt von Gravenberg, Wigalois, hg. G. F. Benecke, 1819; Vers 9894 4 B. Sastrow (1520-1603), Bürgermeister von Stralsund und Histori- ker. Seine "Selbstbiographie" enthält wichtige Angaben zur Ge- schichte von Pommern. - Vgl. Brockhaus/Efron Bd. 28 . 5 J. Wolf, Politische Geschichte des Eichsfeldes I- II, 1792-93. 6 J. F. Schannat, Corpus traditionum Fuldensium, ordine chronolo- gico digestum. ANHANG II Zusammenstellung der Notizen aus Grimms Handexemplar mit Quellenangaben1 oben S.185ff,), sondern nach Themen geordnet, um einen bes seren Überblick Über seine Interessengebiete und das gesammelte Ma terial zu ermöglichen. Die oft zufällige Anordnung im Handexemplar is t in der Faksimileausgabe von Mojasevic/Rehder wiedergegeben; di dort in einem nicht durchnumerierten Anhang abgedruckten losen Zet tel Grimms sind mit meiner Seitenzählung in Klammern angegeben; ebenfalls durch Klammąrn sind meine Ergänzungen der Stichworte Grimms gekennzeichnet. Grimms Unterstreichungen werden beibehalten 2 Gemeint sind Kopitars Rez. der "Pismenica" und des 2. Bd. und Gi R d 1 Bd V k Li d l j t t i KKS I S g g 2 Gemeint sind Kopitars Rez. der "Pismenica" und des 2. Bd. und Grimms Rez. des 1. Bd. von Vuks Liedern, vgl. jetzt in KKS I, S. 310-320, 347-369 und GKS IV, S. 427-436. 3 Literarisches Conversationsblatt hg. Brockhaus, Leipzig. 4 Es handelt sich um die Geschichtsforscher K. W. Böttiger und F. Kruse, vgl. ADB Bd. 3 und 17. , g 5 Morgenblatt für gebildete Stände hg. Cotta, Stuttgart. 6 Allgemeine Zeitung hg. Cotta, Stuttgart, Augsburg, Tübingen. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 00051004 ־ 231 - 1 Gemeint is t Kopitars Rezension "Leben des Archimandriten Zelich" in: Jahrbücher der Literatur 26, Wien 1824 , S. 113-199, vgl. jetzt Spisi II, 1, S. 318-397. 2 Danica. Zabavnik za godinu 1826. Hg. Vuk Stef. Karadziå. 3 Slavin. Hg. J. Dobrovsk^, Prag 1806. 4 CI. Ch. Fauriel, Chants Popul, de la Grèce moderne, Paris 1824-25. 5 s. Anm. 1. 6 ebd. 7 J. Kopitar, Dositej Obradovic. In: Vaterländische Blätter 4, 1811, S. 365-368, vgl. KKS I, S. 113-120. 8 J. Dobrovskÿ, Institutiones... 9 Vgl. S. 231 , Anm. 3. Vera Boji - 9783954793044 [ Zu Siedlungs- und Kulturgeschichte: ] Dobrovsky, Institutiones lingvae slavicae.., Wien 1822 11 Lindenblatts Jahrbücher oder die Chronik von Johannes von Posilge, hg. J. Voigt und F. W. Schubert, Königsberg 1823. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 11 Lindenblatts Jahrbücher oder die Chronik von Johannes von Posilge, hg. J. Voigt und F. W. Schubert, Königsberg 1823. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 232 - ? [Zur Behauptung, polnische und litauische Geistliche schrieben die aksl. Bücher ab.] ? [Zu: "reinersonnene, ungefügere Glagoliza"] Kopitar wien[er] jahrbfücher]1 1824. p. 114: eine kirchen- spräche, die aber nicht, wie bei Griechen und Römern der letzte Schimmer einer reichen literatur, sondern das erste morgenroth einer erst beginnenden cuitur, eben deswegen dex gründe entbehrt, womit man den fortgebrauch des griech. un!d lateins zum theil entschuldigen kann. Kopitar 1. c. pag. 115: es wäre eine frage, der wievielte theil des sprachschatzes in den gottesdienstl[ichen] büchern enthalten sein kann. ־־ die slavischen kirchenbücher machen an 30-50 foliobände aas. ibd. p. 154. 2 [Zur "Hirtensprache":] vgl. Danitza p. 44 über Mraz[ovic] vgl. Slavin p. 312-3183 vgl. Fauriel disc[ours] prêl[iminaire] CXXII.4 Zelich hat viele italismen und w irft selbst den Dalmatinern la tin i ritus vor, daß sie ihre spräche stark mit der ita l. mischen. Kop[itar] 1. c. p. 199. [Zu slavischen Wörtern im Ungarischen:] auch venetianische auch Sophocles haben sie übersetzt. Kopfitar] in Wien[er] jahrb[üchern] 1824. p. 114 der erste neuere schriftsteiler der Illy rie r graeci ritus war der ausgesprungene mönch Dositheus Obradowltsch (sein necrolog in den vaterl[ändischen] blätt[ern] jahrg. 1811. n- 61. p. 365) der zweite war der archimandrit Zelich Житіе - Герасіма ЗелиЬа - нимъ ca^«мъ на свѣтъ издано. Ofen 1823. XVI, 643. 8* . rec. von Kopitar wien[er] jahr[bücher] 1824, vol. 26. p. 113-199. [Zu: Srb] Instit[utiones] p. 154® vgl. Sarvie, Servie (Wigal[ois] ) die östlichen Serben nennen ihren westlichen (dalmatischen) bruder Schijak (dickhals) unirte griechen, deren es viele in Polen gibt, sind solche die den griech. ritus beibehalten, aber sich dem röm. pabst unterwerfen. XI XII IX XIII XIV XV XVI XVII XXIII XXVI XXVII ? [Zur Behauptung, polnische und litauische Geistliche schrieben die aksl. Bücher ab.] pitar wien[er] jahrbfücher]1 1824. p. [ Zu Siedlungs- und Kulturgeschichte: ] 114: eine kirchen- äche, die aber nicht, wie bei Griechen und Römern der zte Schimmer einer reichen literatur, sondern das erste rgenroth einer erst beginnenden cuitur, eben deswegen dex nde entbehrt, womit man den fortgebrauch des griech. un!d eins zum theil entschuldigen kann. Kopitar 1. c. pag. 115: es wäre eine frage, der wievielte theil des sprachschatzes in den gottesdienstl[ichen] büchern enthalten sein kann. ־־ XIII die slavischen kirchenbücher machen an 30-50 foliobände aas. ibd. p. 154. 2 über Mraz[ovic] vgl. Slavin p. 312-3183 vgl. Fauriel disc[ours] prêl[iminaire] CXXII.4 p[ ] p Zu slavischen Wörtern im Ungarischen:] auch venetianisch die östlichen Serben nennen ihren westlichen (dalmatischen) bruder Schijak (dickhals) XXVI unirte griechen, deren es viele in Polen gibt, sind solche die den griech. ritus beibehalten, aber sich dem röm. pabst unterwerfen. XXVII unirte griechen, deren es viele in Polen gibt, sind solche die den griech. ritus beibehalten, aber sich dem röm. pabst unterwerfen. XXVII 1 Gemeint is t Kopitars Rezension "Leben des Archimandriten Zelich" in: Jahrbücher der Literatur 26, Wien 1824 , S. 113-199, vgl. jetzt Spisi II, 1, S. 318-397. 1 Gemeint is t Kopitars Rezension "Leben des Archimandriten Zelich" in: Jahrbücher der Literatur 26, Wien 1824 , S. 113-199, vgl. jetzt Spisi II, 1, S. 318-397. 2 Danica. Zabavnik za godinu 1826. Hg. Vuk Stef. Karadziå. 3 Slavin. Hg. J. Dobrovsk^, Prag 1806. g g 4 CI. Ch. Fauriel, Chants Popul, de la Grèce moderne, Paris 1824-25. 5 s. Anm. 1. 6 ebd 6 ebd. 7 J. Kopitar, Dositej Obradovic. In: Vaterländische Blätter 4, 1811, S. 365-368, vgl. KKS I, S. 113-120. 8 J. Dobrovskÿ, Institutiones... Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 00051004 ־־ 233 - [118] C yrill und Method, von Jos. D[obrovskÿ] für die abh[andlung] der böhmfischen] Ges[ellschaft] Prag 1823. 133 S. 8°. für die abh[andlung] der böhmfischen] Ges[ellschaft] Prag 1823. 133 S. 8°. -s [Zu den reduzierten Vokalen:] goth. vgl. Bopp, vgl. gramm. 339 warum aber zeigt sie ihn nicht in unflektierbaren Wörtern partikéin? 2 asilus, ahd. asilu (gramm. 2, 94) slav. ongilu (angelus) slav. auch das serb. knez verräth das i^ denn es steht offenba f. knezi, russ. knjaz' ebenso zetz (lepus) f. zetzi (aber russ. mjesjatz" f. mjesjatz'?) az (ego) für azi, als аэь, obschon аэъ angegeben wird. 3 Dobrovskjr, Institutiones... 2 J. Grimm, Deutsche Grammatik, T. 2, 1826 1 F. Bopp, Vergleichende Grammatik.., 2. Abt. 1835 2 J. Grimm, Deutsche Grammatik, T. 2, 1826 3 Dobrovskjr, Institutiones... 1 Vuks Danica für das Jahr 1826 [ Zu Siedlungs- und Kulturgeschichte: ] vocalauslautige wurzeln XLVIII Vorrede азъ (ego) f. азь = азі columba f. colunba (m vor b) mensa mit goth mês, slav. misa neugr. <5lvtіціѵоіоѵ pjesma, vgl. litth . giesme labud aus alabud? labe aus alabe? kamen, akm8 sokol, falke labud, alpt na, ana plak, folk prase, porcus (wie decem, octo) ramen, aram brada, part: grad, garto rod? art brod, furt. mleko milch zlato, gold, krai, charl. vladu, waltu buchstabenumsetzung serb. sav, sva, sve f. vas, vsa, vse khi, kheri (filia ) herzeg. schhi (шЬи), schheri böhm. dcÿ, deera russ. dotsch? .. « x . 1_ vgl. gaunerspräche: ottchen russ. çtschela (apis) böhm wcela serb. tschela krain, zhibela f. bizhela ך poln. Iza für slza (Dobr. etymol. p. 45) [Zur Lautlehre:] XXXIII XXV XXXVI 104 [106] XXXII XXII XLIX [106] 1 F. Bopp, Vergleichende Grammatik.., 2. Abt. 1835 2 J Grimm Deutsche Grammatik T 2 1826 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 ־ 234 ־ 2 [In der Alphabet-Tabelle sh verbessert als:] sc 3 [Zu гг] vr gipfel, gen. vria, vr'a 4 и aus 1.: vuk, pun kunem (pag. 88) Danitza p. 521 vgl. litth . dubbus, le tt. dohb merkwürdig serb* dubok (profundus) russ. gluboк böhm. nluboky poln. gleboki dubok scheint f. dlubok, ausgehöhlt v. böhm. dlaubati und hlaubati, grübeln [Zu g - k] g und j aie lieder haben megdan und mejdan, pugnae loccus 7 [Zu: g in ž, in z und in c; z - Ž und in g sind die BeispiLe- le angestrichen; с und leòi sind ausgeklammert] die angestrichenen fälle sind als eine regel darzustellen g vor e wird zh vor i ־z vr9, vrzino sluga (servus) sluzhba (servilium) aber auch sluschkinja (ancilla) слушкив>а knez steht für knezi, russ. knjaz', knjaginja (fürstin böhm. knëz (fürst), кпё£па (fürstin) 8 [Zu: к - 6, in с und in с sind die Beispiele angestrichen, 6 und reci sind ausgeklammert] 9 [Zu -1] мио(carus) f. мила das о gleicht dem krain, v 1 in der mitte aufgelöst Toben p. 4) slav wird in compositis - sav 10 [Zu: s - s sind die Beispiele angestrichen, poslati, pošljem auch unterstrichen] vgl. pag. 14 искати, иштем, postulare [с ־־ с angestrichen] 14 [Zu h : s, s sind die Beispiele angestrichen] vr cacumen vrg cacumen 15 zuweilen setzten sie в (v) für x сув (siccus) russ. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access [ Zu Siedlungs- und Kulturgeschichte: ] сну пас (canis), gen.пса , nom. pl. пси russ. neci, ncà böhm. pes, psa, pl. psy vr cacumen, gen. vr'a böhm. wrch poln. wierzch 31 [Zu lju d i:] dieser gen. - i is t weiblich. II, 194, 1984 [Zur 2. Dekl.] (aurora) (nympha) bemerkenswert!!, daß die Serben im gen. p l. fern, nicht d einsilbigen formen der Böhmen, Polen und Slovenen haben [Zum Lok. ohne -ma:] Danitza p. 655 32 [Zu Dat./Lok. fern.:] (folter) 35 [Korrektur des mißverstandenen Gen. sg. matere (die Müt als:] mati (die Mutter) golijen tibia, crus 00051004 00051004 - 235 - 16 [Zu it , с gegenüber šč sind die Beispiele angestrichen] ? [zur Behauptung, in aksl. Büchern gebe es nirgends 1 , sondern nur ь ] 19 [Zum Akcent:] eàc , omnis;bäc , vos [Zur Deklination der Substantive] XXXIX unterschied zwischen belebten und unbelebten subst. im litth . (Mielke p. 40)1 25 [Zu usi:] v. pag. 35 [Zur 1. Dekl.] ogan1 feuer ognja 26 [durchgestrichen:] gromom tonitribus (ed. 1841, 152) 27 auf j: boj, sclacht ra j, paradies noj, strauß (vogel) kroj, schnitt pokoj, requies 29 двор (aula) дворови 3 ? топ Kanone, pl. топови ? vgl. Danitza p. 118, 16 2 Vuk im W[örter]b[uch] топ fem. grom tonitrua pl. gromovi div gigas, divovi gigantes 30 сан somnus poln. sen, gen. snu gen. сна böhm. - ־־ dat. сну пас (canis), gen.пса , nom. pl. пси russ. neci, ncà böhm. pes, psa, pl. psy vr cacumen, gen. vr'a böhm. wrch poln. wierzch 31 [Zu lju d i:] dieser gen. - i is t weiblich. II, 194, 1984 [Zur 2. Dekl.] (aurora) (nympha) bemerkenswert!!, daß die Serben im gen. p l. fern, nicht d einsilbigen formen der Böhmen, Polen und Slovenen haben [Zum Lok. ohne -ma:] Danitza p. 655 32 [Zu Dat./Lok. fern.:] (folter) 35 [Korrektur des mißverstandenen Gen. sg. matere (die Müt als:] mati (die Mutter) golijen tibia, crus 31 35 [Korrektur des mißverstandenen Gen. sg. matere (die M als:] mati (die Mutter) 1 Ch. G. Mielcke, Litauisch-deutsches und deutsch-litauisches Wör- terbuch, Königsberg 1800. (Zit. A. Kurschat, Lit.-dt.Wb. I, 1968 S XXVIII) 1 Ch. G. Mielcke, Litauisch-deutsches und deutsch-litauisches Wör- terbuch, Königsberg 1800. (Zit. A. Kurschat, Lit.-dt.Wb. I, 1968, S. XXVIII). p j p g S Vuk in Danica 1826, S. [ Zu Siedlungs- und Kulturgeschichte: ] cyxi wovon сушити (sictare) 7 [Zu: v als Ersatzlaut für h:] в in с und ш (ad pag. 15) 1 Vuks Danica für das Jahr 1826 [In der Alphabet-Tabelle sh verbessert als:] sc [Zu гг] vr gipfel, gen. vria, vr'a и aus 1.: vuk, pun kunem (pag. 88) Danitza p. 521 vgl. litth . dubbus, le tt. dohb merkwürdig serb* dubok (profundus) russ. gluboк böhm. nluboky poln. gleboki dubok scheint f. dlubok, ausgehöhlt v. böhm. dlaubati und hlaubati, grübeln [Zu g - k] g und j [Zu: g in ž, in z und in c; z - Ž und in g sind die BeispiLe- le angestrichen; с und leòi sind ausgeklammert] die angestrichenen fälle sind als eine regel darzustelle aber auch sluschkinja (ancilla) слушкив>а knez steht für knezi, russ. knjaz', knjaginja (fürst böhm. knëz (fürst), кпё£па (fürstin) aber auch sluschkinja (ancilla) слушкив>а knez steht für knezi, russ. knjaz', knjaginja (fürstin) böhm. knëz (fürst), кпё£па (fürstin) [Zu: к - 6, in с und in с sind die Beispiele angestrichen, 6 und reci sind ausgeklammert] 9 [Zu -1] мио(carus) f. мила das о gleicht dem krain, v 1 in der mitte aufgelöst Toben p. 4) slav wird in compositis - sav 10 [Zu: s - s sind die Beispiele angestrichen, poslati, pošljem auch unterstrichen] vgl. pag. 14 искати, иштем, postulare [с ־־ с angestrichen] 14 [Zu h : s, s sind die Beispiele angestrichen] vr cacumen vrg cacumen 15 zuweilen setzten sie в (v) für x сув (siccus) russ. cyxi wovon сушити (sictare) 7 [Zu: v als Ersatzlaut für h:] в in с und ш (ad pag. 15) 1004 - 235 - 16 [Zu it , с gegenüber šč sind die Beispiele angestrichen] ? [zur Behauptung, in aksl. Büchern gebe es nirgends 1 sondern nur ь ] 19 [Zum Akcent:] eàc , omnis;bäc , vos [Zur Deklination der Substantive] XXXIX unterschied zwischen belebten und unbelebten subst. im litth . (Mielke p. 40)1 25 [Zu usi:] v. pag. 35 [Zur 1. Dekl.] ogan1 feuer ognja 26 [durchgestrichen:] gromom tonitribus (ed. 1841, 152) 27 auf j: boj, sclacht ra j, paradies noj, strauß (vogel) kroj, schnitt pokoj, requies 29 двор (aula) дворови 3 ? топ Kanone, pl. топови ? vgl. Danitza p. 118, 16 2 Vuk im W[örter]b[uch] топ fem. grom tonitrua pl. gromovi div gigas, divovi gigantes 30 сан somnus poln. sen, gen. snu gen. сна böhm. - ־־ dat. 1 Ch. G. Mielcke, Litauisch-deutsches und deutsch-litauisches Wör- terbuch, Königsberg 1800. (Zit. A. Kurschat, Lit.-dt.Wb. I, 1968, S. XXVIII). ) ł Gemeint sind Vuks Srpske Narodne Pjesme, Bd. I, Wien 3 Vuks Danica für das Jahr 1826, S. 118, Vers 162. 1 Ch. G. Mielcke, Litauisch-deutsches und deutsch-litauisches Wör- terbuch, Königsberg 1800. (Zit. A. Kurschat, Lit.-dt.Wb. I, 1968, S. XXVIII). ł Gemeint sind Vuks Srpske Narodne Pjesme, Bd. I, Wien 1841. 3 Vuks Danica für das Jahr 1826, S. 118, Vers 162. к Vuks Narodne Srpske Pjesme, Bd. II, Leipzig 1823. S Vuk in Danica 1826, S. 65: "ü Srijemu, и Bačkoj i и Banatu kasto se izostavlja ma, i onda dolazi bliže к Slavenskome." Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access p j , , p g S Vuk in Danica 1826, S. 65: "ü Srijemu, и Bačkoj i и Banatu kasto se izostavlja ma, i onda dolazi bliže к Slavenskome." Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM , , к Vuks Narodne Srpske Pjesme, Bd. II, Leipzig 1823. [ Zu Siedlungs- und Kulturgeschichte: ] 65: "ü Srijemu, и Bačkoj i и Banatu kasto se izostavlja ma, i onda dolazi bliže к Slavenskome." Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM i f p j p g S Vuk in Danica 1826, S. 65: "ü Srijemu, и Bačkoj i и Banatu kasto se izostavlja ma, i onda dolazi bliže к Slavenskome." Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 00051004 - 236 - ad a уво (auris) увета паче Entlein патче Hündlein пиле Kücklein сироче Waislein штене Hündlein копиле Bastard 37 [Zur 4. Deki.:] das deutsche epenthetische ir entspricht auch in der bedeu— tung, es sind thiere huindir, varhir, oder hol2 ir (drveta) 38 [Zum Kollektivum auf -ad:] instfitutiones] p. 298. oko (oculus) wird im pl. fern oči (s. 35) uvo (auris) uši ( - ) 1 2 3 vgl. Berlich p. 64 . Murko p. 24 . Kopitar 24 2 (vgl. oben p. XXX, uvo, uho) [Zum Adjektiv:] 17 die indef. gehen wie substantiva pavlov pavlova gen. pavlova pavlovy dat. pavlovu pavlovë p. 48 41 [Zu: "abgeschnitten":] indefinit, abstract мио (carus), fem. мила, neutr.Mítno dies männliche о ist daher nicht flexivisch nb steht das adj. bei leblosen masc. so lautet der acc. ®g. adj. wie der nom. (ganz und abgeschn[itte n ] ) z.b. sav pelen tzrveni pojas (vorhin p. 39) so auch im altslav. sav pelen pobrała I. 123 svega pelena sav Banat Ī l i , 25^ aber svu ruzhu X, 123 acc. fem. жив ти братац! I. nr. 206 4 2 боже мили ! 44 ebenso böhm. petrûv, petrova, petrovo 45 zu sav gehört svaki (omnis)сваки, ка, ко altsTT всАк, quilibet всдческ krain, vsaki (Kopit[ar] p. 298) bulgar, sitschki (omnes) f. vsitschki ^ aber sva (totam) v. bulgar, lieder p. 40 ^ litth . wissas (omnis) auch altpreuB. (Vater p. 89) le tt. wiss, wissa 1 I.A. Brlić, Grammatik der Illyrischen Sprache... Ofen 1833. ad a уво (auris) увета паче Entlein патче Hündlein пиле Kücklein сироче Waislein штене Hündlein копиле Bastard 37 [Zur 4. Deki.:] das deutsche epenthetische ir entspricht auch in der bedeu— tung, es sind thiere huindir, varhir, oder hol2 ir (drveta) 38 [Zum Kollektivum auf -ad:] instfitutiones] p. 298. oko (oculus) wird im pl. fern oči (s. 35) uvo (auris) uši ( - ) 1 2 3 vgl. Berlich p. 64 . Murko p. 24 . Kopitar 24 2 (vgl. [ Zu Siedlungs- und Kulturgeschichte: ] oben p. XXX, uvo, uho) [Zum Adjektiv:] 17 die indef. gehen wie substantiva pavlov pavlova gen. pavlova pavlovy dat. pavlovu pavlovë p. 48 41 [Zu: "abgeschnitten":] indefinit, abstract мио (carus), fem. мила, neutr.Mítno dies männliche о ist daher nicht flexivisch nb steht das adj. bei leblosen masc. so lautet der acc. ®g. adj. wie der nom. (ganz und abgeschn[itte n ] ) z.b. sav pelen tzrveni pojas (vorhin p. 39) so auch im altslav. sav pelen pobrała I. 123 svega pelena sav Banat Ī l i , 25^ aber svu ruzhu X, 123 acc. fem. жив ти братац! I. nr. 206 4 2 боже мили ! 44 ebenso böhm. petrûv, petrova, petrovo 45 zu sav gehört svaki (omnis)сваки, ка, ко altsTT всАк, quilibet всдческ krain, vsaki (Kopit[ar] p. 298) bulgar, sitschki (omnes) f. vsitschki ^ aber sva (totam) v. bulgar, lieder p. 40 ^ litth wissas (omnis) auch altpreuB (Vater p 89) 37 38 1 I.A. Brlić, Grammatik der Illyrischen Sprache... Ofen 1833. 2 A. Murko, Theoretisch-praktische slowenische Sprachlehre,Leib.1832 3 J. Kopitar, Grammatik d. slavischen Sprache in Krain.. ,Leib. 1809. 4 Gem. ist Vuks Liederausgabe von 1823f. 5 Gem. sind die von Vuk hg. bulgarischen Volkslieder ,vgl. GPS II, S. 220-229. Vera Boji 9783954793044 5 Gem. sind die von Vuk hg. bulgarischen Volkslieder ,vgl. GPS II, S. 220-229. Vera Boji 9783954793044 6 J.S. Vater, Die Sprache der alten Preußen. Braunschweig 1821. - Grimm hat darüber e.Rez. verfaßt, vgl. GKS IV, S.160-164. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access ־ 237 ־ ־ 237 ־ [durchgestrichen:] vgl. russ. совершенно , gänzlich, völlig? is t со־вершенно - gehört also nicht hierher Deila Bella und S tulli geben nicht sav, sva, sve sondern vas, sva, sve (Vuk hat im W[örter]b[uch]noch вас) вас in liedern I. pag. 9. II. 152, 150. II. 278, 116. II I . 50, 6. I I I . 96, 18. II. 251, 43. III. 65, 15. I. 123. altsl .весь ,веж , все (omnis) Instit[utiones] p. 340. russ. весь, вся $ все krain, vsi (omnes) ves (omnis) böhm. wssecek; fem. wssecka; neutr. wssecko wssecken; wsse gen. wsseho wssy wsseho altpoln. wszy wsza wsze gen. wszego wszey wszego neupoln. wszystek wszysta wszysto gehört dazu auch wesel lustig, froh? [Zum Komparativ auf - ji: ] nicht aber die auf ет z.b. вет (alt)BeTHjH [Zur unbestimmten und bestimmten Adjektivform:] Bopp vergi [eichende] gramm[atik] p. 333. [ Zu Siedlungs- und Kulturgeschichte: ] [ Die Angaben zur "schwachen*1 und "starken” Deklination sind unterstrichen] analogie des nom. pl. der ganzen (definiten) form im fem. und neutr. (Gretsch p. 208)1 wie auch unsere schwache flex, im neutr. nom. acc. das fem. und neutr. identifiziert. die russ. comparative sowohl def. als indef. Gretsch p. 202 2 Recens. p. 826 : v e lil ist positiv auch der Russe: den' korotok", der tag ist kurz korotkii den1, der kurze tag auch der Böhme wSl gest slepÿ und slepÿ wul - früher wohl: w81 gest slep (Dobr. böhm. gr. p. 333) nb zu vergi [eichen] hierbei das verhältn[is? der weibl.. subst. zu den adj. und die motion 4 zum dat. plur. recens, p. 826 gorod" velik" die stadt ist groß (m) rjeka schirokä der fluß ist breit (f) den' korotok", der tag ist kurz korotkii den1, der kurze tag Puchm[ayer] p. 250. 251. 47 XLI XLII XLVI XLVII [115] 1 N. J. Grec, Frakticeskaja russkaja grammatika, 1827; Prostrannaja russkaja grammatika, 1827, 2.Aufl. 1830. 2 Gemeint ist Grimms Anzeige der "Kleinen serbischen Grammatik" in [durchgestrichen:] vgl. russ. совершенно , gänzlich, völlig? is t со־вершенно - gehört also nicht hierher [durchgestrichen:] vgl. russ. совершенно , gänzlich, völlig? is t со־вершенно - gehört also nicht hierher Deila Bella und S tulli geben nicht sav, sva, sve sondern vas, sva, sve Deila Bella und S tulli geben nicht sav, sva, sve sond vas, sva, sve (Vuk hat im W[örter]b[uch]noch вас) вас in liedern I. pag. 9. II. 152, 150. II. 278, 116. II I . 50, 6. I I I . 96, 18. II. 251, 43. III. 65, 15. I. 123. altsl .весь ,веж , все (omnis) Instit[utiones] p. 340. altsl .весь ,веж , все (omnis) Instit[utiones] p. 340. $ Bopp vergi [eichende] gramm[atik] p. 333. [ Die Angaben zur "schwachen*1 und "starken” Deklination sind unterstrichen] 202 [115] 1 N. J. Grec, Frakticeskaja russkaja grammatika, 1827; Prostrannaja russkaja grammatika, 1827, 2.Aufl. 1830. 2 Gemeint ist Grimms Anzeige der "Kleinen serbischen Grammatik" in den Göttingischen gelehrten Anzeigen 83, S. 820-826, vgl. jetzt GKS IV, 225-229, S. 229. 3 J. Dobrovskÿ, Ausführliches Lehrgebäude der böhmischen Sprache, Prag 1809, 2. Aufl. 1819. 4 s. Anm. 2 5 Puchmayer, A.J., Lehrgebäude der russischen Sprache. Prag 1820. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM 00051004 - 238 - stark dobr", dobrà, dobrô schw. dôbrii , dôbraja, dôbroe das neutr. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access [ Zu Siedlungs- und Kulturgeschichte: ] des starken is t auch adv[erb] malo, wenig böhm. màio rnnőgo, viel mnôho skóro, bald dlauho lange borzo, geschwind boko tie f sljepo blindlings hrubë grob dobfe gut Dobr[ovsk£, Böhmische Grammatik] p. 2122 poln. grubo drogo . einige e, ie Bandtke 157. 158 [durchgestrichen:] a lts l. ш das ш späterer Gramm. 428! ^ ргаѵш neutr. pravo Dobr. p. 4 27ל [1171 w81 slepÿ adv. slepê der mann is t groß muż welkÿ 1 homme est grand la femme est grand la langue est nuette böhm. clowëk gest welikj־ nicht mehr: welik Dobr[ovsk£, Böhmische Grammatlik] p. 333. poln. dobrze bene człowiek iest dobry. grod iest wielki adv. Bandtke p. 156. 157. giupi zwierz das dumme thier piekne drzewo das schone holz [durchgestrichen:] wald [116] die erde war wüste und leer iiemiá była niekszaltowna i prażna żłoto iest wyborne der tag kühle worden war [durchgestrichen] wenn du fromm bist so bistu angenehm ie ś li dobrze czynić bediiesz, nie bedziesz wywyżson? ie wielka była ziosć ludzka iârzyne tielona daß du ein schön weib bis żeś niewiàstå piękna ihre habe war groß byla máietnoêò ich wielka Gen. 1, 30 2, 12 3, 8 4, 7 6, 5 8, 13 9, 3 12, 11 13, 6 2 Dobrovskÿ, Institutiones.., S. 4 27f. 3 M. Luther, B iblia... Das erste Buch Mose. Genesis. stark dobr", dobrà, dobrô schw. dôbrii , dôbraja, dôbroe das neutr. des starken is t auch adv[erb] stark dobr", dobrà, dobrô schw. dôbrii , dôbraja, dôbroe das neutr. des starken is t auch adv[e [1171 [116] 2 Dobrovskÿ, Institutiones.., S. 4 27f. 3 M. Luther, B iblia... Das erste Buch Mose. Genesis. 2 Dobrovskÿ, Institutiones.., S. 4 27f. 3 M. Luther, B iblia... Das erste Buch Mose. Genesis. 3 M. Luther, B iblia... Das erste Buch Mose. Genesis. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 000Б1004 000Б1004 - 239 - Г р. J. Safarlk, Serbische Lesekörner, oder historisch-kritische Beleuchtung der serbischen Mundart, Pesth 1833. tøum Zahlwort:] tøum Zahlwort:] *49 [Zur Deklination von dva - č e tiri:] vgl. Berlic p. 87 anders sloven. Kopitar p. 274. Murko p. 35 [Zum "Gen. sg." nach dva - č e tiri:] vide meine rec[ension] Berlic p. 82. 240 53 [Zu den Multiplikativa:] dies put ist charakteristisch serbisch denn altsl. krat Dobr[ovskÿ, Institutiones] p. 420 (vgl. treputie, trivium, [ebd.] p. 439.) krain, krat Kopfitar] p. 280. böhm. krāt Dobr[ovsk£, Böhmische Grammatik] p. 100. litth . kartas und sykis Mielke p. 62 le tt. reise altpr[euß.] reisan Vater p. 134 ainontinreisan (zumahl) poln. raz (= franz. coup) B[an]dtke p. m. 217 doch ist kroč bekannt (ibd.) russ. гагъ und kratb Puchm[ayer] p. 125 auch bloßes -ъю ibd. [Zum Pronomen:] 56 [Zum poss* Dat. mi, t i: ] inst[ itutiones] p. 607 57 [Zu den Suffixen ־ka, -kar, ־karena:] angehängtes godi - irgend, cumque штогоЬ was aucn, was irgend kojigodj wer auch Vuk [Wörterbuch] s. u. godj also persönliche pronomina; vgl. lat. -met vgl. hernach das зи (pag. 60) vgl. das -iere, - a ille , - ingand im argot und das -noses im dän. rothwelsch auch das nnl. gy-lieden. auch andere partikéin z.b. sad, nunc, sadeka, sadekar, sadekarena . [durchgestrichen:] Schaffarik Lesek[örner] p. 63 58 [Zu môme:] ebenso tvôme, svöme 60 [Zu taj , ovaj, onaj:] dieser, dieser, jener, iste, hic, [Zu tiz i:] instit[utiones] p. 433. auch ibojsH recens [ion von Grimm] p. 824) spur des altsl. russ.cia (hic) vgl. in s tit utiones p. Puchmm[ayer] 298. Übrig im serb.cHHOh = ahd. hînaht vgl. russ.сегодня hodie wofür im gern[einen] leben севодни (sevodni) *49 [Zur Deklination von dva - č e tiri:] vgl. Berlic p. 87 anders sloven. Kopitar p. 274. Murko p. 35 z.b. sad, nunc, sadeka, sadekar, sadekarena [durchgestrichen:] Schaffarik Lesek[örner] p. 63 *49 [Zu môme:] ebenso tvôme, svöme der unterschied sei im deutschen daran erkennbar, daß transitive imperfectiva T־i ihr passiv mit sein und werden bilden: er is t geehrt, geliebt, er wird geehrt, geliebt, gefürcht er war - - wurde - - geehrt sein. geehrt werden. perfectiva aber nur mit werden: er wird getrunken, geschla 104 perf. und imperf. XLIX. L. XLIX [Zur Behauptung, "die üblichen Eintheilungen der Verba in ... Transitiva und Intransitiva" sei "unnütz":] in der formenlehre p. 64 L die deutsche spr[ache] entbehrt der form des imferfects für imperfectiva d.h. sie verwendet ihr imp[erfect] für imp[erfect] und praet[eritum] sie entbehrt der aoristform, d. h. des eigentl[ ichen] perfectums und ersetzt sie durch praet[eritum ]. sie umschreibt das imperfectivum mit fort durch part, praes, и. verb, subst. er is t gehend = geht fort [Viele Unterstreichungen im Text über den Verbalaspekt] LII vgl. unten p. 65. 4 vgl. Thiersch p. 514 perfectiva haben nur ein futurum, kein praesens vgl. p. 64 der Deutsche, dies nicht fühlend sagt: zurück oder ich schieße (st[att] ich werde schießen) recule, ou je te tuerai L III im allg. anz. der D.^ 1827 nr. 254 behauptet ein G aus E. der unterschied sei im deutschen daran erkennbar, daß transitive imperfectiva T־i ihr passiv mit sein und werden bilden: er is t geehrt, geliebt, er wird geehrt, geliebt, gefürchtet er war - - wurde - - geehrt sein. geehrt werden. perfectiva aber nur mit werden: er wird getrunken, geschla- 104 perf. und imperf. XLIX. L. XLIX [Zur Behauptung, "die üblichen Eintheilungen der Verba in ... Transitiva und Intransitiva" sei "unnütz":] in der formenlehre p. 64 die deutsche spr[ache] entbehrt der form des imferfects für imperfectiva d.h. sie verwendet ihr imp[erfect] für imp[erfect] und praet[eritum] sie entbehrt der aoristform, d. h. des eigentl[ ichen] perfectums und ersetzt sie durch praet[eritum ]. ] mschreibt das imperfectivum mit fort p [ ] sie umschreibt das imperfectivum mit fort p p er is t gehend = geht fort g g [Viele Unterstreichungen im Text über den Verbalaspekt] g 1 K. J. Tham, Neues ausführliches und vollständiges deutsch- böhmisches Wörterbuch I, Prag 1799, vollst. Ausg. 1807-1809. 2 M. S. B. Linde, Słownik jeżyka polskiego, Warschau 1807-14. 3 J. M. Hartmann, Anfangsgründe der Hebräischen Sprache, Marburg 1819 4 F. W. Thiersch, Griechische Grammatik vorzüglich des homerischen Dialects. Leipzig 1826. 3 J. M. Hartmann, Anfangsgründe der Hebräischen Sprache, Marburg 1819 4 F. W. [Zu môme:] ebenso tvôme, svöme 60 [Zu taj , ovaj, onaj:] dieser, dieser, jener, iste, hic, [Zu taj , ovaj, onaj:] dieser, dieser, jener, iste, hic, ille [Zu tiz i:] instit[utiones] p. 433. h ib j H [i Gi ] 824) [ ] [ ] p auch ibojsH recens [ion von Grimm] p. 824) spur des altsl. russ.cia (hic) vgl. in s tit utiones p. 493 Puchmm[ayer] 298. Übrig im serb.cHHOh = ahd. hînaht wofür im gern[einen] leben севодни (sevodni) Г р. J. Safarlk, Serbische Lesekörner, oder historisch-kritische Beleuchtung der serbischen Mundart, Pesth 1833. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access [Zum relat. sto:] Wiederholung des persönl. pron. bei relativis, wenn ein lebendiges subject nothwendig; bei leblosen nicht nothw vgl. Beriich § 306 krain. Murko p. 51. oben Kopitar 294-295 [durchgestrichen:] nicht poln. böhm. russ. böhm. Thams Wb. s. и. co poln. Linde2 s. и. co Bandtke § 196 vgl. aber goth. saizei _ hebr. Hartmann p. m. 52 [Im Text sind alle Beispiele unterstrichen.] [Zum relat. sto:] Wiederholung des persönl. pron. bei relativis, wenn e lebendiges subject nothwendig; bei leblosen nicht noth vgl. Beriich § 306 krain. Murko p. 51. oben Kopitar 294-295 [durchgestrichen:] nicht poln. böhm. russ. böhm. Thams Wb. s. и. co poln. Linde2 s. и. co Bandtke § 196 vgl. aber goth. saizei _ hebr. Hartmann p. m. 52 [Im Text sind alle Beispiele unterstrichen.] 62 [durchgestrichen:] nicht poln. böhm. russ. hen:] nicht poln. böhm. russ [Im Text sind alle Beispiele unterstrichen.] 104 perf. und imperf. XLIX. L. XLIX [Zur Behauptung, "die üblichen Eintheilungen der Verba in ... Transitiva und Intransitiva" sei "unnütz":] in der formenlehre p. 64 L die deutsche spr[ache] entbehrt der form des imferfects für imperfectiva d.h. sie verwendet ihr imp[erfect] für imp[erfect] und praet[eritum] sie entbehrt der aoristform d. h. des eigentl[ ichen] perfectums und ersetzt sie durch praet[eritum ]. sie umschreibt das imperfectivum mit fort durch part, praes, и. verb, subst. er is t gehend = geht fort [Viele Unterstreichungen im Text über den Verbalaspekt] LII vgl. unten p. 65. 4 vgl. Thiersch p. 514 perfectiva haben nur ein futurum, kein praesens vgl. p. 64 der Deutsche, dies nicht fühlend sagt: zurück oder ich schieße (st[att] ich werde schießen) recule, ou je te tuerai L III im allg. anz. der D.^ 1827 nr. 254 behauptet ein G aus E. 70 1 Vuks Danica 1826 Vera Boji 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 64 j Downloaded from PubFactory at 01/10/2019 05:52:29AM Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 65 [Zu môme:] ebenso tvôme, svöme Thiersch, Griechische Grammatik vorzüglich des homerischen Dialects. Leipzig 1826. Allgemeiner Anzeiger der Deutschen, red. J. Fr. Hennick 5 Allgemeiner Anzeiger der Deutschen, red. J. Fr. Hennicke, Gotha. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 241 - 2• imperf. haben drei part• pass, praes. geliebt seiend, geliebt werdend, geliebt perfectiva nur eins: geschlagen werdend 2• imperf. haben drei part• pass, praes. geliebt seiend, geliebt werdend, geliebt perfectiva nur eins: geschlagen werdend 3. bei imperf. bedeutet das part, praet. etwas gegenwärtiges bei perf. etwas vergagngenes: geliebt (qui amatus) geschlagen (qui caesum est) (diese bemerkungen sind wenig wert) vgl. Puchmfayer] p. 181 in s tit utiones p. 568 63 [ Zum Passiv:] doch besitzen sie partizipia der passivform p. 69 pjesme II I . p. 299, 18 64 [Zur Einteilung in pf. und ipf. Verben:] Bandtke p. 239 65 [Zur Behauptung, daß "die Handlung eines Perfectivums gar keine Dauer hat": ] d. h. keine gegenwärtige Murko p. 86 70 das part. pass, praes. auf -m fehlt altsl. Dobr[ovskÿ] inst[itutiones] p. 382. russ. Puchm[ayer] p. 181 (bloß im höhern S til Puchm[yer] p. 182.) doch sind in adjectiven spuren übrig p itóm, lakom wie im böhm. Dobr[ovskÿ] böhm[ische] gr[ammatik] p. 183. 71 ausnahmsweise -u: (wie p. 70 gesagt) otju (volo) mogu (possum) [Zum Imperf. und Aorist -h:] in der Herzegow[ ina] dafür g (beispiele oben p. 14.) vgl. Danitza p. 57.^־ 72 andere auxiliaria regelmäßig: morati (müssen), moram. (vgl. Wörterbuch) mogu (possum) mozhesch . mozhe . moresch ^ es more ^ es Voltiggi gibt auch: mozhem und morem (possum) der imperat, hat mozi ! inf. motji (moei) krain, morem (debeo) morati, mogal (Kopitar p. 351) böhm. moKu Qv . ^ , mūžu Possum muzes (potes) poln. mogę (possum) możesz (potes) inf. modz. die slav. sprachen haben kein praesens conj. und keinen ersatz dafür. bl unveränd. Optativ oder conj. (praet.) Glossar h[errn] V[uks] (auch bloß b ': Vuk I. p. 98. 7) böhm. bych, bysi, bys; by; bychom, bychme; byste; by. krain, unveränd. bi (Kop[itar] p. 318. 320) 63 72 die slav. sprachen haben kein praesens conj. und keinen ersatz dafür. 1 Vuks Danica 1826 russ. бы, бъ Puchm[ayer] 201. 237. unveränderlich poln. Bandtke p. 254. 255 verand. bym, bys, by, altsl. бых, бы быхом, быисте, бьшіа Inst[itutiones] p. 391 veränd. russ. [Zu môme:] ebenso tvôme, svöme хочу (volo) böhm. chcy krain, hòzhem sprich ozhenn хочешь (vis) Kop[itar] p. 355. chce krain, n&zhem oder nèzhem (nolo) russ• нехочу (nolo) dela oder de bedeutet: wohlan! age! Voltiggi hat den imp. hotii! böhm. chtj, chtêgj ; chteg! mit пека vgl. böhm. nechey (laß) böhm. chtjti; russ.xoT^Tb ? krain, hotêti; poln. chciêc notëti (nolle) böhm. chtêl poln. chciał krain, hôtel, hòtla, hôtlo oder tel, tia, tio tel, tela, telo. moqи possum (oben p. 70. Glossar p. 409) моли inf. (Gloss. 413) Berlich p. 180. morati müssen is t regelmäßig die erste conj[ugation] hat zwischen wurzel und flexion überall ein a, daher bleibt der wurzelconsonant immer un— afficiert. einzelne verba bilden einzelne tempora nach zweiter conj. und dann schwindet jenes a und die con- s[onant]affectionen treten ein. [Zu "dam":] vgl. dajem, davati [Zu uzeti:] simpl. altsl. ja ti capere vgl. krain, jemlem (ich nehme) jemati, jemta jmem (capio) vsaméin (aufero) in f. vseti (Kopitar p. 346. 347) узети, russ. взять (? ргаев.возму ) otmem, oteti rapere [Zu po-peti:] auch binden, spannen (in die höhe) zuknüpfen, spannen vgl. böhm. pnu, pnauti, heften, knüpfen, dehnen popnu, zapnu (zapjnam) [kunem]f[ür] клнем [donesem] auch несем (gero) inf. нети? нео, нела. изнесем f[ür] нести,несо несла dajem do, davasche dedit (p. 90) [Zu -s ti:] vezem (sticken) vesti muzem (melken) musti тресем (schüttle) c f.Dobr[ovskÿ] inst[itutiones] p. 351. ibd. c it[ie rt] etymologtie] p. 62. russ. бы, бъ Puchm[ayer] 201. 237. unveränderlich poln. Bandtke p. 254. 255 verand. bym, bys, by, altsl. бых, бы быхом, быисте, бьшіа Inst[itutiones] p. 391 veränd. russ. хочу (volo) böhm. chcy krain, hòzhem sprich ozhenn хочешь (vis) Kop[itar] p. 355. chce krain, n&zhem oder nèzhem (nolo) russ• нехочу (nolo) dela oder de bedeutet: wohlan! age! Voltiggi hat den imp. hotii! böhm. chtj, chtêgj ; chteg! mit пека vgl. böhm. nechey (laß) böhm. chtjti; russ.xoT^Tb ? krain, hotêti; poln. chciêc notëti (nolle) böhm. chtêl poln. chciał krain, hôtel, hòtla, hôtlo oder tel, tia, tio tel, tela, telo. moqи possum (oben p. 70. Glossar p. 409) моли inf. (Gloss. 413) Berlich p. 180. morati müssen is t regelmäßig die erste conj[ugation] hat zwischen wurzel und flexion überall ein a, daher bleibt der wurzelconsonant immer un— afficiert. einzelne verba bilden einzelne tempora nach zweiter conj. und dann schwindet jenes a und die con- s[onant]affectionen treten ein. die auf wurzelhaftes p und b schalten im inf. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access [Zu môme:] ebenso tvôme, svöme s ein: zebem, zepsti; grebem, grepsti; dubem, dupsti;tzrpem, tzrpsti; p ; pletem, plesti; metem, mesti; grebem, gresti; predem, presti jedem, jesti; kradem, krasti; bodem,bosti; federn, fcesti; znadem die auf - stem werfen im part, das t vor 1 weg: rasti (crescere) rastern (cresco) ( ) ( ) part, praet. fem. rasla f. rastla (masc. wohl rastao?) russ. rasti (crescere) böhm rosti; part fem rostla ( ) ( ) part, praet. fem. rasla f. rastla (masc. wohl rastao?) russ. rasti (crescere) böhm. rosti; part. fem. rostla böhm. rosti; part. fem. rostla [Zu -nu ti:] кунем (ich fluche) p. 87 клнем кунем, inf. клети(also f . клнем) dirnem, dirnuti [Zu ־c i: ] [dodem] f. do־idem? vgl. Kopitar krain. p. 339 поЬи (gehen) по1)ем, пошао, пошла. отиЬи fortgehen. наЬи (finden) на^ем, нашао, нашла сиЬи (herabgehen) си^ем und сидем, сишао, сишла sihi сиЬи descendere, praet. сишао (Vuk p. 764) auch cahH, са])ем эаЬи (hintergehen) эаЬем заічи aberrare (Vuk p. 22) npohH (Vorbeigehen) прочем praetereo, inf. проЬи, prät. проЬо наІ)ем invenio, - наЬи жежем (иго), inf. жеЬи. жежен (ustus) вучем traho, inf. вуЬи, imperativ вуци стечем,acquiro,comparo стеЬи тучем, tundo ־туЬи течем (fluo) - теЬи &и -n u ti/-ò i:] клеЬи und клекнути (knien) клекнем [Žu -ati : ] лежем, легати казати кажем [durchgestrichen:] полажовати? полаживати полагивати лочем (sorbeo) локати плачем, плакати пушем, пуати blasen пашем пасати umgürten statt иштем sollte stehen ишчем Ьоэп.ишЬем иигге, quaerit, postulat I, 173, 3, 66, 26. böhm. gjskati russ. искат poln. iskać (quaerere) iszczę (quaero) Bandtke p. 267. in слати folglich сл wurzel, ати endung Danitza 18*55 119, 188. 120, 210 auch russ. слать посылаю (mitto) poln. iskać (quaerere) iszczę (quaero) Bandtke p. 267. in слати folglich сл wurzel, ати endung Danitza 18*55 119, 188. 120, 210 auch russ. слать посылаю (mitto) 00051004 - 244 - böhm. ssii, mitto possli (mitto) posÿlâm (mitto) poslati (mittere) berem, brati (lesen, sammeln) brao, brala perem, prati (waschen) 2 eingeschobener vocal колем f. юъем •nfra 90 шал>ем ииьем 7 90 sijem sero, s ijā ti serere [Zu dajem:] (vgl.dam, dadem, dati p. 78) 91 dobijem (lucror) dobiti. Dobr[ovskÿ, Institutiones] 348 92 für jedem auch jem (edo) inf. jesti part, jeo, jrla in I I I . conj[ugation] die 3. pi. praes. auf -e in I. II. auf -и 93 [Zu kunem:] in f. kuniti? 1 Vgl. Anm. 2, S. 235 1 Vuks Danica für das Jahr 1826. 2 M. Höfer, Etymologisches Wörterbuch der in Oberdeutschland ge- sprochenen Mundart, Bd. 1 - 3 , Linz 1815. 3 Die Zahlen hier und im folgenden beziehen sich auf Vuks Narodne Srpske Pjesme, Bd. I - III, Leipzig 1823f. [Zu môme:] ebenso tvôme, svöme (so Voltiggi) 96 [Zu ־i t i : ] водити (führen) водим (duco) voditi, vodim плодим (gigno) бродим (vado, transeo) родим (pario) редим (linum praeparo) молим (precor) гоним (agito) звоним (pulso campanam) носим (gero) косим (foenum seco) росим (irroro) , наресим neue ausg. I. 150 дробим (ich bröckele) drobiti пловим (nato, ich schwimme) кропим (ich sprütze, sprenge) [Zu -im /-je ti] auch рим gorim (ardeo) gorjeti 97 ломим (frango) ломити морим (interficio) морити мерим (metior) мерити 98 [Zu -jim:] 6 0 jaTH (metuere) imp. 60ji [107] verb, subst. 72 dati 78 davati 90 i t j i 88 Relativ p. 62 [Zu den Präpositionen: ] 99 свр ־= a lts l. сверху * с-верху (inst[itutiones] 48) 99 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 00051004 - 245 - 100 [Zur Präpos. и mit Gen.:] dieses у ist a ltsl. oy (instit[utiones] 649) russ. у (Puchm[ayer] 271. ) das andere у - въ ( [Institutiones] 658) russ. въ, во ( [Puchmayer] 27 2.) krain, v', vu, va böhm. w poln. w, we ca висока дела, vom hohen berge Danitza1 p. 112 ( дела f[ür] jmjena) [Zu den Adverbien:] 102 sadma (valde) odma (statim) vetjma (melius) razma praetor, excepto [Zu den Konjunktionen:] би, daß doch, möchte, sollte (ich gäbe) еда! v. gloss[ar] ако, wenn böhm. poln. како, wie gak iak тако, so tak tak mit dakle vgl. dokle, dotle, dovle, donle odakle, odatle, oäavle, odsvakle [Zu den Interjektionen:] 103 ako Bogda! кре, v. gloss[ar] v. крекнути 2 Oesterreich[ische] lockwörter. Höfer 2, 215-17 litth . tisz! hühner weg zu jagen [Zur Kasussyntax:] LXXI spuren des dual I, 225 na dva brda velika auf zwei großen bergen brdo mons, neutr. sagt man dva sokola? oder sokolova l.c 1 V k D i fü d J h 1826 100 [Zur Präpos. и mit Gen.:] dieses у ist a ltsl. oy (instit[utiones] 649) russ. у (Puchm[ayer] 271. ) das andere у - въ ( [Institutiones] 658) russ. въ, во ( [Puchmayer] 27 2.) krain, v', vu, va böhm. w poln. w, we ca висока дела, vom hohen berge Danitza1 p. 112 ( дела f[ür] jmjena) [Zu den Adverbien:] 102 sadma (valde) odma (statim) vetjma (melius) razma praetor, excepto [Zu den Konjunktionen:] би, daß doch, möchte, sollte (ich gäbe) еда! v. gloss[ar] ако, wenn böhm. poln. [Zu môme:] ebenso tvôme, svöme како, wie gak iak тако, so tak tak mit dakle vgl. dokle, dotle, dovle, donle odakle, odatle, oäavle, odsvakle [Zu den Interjektionen:] 103 ako Bogda! кре, v. gloss[ar] v. крекнути 2 Oesterreich[ische] lockwörter. Höfer 2, 215-17 litth . tisz! hühner weg zu jagen [Zur Kasussyntax:] LXXI spuren des dual I, 225 na dva brda velika auf zwei großen bergen brdo mons, neutr. sagt man dva sokola? oder sokolova l.c 100 1 Vuks Danica für das Jahr 1826. 2 M. Höfer, Etymologisches Wörterbuch der in Oberdeutschland ge- sprochenen Mundart, Bd. 1 - 3 , Linz 1815. 3 Die Zahlen hier und im folgenden beziehen sich auf Vuks Narodne Srpske Pjesme, Bd. I - III, Leipzig 1823f. 1 Vuks Danica für das Jahr 1826. 2 M. Höfer, Etymologisches Wörterbuch der in Oberdeutschland ge- sprochenen Mundart, Bd. 1 - 3 , Linz 1815. 3 Die Zahlen hier und im folgenden beziehen sich auf Vuks Narodne Srpske Pjesme, Bd. I - III, Leipzig 1823f. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00061004 • 246 - [112 ] I. n- 137 dva se draga vrlo milovala 147 dva se draga и selu gledaTa 219 dva se draga ljube 222 tri devojke govorile - 287 tri devojke tzvetscEe posejale 289 dva tzvijeta и bostanu rasla 312 tri putnika 319 dve se vode puteam pretitzale 320 pile su tri jetrvitze , [zwei Wörter unleserlich] 405 dva su bora na poredo rasla m[sk.] dva bora zelena [113] 3, 95 trideset i tschetiri druga 123 tridest кареtana 326 polehela dva vrana gavrana 2, 10,1 grad gradila 2, 64,1 sastala se tschetiri tabora 2,105,7 tri voivode srpske conf. ahd. wir zwei m[ask.] et f[en-] I. n- 110. dvije seje brata ne imale [114] III. 286. II. 152. dva vrana gavrana obadva zara aber II. 10,1 t r i brata romena v. romeni germanus I 305,5 dva brata romena I, 310 sedam perischana t r i kavada zhuta, drei gelbe Röcke t r i zlatna pojasa drei goldne Gürtel tr i, tschetiri - puta, drei, viermal t)ece obadvoje, beide Kinder II. 166,10 dvje srpske voivode 22 devet schura, Schwäger 11,21 oboje beide 60 II, 74, 256 dok je suntza i dok je mjesetza I. vorr[ede] LI. dok mi je Danitza p. 1 Leider gelang es trotz freundlicher Unterstützung von M. Mojaševic, für die ich ihm danken möchte, nicht, diese Stelle in Grimms Hand- schrift zu lesen. 2 Vuks Danica für das Jahr 1826. 2 Vuks Danica für das Jahr 1826. [Zu môme:] ebenso tvôme, svöme 108^ dok je mene II, 154,198 dok je ljudi i dok je Kosova II, 244, 33 dok je moje na ramenu glave III,269,179 dok je nama zdravlja i pameti I. Vorrede p. XXI dok je moje... verte II. 53,167 be si mene? (wo bist du mir? wo bleibst du mir? 2 Vuks Danica für das Jahr 1826. [112 ] I. n- 137 147 219 222 - 287 289 312 319 320 405 60 Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access 00051004 - 247 - LITERATURVERZEICHNIS 1. Jacob Grimms und Vuk Karadzics Schriften und Briefwechsel 1. Jacob Grimms und Vuk Karadzics Schriften und Briefwechsel Grimm, Jacob: Deutsche Grammatik T. 1 - 4 , Göttingen 1819ff. T. 1, 1819, 2.Aufl. 1822, 3. Aufl. 1840; T. 2, 1826; T. 3, 1831; T. 4, 1837. 2. Aug., neuer vermehrter Abdruck T. 1-4, Gütersloh 1878 - 1898. Deutsches Wörterbuch. Von Jacob und Wilhelm Grimm. Bd. 1 - 16, . 1960 ־ 1852 Geschichte der deutschen Sprache. 4. Aufl. Leipzig 1880. Kleinere Schriften Bd. 1 - 8 , Berlin 1863 1890 ( .־Bd. 8, Gütersloh). Karadzic, Vuk Stefanovic: Mala prostonarodnja slaveno-serbska pjesnarica. Wien 1814. (Sabrana delà Bd. I). Narodna srbska pjesnarica, Wien 1815. (Sabrana delà Bd. I ) . Srpski Rječnik istolkovan njemačkim i latinskim riječma. Narodna srbska pjesnarica, Wien 1815. (Sabrana delà Bd. I ) Narodna srbska pjesnarica, Wien 1815. (Sabrana delà Bd. I ) . Srpski Rječnik istolkovan njemačkim i latinskim riječma. Wien 1818. (Sabrana delà Bd. I I ) . Srpski Rječnik istolkovan njemačkim i latinskim riječma. Wien 1818. (Sabrana delà Bd. I I ) . Kleine serbische Grammatik übersetzt und mit einer Vorrede von Jacob Grimm (1824). Neu hrg. und eingel. von M. Mojaie- viè und P. Rehder. Belgrad/München 1974. (Sagners slavisti- sehe Sammlung Bd. 1). Danica. Zabavnik za godinu 1826. Wien 1826. (Sabrana dela Bd. V III). Novi zavjet Gospoda nasega Isusa Hrista. Wien 1847. (Sabra- na delà Bd. X). Srpski Rječnik istumačen njemackijem i latinskijem riječima. Wien 1852. Skupljeni gramatički i polemički spisi I - II I , hrg. P. P. Bordevic (državno izdanje), Belgrad 1894. brana dela Vuka Karadžica, Bd. I ff., Belgrad 1965 ff. Sabrana dela Vuka Karadžica, Bd. I ff., Belgrad 1965 ff. Rezension von L. Mušickis **Glas narodol jupea", Archiv SANU, Signatur 8552/14. Brief an J. Stejic, Archiv SANU, Signatur 8552/30(2). j , , g ( ) Materialien über die serbischen Verben, Archiv SANU 8552/11-12. Aufzeichnungen der Merkmale des kajkavischen Dialekts, Archiv SANU 8552/30(27). Leitzmann, A. (Hrg.): Briefwechsel der Brüder Jacob und Wilhelm Grimm mit Karl Lachmann, Bd. I- II, Jena 1927. Leitzmann, A. (Hrg.): Briefwechsel der Brüder Jacob und Wilhelm Grimm mit Karl Lachmann, Bd. I- II, Jena 1927. Sauer, A. (Hrg.): Aus Jacob Grimms Briefwechsel mit slavischen Ge- lehrten. In: Prager deutsche Studien 8, Prag 1908, S. 585 - 629 . Steig, R. (Hrg.): Briefwechsel zwischen Jacob Grimm und Therese von Jacob. In: Preußische Jahrbücher 76, Berlin 1894, S. LITERATURVERZEICHNIS 345 - 366. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Steig, R. (Hrg.): Briefwechsel zwischen Jacob Grimm und Therese von Jacob. In: Preußische Jahrbücher 76, Berlin 1894, S. 345 - 366. Vera Boji - 9783954793044 Steig, R. (Hrg.): Briefwechsel zwischen Jacob Grimm und Therese von Jacob. In: Preußische Jahrbücher 76, Berlin 1894, S. 345 - 366. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM Steig, R. (Hrg.): Briefwechsel zwischen Jacob Grimm und Therese von Jacob. In: Preußische Jahrbücher 76, Berlin 1894, S. 345 - 366. Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access Stojanovic, Lj. (Hrg.): Vukova prepiska, Bd. 1 - 7, (državno iz- danje), Belgrad 1907 - 1913. Vasmer, M. (Hrg.): В. Kopitars Briefwechsel mit Jakob Grimm, Berliin 1938. (Abh. der Preuß. Akad. der Wiss., Jg. 1937, Phil.-hist.. Klasse Nr. 7). Bausteine zur Geschichte der deutsch slavischen geistigen Be Vasmer, M. (Hrg.): В. Kopitars Briefwechsel mit Jakob Grimm, Berliin 1938. (Abh. der Preuß. Akad. der Wiss., Jg. 1937, Phil.-hist.. Klasse Nr. 7). 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Düsseldorf 1954 - 1959. Wiese, B. von: Herder. Grundzüge seines Weltbildes. Leipzig 1939. Wilson, D.: The Life and Times of Vuk Stefanoviò Karadzic 1787 - 1864. Literacy, Literature and Nation Indenpendence in Serbia. Oxford 1970. Wirnt von Gravenberg, Wigalois. Hrg. G. F. Benecke 1819. Wissenschaftliche Zeitschrift der Humboldt-Universität Berlin, Ge- sellsch.-sprachwiss. Reihe Bd. XIV, 1965, S. 447 - 525 (11 Aufsätze gewidmet Grimm). g ) Wolf, J .: Politische Geschichte des Eichsfeldes I - II, 1792 - 1793. Zbornik za filoloģiju i lingvistiku, hrg. Matica srpska, književno odeljenje. Bd. I ff. , Novi Sad 1957 ff. Zbornik za filoloģiju i lingvistiku, hrg. NAMENREGISTER NAMENREGI Adamovic, P. 17 2 Adelung, J. Ch. 10, 51 53 ־ Adrian, J. V. 172 Arens, H. 13, 14, 21, 33 Arndt, E. 21, 33 Arnim, A. von 13 Bandtke, G. S. 238 Barnard, F. 48 Belic, A. 12, 38, 39, 54, 70, 77f., 87f ., 89f 104 ,94 , ,״ 106, 143, 152, 168 Beneš, B. 13, 29, 31, 194 Biterolf und Dietleib 175, 231 Boehlich, W. 11 Böttiger, K. W. 173, 230 Зодісеѵіс, J., 111 Sondzio, W. 23, 24, 25, 26 Bopp, F. 176, 186, 233 Borst, A. 53 Boškovic, R. 168 Brlic, I. A. 163, 186, 236 Butler, Th. 69 Daničic, В. 62, 79, 102, 104, 123£., 167, 200 Demeter, D. 58 Denecke, L. l l f . , 13, 21 Deretić, J. 66 Dobrovsky, J. 40, 44f ., 69, 112 148, 150, 153, 157, 161, 167 185, 231f., 233, 237f. Bordevic, P.P. 54, 142, 168 Borflic, P. 78 Fick, A. 167 Fiesel, E. 15, 30 Gallis, A. 154 Adamovic, P. 17 2 Adelung, J. Ch. 10, 51 53 ־ Adrian, J. V. 172 Arens, H. 13, 14, 21, 33 Arndt, E. 21, 33 Arnim, A. von 13 Bandtke, G. S. 238 Barnard, F. 48 Belic, A. 12, 38, 39, 54, 70, 77f., 87f ., 89f 104 ,94 , ,״ 106, 143, 152, 168 Beneš, B. 13, 29, 31, 194 Biterolf und Dietleib 175, 231 Boehlich, W. 11 Böttiger, K. W. 173, 230 Зодісеѵіс, J., 111 Sondzio, W. 23, 24, 25, 26 Bopp, F. 176, 186, 233 Borst, A. 53 Boškovic, R. 168 Brlic, I. A. 163, 186, 236 Butler, Th. 69 Daničic, В. 62, 79, 102, 104, 123£., 167, 200 Demeter, D. 58 Denecke, L. l l f . , 13, 21 Deretić, J. 66 Dobrovsky, J. 40, 44f ., 69, 112 148, 150, 153, 157, 161, 16 185, 231f., 233, 237f. Bordevic, P.P. 54, 142, 168 Borflic, P. 78 Fick, A. 167 Fiesel, E. 15, 30 Gallis, A. 154 Adamovic, P. 17 2 Adelung, J. Ch. 10, 51 53 ־ Adrian, J. V. 172 Arens, H. 13, 14, 21, 33 Arndt, E. 21, 33 Arnim, A. von 13 Bandtke, G. S. 238 Barnard, F. 48 Belic, A. 12, 38, 39, 54, 70, 77f., 87f ., 89f 104 ,94 , ,״ 106, 143, 152, 168 Beneš, B. 13, 29, 31, 194 Biterolf und Dietleib 175, 231 Boehlich, W. 11 Böttiger, K. W. ovic, M.: Za i protiv Vuka. Belgrad 1967. Matica srpska, književno odeljenje. Bd. I ff. , Novi Sad 1957 ff. Živančevic, M.: Vukovi p rija te lji ilir c i. In: Vukov zbornik 1966, S. 229 - 258. Živkovic, D.: Silerovska i herderovska koncepcija poezije i Vukova borba za narodni jezik. Evropski okviri srpske književnosti, Belgrad 1970, S. 119 - 146. Odjeci sentimentalizma и srpskoj književnosti XVIII i prve poiovine XIX veka. Evropski okviri srpske književnosti, Belgrad 1970, S. 79 - 104. Živkovic, D.: Silerovska i herderovska koncepcija poezije i Vukova borba za narodni jezik. Evropski okviri srpske književnosti, Belgrad 1970, S. 119 - 146. Odjeci sentimentalizma и srpskoj književnosti XVIII i prve poiovine XIX veka. Evropski okviri srpske književnosti, Belgrad 1970, S. 79 - 104. ER Goethe, J. W. von 25, 194, 201 Grickat, I. 38, 77, 86 Grimm, H. 13 Grimm, J. 9 - 37, 44f., 47, 52, 63f., 72, 79, 90, 92, 94f., 96f., 98, 100, 103, 107, 110 202 - 171 , 166 ־ 112 Gruenter, R. 11 Hadíiè, J. 57, 61, 70, 74f., 76, 98 Hamann, J. G. 49 Hartmann, J* M. 240 Herder, J. G. 10, 17, 28 f., 43f 46 - 53, 59, 64, 72, 101, 196, 201f• Hermann, L. 13 Hiehle, K. 34, 37 Höfer, M. 245 Hraste, M. 108 Humboldt, W. von 16, 18 - 20, 191, 192 Ivic, A. 39 Ivic, M. 61, 63, 64 Ivic, P. 10, 12, 42, 71f. , 73, 87f. , 90f., 93, 95, 98f., 103, 108ff., 134, 137, 156, 184 NAMENREGISTER 46 Smotrickyj, M. 34 Sreznevskij, I. I- 69 Starcevié, A. 105 Steig, R. 13 Stejic, J. 58, 77, 79f., 85, 92 Vera Boji 9783954793044 Obradovié, Dositej 39 - 41, 44, 50, 55, 58, 61, 70 Orfelin Z, 39, 58 Ottokar von Steier 175, 331 Pavié, M. 39 Pavlovié, M. 62 Petrê,F. 42f., 46f. Petrovskij, N. M. 4 2f. Peukert, H. 146, 150, 173 Polheim, K. K. 65 Popovié, J. Sterija 61 Popovic, M. 46, 78 Popovié, P. 66 PreŠern, F. 43 Puchmayer, A. J. 237 Radičevic, В- 62 Rajic, J. 39, 41 Rehder, P. 146, 172, 230 Reljkovié, M. 59 Rousseau, J. J. 49, 50, 64, 196 Sastrow, B. 174, 231 Saussure, F. de 19, 32 Schannat, J. F. 174, 231 Scherer, W. 11, 33 Schiller, F. von 25, 194, 201 Schlegel, A. W. 30, 191 Schlözer, A. L. 43, 45 Schoof, W. 16 Selimovic, M. 41 Skerlic, J. 67, 79 Skok, P. 176f., 184 Slodnjak, A. 46 Smotrickyj, M. 34 Sreznevskij, I. I- 69 Starcevié, A. 105 Steig, R. 13 Stejic, J. 58, 77, 79f., 85, 92 Kicovic, M. 74 Kolb, H. 11 Konrad, G. 48 Kopitar, J. 10, 34, 40, 42 ־ 47, 51f., 71f., 107, 110f., , 137 , 124 ־ 122 , 115 , 112 , 165 , 161 , 158 , 148 , 146 , 232 , 230 , 188 , 186 , 172 236 Kravar, M. 10 Kruse, F. 173, 230 Ladevic, M. 62 Lang, K. H. Ritter von 174, 231 Linde, M. S. B. 246 Linhart, A. 45 Lord, A. 196 Luther, M. 25, 238 Maretic, T. 118 Mielcke, Ch. G. 235 Mihajlovic, V. 87 Miklosich, F. 104, 147f•, 149, 185, 187 Milovic, J. 188 Mirijevski, Teodor Jankovic 39 Mladenovic, A. 10, 11 MojaSevic, M. 12, 111, 146, 172 230, 246 Kravar, M. 10 Kruse, F. 173, 230 Ladevic, M. 62 Kruse, F. 173, 230 Ladevic, M. 62 Lang, K. H. Ritter von 174, 231 Linde, M. S. B. 246 Linhart, A. 45 Radičevic, В- 62 Rajic, J. 39, 41 Rehder, P. 146, 172, 230 Reljkovié, M. 59 Mielcke, Ch. G. 235 Rousseau, J. J. 49, 50, 64, 196 Mihajlovic, V. 87 Sastrow, B. 174, 231 Miklosich, F. 104, 147f•, 149, Miklosich, F. 104, 147f•, 149, Saussure, F. de 19, 32 185, 187 Schannat, J. F. 174, 231 Milovic, J. 188 Scherer, W. 11, 33 Mirijevski, Teodor Jankovic 39 Schiller, F. NAMENREGISTER 173, 230 Зодісеѵіс, J., 111 Sondzio, W. 23, 24, 25, 26 Bopp, F. 176, 186, 233 Borst, A. 53 Boškovic, R. 168 Brlic, I. A. 163, 186, 236 Butler, Th. 69 Daničic, В. 62, 79, 102, 104, 123£., 167, 200 Demeter, D. 58 Denecke, L. l l f . , 13, 21 Deretić, J. 66 Dobrovsky, J. 40, 44f ., 69, 112 148, 150, 153, 157, 161, 16 185, 231f., 233, 237f. Bordevic, P.P. 54, 142, 168 Borflic, P. 78 Fick, A. 167 Fiesel, E. 15, 30 Gallis, A. 154 Barnard, F. 48 Hamann, J. G. 49 Hamann, J. G. 49 Hartmann, J* M. 240 Hartmann, J* M. 240 Biterolf und Dietleib 175, 231 Herder, J. G. 10, 17, 28 f., 43f., Boehlich, W. 11 46 - 53, 59, 64, 72, 101, Böttiger, K. W. 173, 230 196, 201f• Hermann, L. 13 Зодісеѵіс, J., 111 Hiehle, K. 34, 37 Sondzio, W. 23, 24, 25, 26 Höfer, M. 245 Bopp, F. 176, 186, 233 Borst, A. 53 Hraste, M. 108 191, 192 Ivic, A. 39 Demeter, D. 58 Denecke, L. l l f . , 13, 21 Deretić, J. 66 Ivšic, S. 108f. Dobrovsky, J. 40, 44f ., 69, 112, 148, 150, 153, 157, 161, 167, 185, 231f., 233, 237f. Jagič, V. 40, 149, 185 185, 231f., 233, 237f. Jakobson, R. 196 Johannes von Posilge (Lindenblatt), 174, 231 Kanne, A. 13f., 191, 231 Karadzifc, Vuk St. 9 29 ־ 26 , 12 ־ , 34f , 38 - 42, 44, 46 202 ־ Bordevic, P.P. 54, 142, 168 Borflic, P. 78 Gallis, A. 154 00051004 00051004 256 ־ Obradovié, Dositej 39 - 41, 44, 50, 55, 58, 61, 70 Orfelin Z, 39, 58 Ottokar von Steier 175, 331 Pavié, M. 39 Pavlovié, M. 62 Petrê,F. 42f., 46f. Petrovskij, N. M. 4 2f. Peukert, H. 146, 150, 173 Polheim, K. K. 65 Popovié, J. Sterija 61 Popovic, M. 46, 78 Popovié, P. 66 PreŠern, F. 43 Puchmayer, A. J. 237 Radičevic, В- 62 Rajic, J. 39, 41 Rehder, P. 146, 172, 230 Reljkovié, M. 59 Rousseau, J. J. 49, 50, 64, 196 Sastrow, B. 174, 231 Saussure, F. de 19, 32 Schannat, J. F. 174, 231 Scherer, W. 11, 33 Schiller, F. von 25, 194, 201 Schlegel, A. W. 30, 191 Schlözer, A. L. 43, 45 Schoof, W. 16 Selimovic, M. 41 Skerlic, J. 67, 79 Skok, P. 176f., 184 Slodnjak, A. NAMENREGISTER von 25, 194, 201 Mladenovic, A. 10, 11 Schlegel, A. W. 30, 191 MojaSevic, M. 12, 111, 146, 172, 230, 246 Moser, H. 37 Mrazovic, A. 69 Murko, A. 185, 236 MuSicki, L. 47, 50, 57, 66, MojaSevic, M. 12, 111, 146, 172, 230, 246 Moser, H. 37 Mrazovic, A. 69 Murko, A. 185, 236 MuSicki, L. 47, 50, 57, 66, 70, 110 Nedic, V. 47 Novak, V. 46, 105f. Schlözer, A. L. 43, 45 Schoof, W. 16 Moser, H. 37 Nedic, V. 47 Novak, V. 46, 105f. Novak, V. 46, 105f. NjegoS, P. P. 66 NjegoS, P. P. 66 NjegoS, P. P. 66 Novakovié, S. 112f. Novakovié, S. 112f. Novakovié, S. 112f. 00051004 00051004 ־ 257 - Vasmer, M. 17, 46, 147, 174 Vater, J. 110, 236 Vencloviè, Gavril Stefanovic, 39, 41 Vidakovic, M. 38, 40, 55, 60, 62, 65ff., 67, 71, 88ff• Vraz, S. 58 Weisgerber, L. 29, 31 Wiese, B. von 49 Wirnt von Gravenberg 175, 231 Wolf, J. 174, 231 Zivancevic, M. 46 Zivkovic, D. 50 Stojanovic, L j. 10, 12, 42, 4 3f., 107, 156, 167f• Safarîik, P. J. 104, 148f., 187, 238 Siikov, A. S. 60, 65, 70, 197 Šulek, B. 105 Talvj (Therese Albertine Luise von Jacob) 31, 189 Tham, K. 240 Thiersch, F. W. 183, 240 Tirol, D. 11 142 ,125 ,114 ,־ 143 - 145, 162, 172 Trops, S. 188f. Stojanovic, L j. 10, 12, 42, 4 3f., 107, 156, 167f• Safarîik, P. J. 104, 148f., 187, 238 Siikov, A. S. 60, 65, 70, 197 Šulek, B. 105 Talvj (Therese Albertine Luise von Jacob) 31, 189 Tham, K. 240 Thiersch, F. W. 183, 240 Tirol, D. 11 142 ,125 ,114 ,־ 143 - 145, 162, 172 Trops, S. 188f. Weisgerber, L. 29, 31 Wiese, B. von 49 143 - 145, 162, 172 Vera Boji 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access j Downloaded from PubFactory at 01/10/2019 05:52:29AM ABKÜRZUNGSVERZEICHNIS ABKÜRZUNGSVERZEICHNIS Anali Filološkog fakulteta и Beogradu J. Grimm, Kleinere Schriften Godišnjak Filozofskog fakulteta и Novom Sadu V. Karadzic, Skupljeni gramatički i polemicki spisi Jugoslavenska akadēmija znanosti i umjetnisti Južnoslovenski filolog В. Kopitar, Kleinere Schriften Matica srpska Naučni sastanak slavista и Vukove dane. Refe- ra ti i saopštenja Jagic (Hrg.), Pis'ma Dobrovskago i Kopitara Srpska akadēmija nauka i umetnosti V. Karadzic, Sabrana dela J. Kopitar, Spisi II M. Vasmer (Hrg.), B. Kopitars Briefwechsel mit Jakob Grimm Zbornik za filolo ģiju i lingvistiku Anali FF GKS Godišnjak FFNS GPS JAZU JF KKS MS Naučni sastanak Pis1ma SANU SD Spisi Vasmer Zbornik FL Anali FF GKS Godišnjak FFNS GPS JAZU JF KKS MS Naučni sastanak Pis1ma SANU SD Spisi Vasmer Zbornik FL Anali Filološkog fakulteta и Beogradu Anali Filološkog fakulteta и Beogradu J. Grimm, Kleinere Schriften Godišnjak Filozofskog fakulteta и Novom Sadu V. Karadzic, Skupljeni gramatički i polemicki spisi Jugoslavenska akadēmija znanosti i umjetnisti Južnoslovenski filolog В. Kopitar, Kleinere Schriften Matica srpska Naučni sastanak slavista и Vukove dane. Refe- ra ti i saopštenja Jagic (Hrg.), Pis'ma Dobrovskago i Kopitara Srpska akadēmija nauka i umetnosti V. Karadzic, Sabrana dela J. Kopitar, Spisi II M. Vasmer (Hrg.), B. Kopitars Briefwechsel mit Jakob Grimm Zbornik za filolo ģiju i lingvistiku J. Grimm, Kleinere Schriften Godišnjak Filozofskog fakulteta и Novom Sadu V. 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Leitner, A.: Die Erzählungen Fedor Sologubs. 1976. 249 S. 101. Leitner, A.: Die Erzählungen Fedor Sologubs. 1976. 249 S. 102. Lenga, G.: Zur Kontextdeterminierung des Verbalaspekts im modernen Polnisch. 1976. VIII, 233 S. 03. Zlatanova, R.: Die Struktur des zusammengesetzten Nom im Altbulgarischen. 1976. VIII, 220 S. 104. Krupka, P.: Der polnische Aphorismus. Die "Unfrisierten Gedanken” von Stanisław Jerzy Lec und ihr Platz in der polnischen Aphoristik. 1976. 197 S. ABKÜRZUNGSVERZEICHNIS ł -V ?י SI• ־4 ן * •?יi 1*■ * i 4 >־+-■ ■У i1 ’ l ^ v f e & ' - *" י ־ ־״Ч ־ Л־ וו * * ■ I , J é — * ■1« гг־ г ^ЦГ f ־י я 4 י-г'й^гл JC~ * **'י ־ ąrfuu F %? 1״п1 Vs а ־1 fn 1 * І -I .■ 1 - i f ' .JFÍ 4Ь I Л?-1і * « = >יוכ II 3 ! f ״ = 1׳ » , !i4 .« ,ע *jI* 1 j ־i/ ־ •ГЧ';׳׳ Ж і Т Ч ^ ־ ־ ' *A ?* ״־1 ! י ?Д ; 1 “ ЧМ - - 11 ..*׳-.H J- ài■ i lb 00051004 S l a v i s t i s c h e B e i t r ä g e 83. Baumann, W.: Die Sage von Heinrich dem Löwen bei den Slaven. 1975. 185 S. 83. Baumann, W.: Die Sage von Heinrich dem Löwen bei den Slaven. 1975. 185 S. 83. Baumann, W.: Die Sage von Heinrich dem Löwen bei den Slaven. 1975. 185 S. 84. Everts-Grigat, S.: V. V. Majakovskij: Pro êto. Übersetzung und Interpretation. 1975. 262 S. 84. Everts-Grigat, S.: V. V. Majakovskij: Pro êto. Übersetzung und Interpretation. 1975. 262 S. 84. Everts-Grigat, S.: V. V. Majakovskij: Pro êto. Übersetzung und Interpretation. 1975. 262 S. 85. Mirsky, S.: Der Orient im Werk Velimir Chlebnikovs. 1975. V III, 112S. 85. Mirsky, S.: Der Orient im Werk Velimir Chlebnikovs. 1975. V III, 112S. 86 Ditterich M Unters ch ngen m altr ssischen Ak ent an 86. Ditterich, M.: Untersuchungen zum altrussischen Akze hand von Kirchengesangshandschriften. 1975. 147 S. 87. Cummins, G. M.: The Language of the Old Czech L e g e n d a о s v a - cé K a t e t i n ë . 1975. V ili, 371 S. 88. Földeák, H.: Neuere Tendenzen der sowjetischen Science Fic- t ion. 1975. VI, 208 S. 89. Drews, P.: Devêtsil und Poetismus. Künstlerische Theorie und Praxis der tschechischen literarischen Avantgarde am Beispiel Vltëzslav Nezvals, Jaroslav Seiferts und JiM Wolkers. 1975. 330 S. 90. SchÖnle, P. W.: Zur Wortbildung im modernen Russisch. 1975. VIII, 195 S. 91. Okuka, M.: Sava Mrkalj als Reformator der serbischen Kyrii- liza. Mit einem Nachdruck des S a lo d e b e l o g a jera l i bo A z b u k o - p r o t r e s . 1975. 123 S. p 92. Neuhäuser, R.: The Romantic Age in Russian Literature: Poe- tic and Esthetic Norms. An Anthology of Original Texts (1800- 1850). 1975. VIII, 300 S. 93. Döring, J. R. (Hrg.): Literaturwissenschaftliches Seminar: Zur Analyse dreier Erzählungen von VI. I. Dal'. Mit einem me- thodologischen Geleitwort von Johannes Holthusen.1975. 203 S. 93. Döring, J. R. (Hrg.): Literaturwissenschaftliches Seminar: Zur Analyse dreier Erzählungen von VI. I. Dal'. Mit einem me- thodologischen Geleitwort von Johannes Holthusen.1975. 203 S. 94. Alexander, R.: Toriak Accentuation. 1975. XVI, 806 S. g 94. Alexander, R.: Toriak Accentuation. 1975. XVI, 806 95. Schenkowitz, G.: Der Inhalt sowjetrussischer Vorlesestoffe für Vorschulkinder. Eine quantifizierende Corpusanalyse unter Benutzung eines Computers. 1976. 767 S. 95. Schenkowitz, G.: Der Inhalt sowjetrussischer Vorlesestoffe für Vorschulkinder. S l a v i s t i s c h e B e i t r ä g e Eine quantifizierende Corpusanalyse unter Benutzung eines Computers. 1976. 767 S. 95. Schenkowitz, G.: Der Inhalt sowjetrussischer Vorlesestoffe für Vorschulkinder. Eine quantifizierende Corpusanalyse unter Benutzung eines Computers. 1976. 767 S. g p 96. Kitch, F. C. M.: The Literary Style of Epifanij Premudryj. P l e t e n i j e sloves. 1976. 298 S. 96. Kitch, F. C. M.: The Literary Style of Epifanij Premudryj. P l e t e n i j e sloves. 1976. 298 S. 97. Eschenburg, B.: Linguistische Analyse der Ortsnamen der ehe- maligen Komitate Bäcs und Bodrog von der ungarischen Landnahme (896) bis zur Schlacht von Mohács (1526). 1976. 156 S. 3 Kt. 97. Eschenburg, B.: Linguistische Analyse der Ortsnamen der ehe- maligen Komitate Bäcs und Bodrog von der ungarischen Landnahme (896) bis zur Schlacht von Mohács (1526). 1976. 156 S. 3 Kt. 98. Lohse, H.: Die Ikone des hl. Theodor Stratilat zu Kalbenstein- berg. Eine philologisch-historische Untersuchung. 1976.XX,242 S. 99. Erbslöh, G.: "Pobeda nad solncem". Ein futuristisches Drama von A. Kruäenych. Übersetzung und Kommentar. (Mit einem Nachdruck 97. Eschenburg, B.: Linguistische Analyse der Ortsnamen der ehe- maligen Komitate Bäcs und Bodrog von der ungarischen Landnahme (896) bis zur Schlacht von Mohács (1526). 1976. 156 S. 3 Kt. (896) bis zur Schlacht von Mohács (1526). 1976. 156 S. 3 Kt. 98. Lohse, H.: Die Ikone des hl. Theodor Stratilat zu Kalbenstein- berg. Eine philologisch-historische Untersuchung. 1976.XX,242 S. 99. Erbslöh, G.: "Pobeda nad solncem". Ein futuristisches Drama von ( ) ( ) 98. Lohse, H.: Die Ikone des hl. Theodor Stratilat zu Kalbenstein- berg. Eine philologisch-historische Untersuchung. 1976.XX,242 S. 99. Erbslöh, G.: "Pobeda nad solncem". Ein futuristisches Drama von A. Kruäenych. Übersetzung und Kommentar. (Mit einem Nachdruck der Originalausgabe.) 1976. 121 S. 99. Erbslöh, G.: "Pobeda nad solncem". Ein futuristisches Drama von A. Kruäenych. Übersetzung und Kommentar. (Mit einem Nachdruck der Originalausgabe.) 1976. 121 S. g g ) 100. Koszinowski, K.: Die von präfigierten Verben abgeleiteten Sub- stantive in der modernen serbokroatischen Standardsprache. Eine Untersuchung zu den Präfixen do, iz, na, za. 1976. 271 S. 100. Koszinowski, K.: Die von präfigierten Verben abgeleiteten Sub- stantive in der modernen serbokroatischen Standardsprache. Eine Untersuchung zu den Präfixen do, iz, na, za. 1976. 271 S. , p g g stantive in der modernen serbokroatischen Standardsprache. Eine Untersuchung zu den Präfixen do, iz, na, za. 1976. 271 S. 101. Leitner, A.: Die Erzählungen Fedor Sologubs. 1976. 249 S. 102. 19 7 7 105. Pogačnik, J .: Von der Dekoration zur Narration. Zur Entstehungsge- schichte der slovenischen Literatur. 1977. 165 S. 106. Bojić, V.: Jacob Grimm und Vuk KaradŽic. Ein Vergleich ihrer Sprach- auffassungen und ihre Zusammenarbeit auf dem Gebiet der serbischen Grammatik. 1977. 257 ^ Bayerische ļ Vera Boji - 9783954793044 Downloaded from PubFactory at 01/10/2019 05:52:29AM via free access
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Unsupervised Graph Deep Learning Reveals Emergent Flood Risk Profile of Urban Areas
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Texas A&M University-College Station kai kai Texas A&M University https://orcid.org/0000-0002-4440-4846 Unsupervised Graph Deep Learning Reveals Emergent Flood Risk Pro¦le of Urban Areas Ali Mostafavi Ali Mostafavi Texas A&M University-College Station kai kai Abstract Urban flood risk emerges from complex and nonlinear interactions among multiple features related to flood hazard, flood exposure, and social and physical vulnerabilities, along with the complex spatial flood dependence relationships. Existing approaches for characterizing urban flood risk, however, are primarily based on flood plain maps, focusing on a limited number of features, primarily hazard and exposure features, without consideration of feature interactions or the dependence relationships among spatial areas. To address this gap, this study presents an integrated urban flood-risk rating model based on a novel unsupervised graph deep learning model (called FloodRisk-Net). FloodRisk-Net is capable of capturing spatial dependence among areas and complex and nonlinear interactions among flood hazards and urban features for specifying emergent flood risk. Using data from multiple metropolitan statistical areas (MSAs) in the United States the model characterizes their flood risk into six distinct city-specific levels Urban flood risk emerges from complex and nonlinear interactions among multiple features related to flood hazard, flood exposure, and social and physical vulnerabilities, along with the complex spatial flood dependence relationships. Existing approaches for characterizing urban flood risk, however, are primarily based on flood plain maps, focusing on a limited number of features, primarily hazard and exposure features, without consideration of feature interactions or the dependence relationships among spatial areas. To address this gap, this study presents an integrated urban flood-risk rating model based on a novel unsupervised graph deep learning model (called FloodRisk-Net). FloodRisk-Net is capable of capturing spatial dependence among areas and complex and nonlinear interactions among flood hazards and urban features for specifying emergent flood risk. Using data from multiple metropolitan statistical areas (MSAs) in the United States, the model characterizes their flood risk into six distinct city-specific levels. The model is interpretable and enables feature analysis of areas within each flood-risk level, allowing for the identification of the three archetypes shaping the highest flood risk within each MSA. Flood risk is found to be spatially distributed in a hierarchical structure within each MSA, where the core city disproportionately bears the highest flood risk. Multiple cities are found to have high overall flood-risk levels and low spatial inequality, indicating limited options for balancing urban development and flood-risk reduction. Relevant flood-risk reduction strategies are discussed considering ways that the highest flood risk and uneven spatial distribution of flood risk are formed. Unsupervised Graph Deep Learning Reveals Emergent Flood Risk Profile of Urban Areas Kai Yin1, Ali Mostafavi1* 1 Urban Resilience.AI Lab, Zachry Department of Civil and Environmental Engineering, Texas A&M University, College Station, United States. * Corresponding author: Ali Mostafavi, amostafavi@civil.tamu.edu. Unsupervised Graph Deep Learning Reveals Emergent Flood Risk Profile of Urban Areas Kai Yin1, Ali Mostafavi1* 1 Urban Resilience.AI Lab, Zachry Department of Civil and Environmental Engineering, Texas A&M University, College Station, United States. * Corresponding author: Ali Mostafavi, amostafavi@civil.tamu.edu. Kai Yin1, Ali Mostafavi1* 1 Urban Resilience.AI Lab, Zachry Department of Civil and Environmental Engineering, Texas A&M University, College Station, United States. * Corresponding author: Ali Mostafavi, amostafavi@civil.tamu.edu. Article Article Keywords: Urban §ood risk rating model, Unsupervised graph deep learning, Flood risk map, Hierarchica spatial distribution, Spatial inequality Posted Date: March 21st, 2024 DOI: https://doi.org/10.21203/rs.3.rs-3915689/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: (Not answered) Additional Declarations: (Not answered) Keywords: Urban flood risk rating model, Unsupervised graph deep learning, Flood risk map, Hierarchical spatial distribution, Spatial inequality Introduction Urban flooding has been the cause of tremendous social, economic, and ecological impacts throughout the past several decades1–5. Catastrophic floods in urban areas, such as those caused by Hurricane Katrina in New Orleans in 2005, Superstorm Sandy in New York in 2012, and Hurricane Harvey in Houston in 2017, have resulted in hundreds of casualties, billions of dollars in property damage, and millions of people adversely affected6–8. What is worse, populations and assets in flood-prone areas continue to expand due to uncontrolled urban planning1,9, surface water runoff is increasing as a result of the growth of impervious surfaces under urbanization, and the precipitation is becoming more frequent and intense due to climate change1,10,11. These changes have collectively increased the flood risk in urban areas in particular. p The tremendous impacts caused by floods and the increasing magnitude of flood risk in urban areas bring into sharp focus the importance of characterizing flood risk across areas in cities to identify areas with the greatest risk of floods and to analyze the underlying urban characteristics that give rise to high-flood-risk areas. Identification of high-flood-risk areas and understanding of spatial distribution of flood risk in cities are critical inputs for integrated urban design to achieve flood risk reduction. The existing approaches for rating urban flood risk are incomplete, inaccurate, and unreliable1,12. In the United States, Flood Insurance Rate Maps (FIRMs) provided by Federal Emergency Management Agency (FEMA) are usually used to analyze urban flood risk1. FIRMs define flood risk by identifying areas with a 1% chance of being inundated by floods in any given year13. Stakeholders merely intersect flood exposure component location information with FIRMs to evaluate the locational flood risk. Several studies14–17 have revealed, however, that FIRMs perform poorly in evaluating locational flood risk. Datasets from past flood events have also proved inaccurate in predicting urban flood risk. For example, about half the homes in Houston that suffered from flooding caused by Hurricane Harvey, which is a flood event that occurs 3000 to 2000 years, were outside the mapped 500-year floodplains1. Relying solely on FIRMs to evaluate urban flood risk causes misunderstanding of locational flood risk, which further leads to the failure of flood risk reduction strategies. Abstract The model enables characterizing urban flood risk as an emergent property arising from non-linear interactions among flood hazard and urban features, and integration of spatial flood dependence relationships among areas. Accordingly, this study’s outcomes provide a novel perspective and better understanding of urban flood-risk profiles by harnessing machine intelligence and urban big data. Keywords: Urban flood risk rating model, Unsupervised graph deep learning, Flood risk map, Hierarchical spatial distribution, Spatial inequality 1 Introduction Furthermore, FIRMs provide hardly any interpretable information for stakeholders (e.g., communities, insurance companies, city managers and planners, and policy makers) to understand underlying determinants for shaping their flood risk, which hinders them from taking protective actions, such as home improvements, insurance plans, or evacuation during an event. p p g In the field of flood risk modeling, researchers have made progress in flood risk rating by incorporating flood vulnerability, in contrast to the simplistic real-world approach that merely intersects FIRMs with its exposed components. There are two main streams of methods related to flood risk rating: monetary loss-based and flood risk-related-indicators-based. For the first stream of method, researchers18–21 generally intersect flood hazard maps with asset location information and their vulnerability function (depth-damage function) to calculate monetary loss caused by floods (e.g., the average annual loss) that is further referred to flood risk. This approach, although widely used, considers only risks associated to limited physical assets (e.g., properties)22. It actually measures property damage caused by floods without the ability to rate flood risk considering other aspects of physical and social vulnerabilities. In addition to properties, vulnerability of other physical infrastructure and facilities to floods should be captured. Also, multiple studies1,23–26 have revealed that floods disproportionately impact socially disadvantaged communities due to their limited capacity to prepare for, resist, and recover from flood damage. Hence, a proper urban flood-risk rating approach should also consider various urban characteristics related to flood hazards, exposure, as well as physical and social vulnerability. Existing methods proposed for capturing the combined effects of flood hazard, exposure, and vulnerability characteristics in calculating flood risk can be summarized by ∑𝑤𝑖∗𝑥𝑖, where 𝑥𝑖 represents flood risk-related features. These approaches focus mainly on determination of the optimized weight value (𝑤𝑖) of each feature using methods, such as the analytic hierarchy process, scoring by experts, set pair analysis, and fuzzy comprehensive evaluation27. These weight determination methods are mostly based on expert experience and 2 knowledge28, resulting in a subjective assessment of flood risk.27. Furthermore, cities in nature are complex systems with features related to the landscape, populations, and infrastructures that nonlinearly interact with each other given rise to different urban phenomena29. Introduction Flood hazards in cities interact with other urban features (exposed people and infrastructures, the socially vulnerable communities, and the physically vulnerable infrastructures), which makes the urban flood risk an emergent property arising from complex and nonlinear interactions between flood hazards and urban features. This emergent nature of urban flood risk profiles also implies that independent variables interact with each other30. Hence, capturing the complex and non-linear interactions among various urban features is essential for mapping the flood-risk profiles. The existing indicators-based flood risk assessment methods, however, lack the consideration of interactions among independent variables (features). Also, indicator-based methods, in essence, calculate flood risk in a linear way, which is inadequate to capture the nonlinear relationships among various hazards and urban features and their intersections that combine to form flood risk. Another major constraint of the existing approaches for urban flood risk rating is limited consideration of spatial flood dependence relationships among areas of cities. Most of these studies simply assume that the flood risk at a location is independent of all other locations without considering spatial interactions. However, as floods generally spread across many regions, flood risk assessment needs to account for the interrelations of floods between regions34. Spatial flood dependence is defined by Brunner et al. as “the degree to which flood risk at one location is related to flood risk at other locations.” 31 Flood frequency in one location will be directly impacted by other locations that have significant spatial flood dependence with it32. Hence, the flood exposure and flood vulnerability in one location will be indirectly impacted by spatially dependent locations. Therefore, consideration of spatial flood dependence is crucial in determining the flood risk of each location31,33,34. Lack of consideration of spatial flood dependence among areas would lead to the underestimate or overestimation of the potential losses caused by flood hazards34,35. The purpose of flood risk rating across areas of cities is to identify high risk areas and also enable evaluation of the extent to which urban features related to urban development and growth patterns could shape the flood risk profiles of cities. However, the existing approaches to flood risk rating in cities have major limitations in these aspects. Most of the flood maps are not updated regularly as urban features evolve. An alternative approach would allow updating of flood risk rating of urban areas as the underlying features change over time. Results Three-layer unsupervised deep learning model for urban flood risk rating. The main tasks associated with urban flood risk rating through machine intelligence and urban big data are: (1) modeling spatial flood dependence relationships and integrating these relationships into flood risk ratings; (2) capturing complex and nonlinear interactions among flood risk-related features; (3) addressing the unavailability of ground truthing labels for flood-risk levels during model training. To address these tasks, a three-layer unsupervised flood-risk rating model, FloodRisk-Net is developed and validated (Fig.1; see Methods for details). In the first layer, spatial dependence relationship learner, spatial flood dependence relationships among different locations (grid cells) are modeled in a network data structure. The node in the spatial dependence graph is each grid cell, and the weight of the edge connecting two nodes is the spatial dependence strength between them. An unsupervised graph structure learning (GSL) approach is implemented to learn the optimal spatial flood dependence network. The adjacency matrix is modeled by a parameterized graph learner based on the pairwise similarity of node embeddings, considering that spatial flood dependence measures “the degree to which flood risk at one location are related to flood risks at other locations”31. Parameters of the graph learner are learned using a contrastive learning framework with training signals obtained from the data itself. In the second layer, node representation leaner, a node embedding module composed of a deep neural network (DNN) and a graph convolution network (GCN) is implemented to jointly capture complex and nonlinear interactions among flood risk-related features and to incorporate spatial flood dependence relationships. These relationships are incorporated into flood risk rating by propagating and aggregating node attributes from neighbors on the optimal spatial flood dependence network using GCN. This approach originates from the recognition that flood hazard, exposure, and vulnerability in one location would be impacted by the features of other locations upon which it is spatially dependent (see Methods for details). DNN is adopted to capture complex and nonlinear interactions among flood risk-related features in each grid cell. Node embeddings learned by DNN and GCN are combined to obtain the final informative node embeddings. Introduction Finally, the existing methods provide a one-size-fits-all rating for the extent of flood risk in cities. The flood risk extent of areas in a city should be determined relative to other areas in the same city. An analogy is rating people’s height as “tall” across countries. This limitation can be addressed using city-specific rating of flood-risk levels in which a high-risk area in one city would signify a different extent of flood risk than a high-risk area in another city. City-specific flood-risk level rating enables a better understanding of the flood risk profile across areas of a city. Recognizing the importance of specifying and characterizing emergent flood risk across urban areas and considering limitations of the existing methods in providing updated flood risk rating, we propose an integrated three-layer unsupervised urban flood risk rating model (called FloodRisk-Net) to characterize emergent flood risk in cities using urban big data and machine intelligence. FloodRisk-Net is capable of discovering spatial flood-dependence structure, incorporating it into flood-risk rating, and capturing complex and nonlinear interactions among individual flood risk-related features, including flood hazard, exposure, and social and physical vulnerabilities. The five most populous metropolitan statistical areas (MSAs) in the United States—are selected as study regions: Greater New York, Los Angeles, Chicago, Dallas, and Houston. The model rates the flood-risk level of grid cells created within each MSA. The main contribution of this study is mining heterogenous feature information related to urban development and flood hazards and integrating spatial flood dependence relationships using unsupervised graph neural network to reveal emergent property flood risk in urban areas. The model and findings will be particularly valuable to various academic disciplines and diverse stakeholder practitioners: (1) FloodRisk-Net provides a novel method for urban scientists and flood researchers to quantify and analyze the flood risk profile of cities based on 3 heterogenous urban development and flood hazard features, and their non-linear interactions; (2) discovering the hierarchical and uneven spatial distribution patterns of flood risk in urban areas informs geo-spatial scientists and urban planners about the spatial autocorrelation and spatial inequality in flood-risk levels and inform integrated urban design strategies in cities; (3) the evaluation of characteristics in high flood-risk-level clusters enables city planners and flood risk managers to develop effective flood risk reduction strategies for balancing urban development and growth and flood-risk reduction. Results The binary flood occurrence matrix (𝑩𝑭), measuring whether there is a flood event in grid cell 𝑖 during week 𝑗, is taken as the input data to learn the spatial flood dependence network (𝑨∗) (See Data processing for the construction of 𝑩𝑭.) The flood risk features matrix (𝑭𝑹), which includes flood hazard, flood exposure, and social and physical vulnerabilities information of each grid cell, is constructed (Table 1 See the Data processing section for the construction of 𝑭𝑹) 𝑭𝑹is standardized Fig. 1 | The overall framework of the proposed three-layer urban flood risk-rating model. Fig. 1 | The overall framework of the proposed three-layer urban flood risk-rating model. Each MSA is divided into grid cells of equal size. Areas in each grid cell are assumed to have the same flood-risk level. The binary flood occurrence matrix (𝑩𝑭), measuring whether there is a flood event in grid cell 𝑖 during week 𝑗, is taken as the input data to learn the spatial flood dependence network (𝑨∗) (See Data processing for the construction of 𝑩𝑭.) The flood risk features matrix (𝑭𝑹), which includes flood hazard, flood exposure, and social and physical vulnerabilities information of each grid cell, is constructed (Table 1, See the Data processing section for the construction of 𝑭𝑹,) 𝑭𝑹 is standardized using z-score standardization. The standardized 𝑭𝑹 is treated as the node attributes, and the optimal adjacency matrix of spatial flood dependence network (𝑨∗) is regarded as the graph structure to capture nonlinear interactions among flood risk-related features and to integrate flood spatial dependence relationships. Notations of core parameters used in this model are illustrated in Table S.4 in the Supplementary material. Fig. 1 | The overall framework of the proposed three-layer urban flood risk-rating model. Each MSA is divided into grid cells of equal size. Areas in each grid cell are assumed to have the same flood-risk level. The binary flood occurrence matrix (𝑩𝑭), measuring whether there is a flood event in grid cell 𝑖 during week 𝑗, is taken as the input data to learn the spatial flood dependence network (𝑨∗) (See Data processing for the construction of 𝑩𝑭.) The flood risk features matrix (𝑭𝑹), which includes flood hazard, flood exposure, and social and physical vulnerabilities information of each grid cell, is constructed (Table 1, See the Data processing section for the construction of 𝑭𝑹,) 𝑭𝑹 is standardized using z-score standardization. Results Table 1 Selection of flood risk-related features Flood risk aspects Related features Data source Hazard Flood intensity FEMA flood insurance claim dataset Flood frequency Exposure Population number US Census Bureau dataset Building area National Structure Inventory (NSI) dataset Social vulnerability Poverty rate The CDC/ATSDR Social Vulnerability Index (SVI) dataset Disability rate Limited English rate Physical vulnerability POI number SafeGraph Building age National Structure Inventory (NSI) dataset Reciprocal foundation height In the third layer, flood-risk-level determination, grid cells with similar flood risk are clustered, into the same group ensuring grid cells in different groups have as much difference in flood risk as possible by implementing a deep clustering framework on the learned node embeddings. The aggregated flood-risk value of each cluster is calculated by multiplying values of flood hazard, exposure, and vulnerability of each cluster. These aggregated flood risk values are ranked to obtain the flood-risk level of each cluster. 5 Fig. 1 | The overall framework of the proposed three-layer urban flood risk-rating model. Each MSA is divided into grid cells of equal size. Areas in each grid cell are assumed to have the same flood-risk level. The binary flood occurrence matrix (𝑩𝑭), measuring whether there is a flood event in grid cell 𝑖 during week 𝑗, is taken as the input data to learn the spatial flood dependence network (𝑨∗) (See Data processing for the construction of 𝑩𝑭.) The flood risk features matrix (𝑭𝑹), which includes flood hazard, flood exposure, and social and physical vulnerabilities information of each grid cell, is constructed (Table 1, See the Data processing section for the construction of 𝑭𝑹,) 𝑭𝑹 is standardized using z-score standardization. The standardized 𝑭𝑹 is treated as the node attributes, and the optimal adjacency matrix of spatial flood dependence network (𝑨∗) is regarded as the graph structure to capture nonlinear interactions among flood risk-related features and to integrate flood spatial dependence relationships. Notations of core parameters used in this model are illustrated in Table S.4 in the Supplementary material. Fig. 1 | The overall framework of the proposed three-layer urban flood risk-rating model. Each MSA is divided into grid cells of equal size. Areas in each grid cell are assumed to have the same flood-risk level. Results The standardized 𝑭𝑹 is treated as the node attributes, and the optimal adjacency matrix of spatial flood dependence network (𝑨∗) is regarded as the graph structure to capture nonlinear interactions among flood risk-related features and to integrate flood spatial dependence relationships. Notations of core parameters used in this model are illustrated in Table S.4 in the Supplementary material. We validated the model by calculating cosine similarity between each grid cell’s embedding. We find that inner-cluster similarities are significantly larger than between-cluster similarities. These inner-cluster similarities validate that we successfully clustered grid cells with similar flood-risk levels within the same group and ensure the differences between groups as much as possible. Between-cluster similarities are found to be larger among similar flood- risk level clusters than those among different flood-risk level clusters. For example, similarities between levels 6 and 5 are significantly larger than those of levels 6 and 1. Areas with similar (but not the same) flood-risk levels would show larger similarities, making similar risk level clusters show larger between-cluster similarities. As we rank data samples in the similarity matrix based on the calculated flood-risk level, this between-cluster similarity could validate the calculation of the flood-risk level for each cluster. Otherwise, if our calculation of flood- risk level is not scientific, the between-cluster similarities would not be significantly larger among similar flood-risk level clusters. 6 Fig. 2 | Similarities between each grid cell’s embedding for all five MSAs. Grid cells are listed based on their flood-risk level. Similarities between grid cell’s embeddings in (a) Greater New York, (b)Greater Los Angeles, (c) Greater Chicago, (d) Greater Dallas, and (e) Greater Houston. Revealing six flood-risk levels within each MSA under interactions of flood risk-related features and integration of spatial dependence relationships. FloodRisk-Net yields six flood-risk levels, with level 1 being the lowest flood risk, and level 6, the greatest. (Fig. 3). These outputs provide a spatial profile of flood risk across different areas of cities in a much i bl d i h h i i fl d l i h E h l f i k Fig. 2 | Similarities between each grid cell’s embedding for all five MSAs. Grid cells are listed based on their flood-risk level. Similarities between grid cell’s embeddings in (a) Greater New York, (b)G t L A l ( ) G t Chi (d) G t D ll d ( ) G t H t Fig. Results 2 | Similarities between each grid cell’s embedding for all five MSAs. Grid cells are listed based on their flood-risk level. Similarities between grid cell’s embeddings in (a) Greater New York, (b)Greater Los Angeles, (c) Greater Chicago, (d) Greater Dallas, and (e) Greater Houston. 7 Revealing six flood-risk levels within each MSA under interactions of flood risk-related features and integration of spatial dependence relationships. FloodRisk-Net yields six flood-risk levels, with level 1 being the lowest flood risk, and level 6, the greatest. (Fig. 3). These outputs provide a spatial profile of flood risk across different areas of cities in a much more interpretable way compared with the existing flood plain approaches. Each cluster of risk 7 Revealing six flood-risk levels within each MSA under interactions of flood risk-related features and integration of spatial dependence relationships. FloodRisk-Net yields six flood-risk levels, with level 1 being the lowest flood risk, and level 6, the greatest. (Fig. 3). These outputs provide a spatial profile of flood risk across different areas of cities in a much more interpretable way compared with the existing flood plain approaches. Each cluster of risk 7 levels has distinct feature values related to the extent of hazards, exposure, and physical and social vulnerability enabling the interpretation of each flood-risk level (Fig. 4). For example, the greatest flood-risk level areas in Greater Houston are found to have the highest flood hazard, flood exposure, and suffer from the highest social and physical vulnerabilities. Areas with the highest flood risks are situated in or near the Houston city which is located in flat, low-lying regions with high rainfall and clay-based soils1, characterizing these areas as having the highest flood hazard in the MSA. Furthermore, the largest number of residences and buildings within Greater Houston are located in these areas, resulting in the highest flood exposure. Residents in these areas have the highest poverty rate, the highest limited English speaking, and infrastructures in these areas are the oldest with the lowest foundation height and the largest attraction of people (the largest POI number), which make these areas have the highest social and physical vulnerabilities. 8 and physical vulnerabilities. Fig. 3 | Geographical distribution of flood-risk level in all five MSAs. Geographical distribution of flood-risk levels in (a) Greater New York, (b) Greater Los Angeles, (c) Greater Chicago, (d) Greater Dallas, (e) Greater Houston. Fig. Results 3 | Geographical distribution of flood-risk level in all five MSAs. Geographical distribution of flood-risk levels in (a) Greater New York, (b) Greater Los Angeles, (c) Greater Chicago, (d) Greater Dallas, (e) Greater Houston. 8 Spatial flood dependence plays a key role in the formation and spatial distribution of the six levels of flood risk. Global Moran’s I index is calculated by taking spatial dependence network as spatial weight matrix (see S7 in Supplementary material for its calculation.) The large positive values of Global Moran’s I with the small enough 𝑝-values (Table 2) quantitively illustrate that grid cells with spatial dependence relationships tend to have similar flood-risk levels. This result shows the presence of spatial autocorrelation of areas with similar flood risk levels, which further suggests that flood risk reduction measures in one area would have possible spillover effects and also reduce the flood risk of neighboring areas. Conversely, development patterns in one area could exacerbate flood-risk level in the neighboring areas. Table 2. Results of Global Moran’s I index taking spatial dependence network as weight matrix in all five MSAs MSAs Greater New York Greater Los Angeles Greater Chicago Greater Dallas Greater Houston Moran’s I value 0.792*** 0.763*** 0.830*** 0.708*** 0.930*** ***significant at 0.01 level Table 2. Results of Global Moran’s I index taking spatial dependence network as weight matrix in all five MSAs Hierarchical and uneven spatial distribution of flood risk. Flood risk is distributed hierarchically from the highest to the lowest level, with the highest flood risk areas being typically encompassed by those with the second-highest risk areas, which are further surrounded by areas with the third-highest, and so forth until the boundary of MSA is reached (Fig. 3). This result indicates that the hierarchical development of cities combined with flood hazards would shape a hierarchical flood risk profile in cities. These results also show that the spatial gradient of flood-risk levels is anisotropic in cities, and the rate of change in flood-risk levels is different in different directions moving from the city core. An anisotropic spatial distribution of flood risk suggests that the risk of flooding varies depending on the direction from the city core. This can be due to city topography, land use, and urban sprawl patterns. The overlapping of the flood risk map and the core city boundary map show that the high flood risk disproportionately falls on the core city (Fig. 4). Results The flood risk-related features (except for the disability rate) in the core city are larger than those in other areas in the MSA (Fig. 5). This means that the core city in each MSA suffers from greater flood hazards, and more people and infrastructure are exposed to flood hazards with a larger proportion being socially or physically vulnerable. This result signifies the relationship between urban sprawl and development patterns that shape hierarchical structure and the flood risk profile of cities. In a city such as Houston without land use plans and policies, the random development and growth patterns are likely to exacerbate situations in high-flood-risk areas and lead to the cascade of flood-risk levels to the surrounding areas, thus increasing the flood-risk level of areas outside the city core. 9 Fig. 4 | Overlapping the boundary of each MSA’s core city and the flood risk map. Core cities for these five MSAs are (a) New York city, (b) Los Angeles city, (c) Chicago city, (d) Dallas city, and (e) Houston city. Fig. 4 | Overlapping the boundary of each MSA’s core city and the flood risk map. Core cities for these five MSAs are (a) New York city, (b) Los Angeles city, (c) Chicago city, (d) Dallas city, and (e) Houston city. 10 Fig. 5 | Characteristics of feature distribution in core city and other areas in Greater Houston. The other four MSAs are shown in Supplementary Figs. 5–8. Fig. 5 | Characteristics of feature distribution in core city and other areas in Greater Houston. The other four MSAs are shown in Supplementary Figs. 5–8. In each metropolitan area, cities with a population larger than 25,000 are further selected to examine flood risk distribution within the city boundary. The average flood-risk level and spatial inequality of each selected city are calculated (see S8 in Supplementary materials for calculations.) The large negative person correlation between average flood risk and spatial inequality of selected cities combined with small enough 𝑝−𝑣𝑎𝑙𝑢𝑒𝑠 means that cities with higher average flood-risk levels tend to have smaller spatial inequality (Fig. 6). This indicates that less significant spatial variations of flood risk distribution could be found in cities with higher flood-risk levels. As shown in Figure S.13, some cities are found to have high flood-risk levels while also having low spatial inequality (with the threshold of average flood risk ≥5 and spatial inequality ≤0 .1). Results Such high average flood-risk levels and small spatial inequality should be noted because it indicates that there are few low-risk areas in this city for further urban growth and development. In fact, the spatial inequality of flood-risk levels could be an indicator of the extent to which urban growth and development could be made without exacerbating the overall flood risk of cities. Due to low spatial inequality, further urban development would exacerbate the overall flood risk of the cities since there are limited available low-flood-risk areas. In cities with high risk levels and low spatial inequality, low impact development strategies are essential for balancing urban growth and flood risk reduction strategies. 11 Fig. 6 | Relation between average flood risk and spatial inequality of cities within Greater Houston. The other four MSAs are shown in Supplementary Figures 9–12. The person correlation and p-values for average flood risk and spatial inequality of cities in Greater New York, Greater Los Angeles, Greater Chicago, Greater Dallas, and Greater Houston are (−0.461, 6.404 × 10−5), (−0.507, 2.861 × 10−8), (-0.693, 1.454× 10−12), (-0.653, 6.426× 10−7), and (-0.772, 4.421× 10−5), respectively. Fig. 6 | Relation between average flood risk and spatial inequality of cities within Greater Houston. The other four MSAs are shown in Supplementary Figures 9–12. The person correlation and p-values for average flood risk and spatial inequality of cities in Greater New York, Greater Los Angeles, Greater Chicago, Greater Dallas, and Greater Houston are (−0.461, 6.404 × 10−5), (−0.507, 2.861 × 10−8), (-0.693, 1.454× 10−12), (-0.653, 6.426× 10−7), and (-0.772, 4.421× 10−5), respectively. Fig. 6 | Relation between average flood risk and spatial inequality of cities within Greater Three archetypes shaping the highest flood-risk level. The underlying determinants shaping Three archetypes shaping the highest flood-risk level. The underlying determinants shaping the highest flood-risk levels vary across cities, but fall into three archetypes, discussed below. In Greater Houston, the highest flood-risk level areas fall into the first archetype: high flood hazards, high exposure, and high social and physical vulnerabilities (Fig. 7). In these highest flood risk areas, the largest flood hazard combined with the largest flood exposures cause significant direct impacts (e.g., damage to infrastructure, loss of life). The impact on vulnerable populations and infrastructure is exacerbated due to their limited ability to cope with, resist and recover from adverse impacts caused by floods24. Results The highest flood risk areas in Greater New York and Dallas are similar to those of Greater Houston (Fig.S4). The highest flood risk areas in Greater Chicago fall into the second archetype (Fig. S2) indicating that even if flood events in densely developed areas are neither intense nor frequent, they would still cause great risks due to the large number of people and assets exposed and their limited abilities to cope with, resist, and recover from flood damage. The highest flood risk areas in Greater Los Angeles fall into the third archetype (Fig S3), in which the largest flood hazards combined with the medium flood exposure and vulnerabilities result in the highest flood risk. These results show the importance of city-specific rating of flood risks since the areas with highest flood-risk levels could have different determinants in different cities. For example, if flood-risk levels in other cities are evaluated based on feature values in high flood- risk levels in Greater Houston, their flood-risk levels would be mischaracterized and underestimated. 12 Fig.7 | Characteristics of clusters in Greater Houston. Flood risk comprised the characteristics of (a) flood frequency, (b) flood intensity, (c) poverty rate, (d) POI number, (e) population number, (f) building area, (g) disability rate, (h) building age, (I) the percentage of limited English speaking, (j) reciprocal foundation height. (The other four MSAs are shown in Supplementary Figures 1–4, respectively.) Fig.7 | Characteristics of clusters in Greater Houston. Flood risk comprised the characteristics of (a) flood frequency, (b) flood intensity, (c) poverty rate, (d) POI number, (e) population number, (f) building area, (g) disability rate, (h) building age, (I) the percentage of limited English speaking, (j) reciprocal foundation height. (The other four MSAs are shown in Supplementary Figures 1–4, respectively.) Suitable flood risk reduction strategies (summarized in Table S3) are examined based on underlying determinants shaping the highest flood-risk levels. Considering the high flood hazards in Level 6 areas, comprehensive stormwater management strategies are necessary to collect, redirect, or hasten the flow of water to reduce flood hazards. Strategies to improve stormwater management include widening ditches, upgrading drainage systems, implementing green infrastructure, and constructing retention ponds. The high flood exposure also calls for the land use planning and policy to limit urban development in high flood-prone areas, or the government’s buyout of flood-prone areas. These strategies are important to control the increased exposure in high flood-hazard area. Discussion In this study, we presented an integrated three-layer graph deep learning-based model, FloodRisk-Net, for examining the emergent flood risk profiles of cities. FloodRisk-Net includes three components: 1) spatial flood dependence relationships unveiling within urban areas, 2) node representation learning, and 3) flood-risk-level determination. Using FloodRisk-Net, city- specific flood risk ratings are obtained by mining entangled relationships among heterogenous features related to urban development and flood hazards and the consideration of spatial flood dependence relationships. This study’s findings provide multiple important contributions to the characterization and analysis of urban flood risk rating. First, departing from the current approaches with binary characterization of flood risk (based on flood-plain maps), the results of this study reveal six distinct city-specific levels of flood risk to evaluate the flood-risk profile of cities. Second, the characterization of flood-risk levels obtained from FloodRisk-Net capture the complex nonlinear interactions about heterogenous flood hazard and urban development features, as well as spatial dependence of areas, that lead to the emergence of flood risk profiles of cities. This characterization enables a system-of-systems approach to urban flood risk reduction by applying a holistic and integrative strategy that considers the interdependencies and interactions among various components of an urban environment. It acknowledges that urban flood risk reduction is not just about managing water flows; it also involves land use planning, infrastructure design, and the ramifications of development patterns. By understanding and addressing the complex and non-linear connections among these components, cities can build more resilient and adaptive systems to mitigate flood risks. Third, a major burden in planning and implementation of flood risk reduction measures is lack of updated information about flood risk in different areas of cities. In the United States, most floodplain maps are outdated. Significant cost and effort are necessary to produce updated flood hazard maps based on physics-based hydraulic and hydrological models. FloodRisk-Net, however, can be implemented conveniently every few years based on updated information about urban development patterns to yield updated flood risk profiles for cities. Fourth, the interpretation of feature values within each flood-risk level, as well as patterns of spatial inequality of flood-risk levels among different urban areas, inform about suitable strategies for balancing urban development and flood risk reduction. Results Green infrastructure, such as rain gardens and green roofs, can help to absorb and manage stormwater runoff. In these areas, these strategies can be incorporated into the design of new construction and into the retrofitting of existing buildings. The social vulnerability in these areas is very high with a large percentage in poverty and with limited English proficiency. Financial support should be made available to subsidize flood insurance premiums for poor residents and to assist in the rebuilding of flood-damaged homes or to migrate to less flood-prone areas after floods. Flood information should be translated into languages familiar to the populations to help those with limited English skills become familiar with local flood risks. The physical vulnerability in these areas is also very high, with a large number of POIs, aging buildings, and low foundation heights. Engineering solutions, such as elevating buildings above flood levels, installing flood barriers, and retrofitting buildings, are thus crucial for reinforcing buildings in these areas. Given the large number of POIs in these areas, it is a high priority to identify and protect the most essential infrastructure, such as hospitals, schools, and emergency services, against flood risks. Also, future developments should focus on decentralizing facility distribution to areas with less flood exposure. Urban planning policies that prioritize the allocation of certain essential POIs to low- risk areas are also meaningful for ensuring the continued provision of essential services to the public. 13 Methods Data processing. For each MSA, using FEMA historical flood claim dataset, a binary flood occurrence matrix (𝑩𝑭∈𝑅𝑚∗𝑑𝐵𝐹, 𝑚 being the number of grid cells in each MSA and 𝑑𝐵𝐹, the number weeks during study period) is constructed indicating whether there is a flood event in grid cell 𝑖 during week 𝑗36. Details of the construction of BF are described in S1 in Supplementary Notes. pp y Ten flood risk-related features, selected from aspects of flood hazard, exposure, and vulnerability following de Moel et al37., UNISDR38, and Wing et al21.’s work, shown in Table 1, are adopted to construct the flood risk feature matrix (𝑭𝑹∈𝑅𝑚∗10). These flood risk-related features are in different scales ranging from census-tract level to highly-detailed geographic coordinates at the resolution of seven decimals. The study area is divided into grid cells of equal size. Flood risk-related features are aggregated into the grid-cell level. The resolution of the grid cell is set to be 2 km ×2 km considering both the computation cost and the need for proper scale of analysis related to urban flood risk profiles. Each column in FR represents a feature and every row records flood risk features for a grid cell. Flood frequency and intensity measure flood hazard using the FEMA historical flood claim dataset36. Population and building area, representing the valuated social components (e.g., people and buildings) exposed to flood hazards, are adopted to measure flood exposure using Census Bureau dataset39 and National Structure Inventory (NSI)40 dataset, respectively. Flood vulnerability is measured from physical and social aspects. Poverty rate, disability rate, and limited percentage of English-speaking persons are selected to measure social vulnerability using Centers for Disease Control/Agency for Toxic Substances and Disease Registry Social Vulnerability Index (SVI) dataset41. POI number, building age, and the foundation height of the building are used to measure physical vulnerability using SafeGraph42 and National Structure Inventory40 dataset. Reasons for selecting these features to measure flood risk and details of the construction of FR are described in S2 in Supplementary material. Graph structure learner. We obtain node embedding (E) from the input data 𝑩𝑭 through an embedding network and model the network as the pair-wise similarity between node embeddings as shown in Equation 1. We try to learn optimized graph structures by calculating pair-wise similarities between node embeddings instead of relying on raw node features. Discussion For example, areas whose high flood-risk levels are due to high flood hazard and high physical and social vulnerability could be targets for flood retreat, strategies such as buyouts, zoning policies to restrict or discourage new development in flood-prone areas, and easements. Areas with high flood hazards, high physical vulnerability, and low social vulnerability can be targets for flood infrastructure improvements and flood-proofing requirements (e.g., raising building elevation). Low-impact development strategies are essential for further development in areas with levels 5 and 6 flood risk. While areas with lower flood-risk levels are possible candidates for further development, the spatial autocorrelation of flood risk found in this study suggests that development in areas with lower flood-risk levels would have spillover effect and increase the flood risk of other areas. FloodRisk-Net establishes a new paradigm for understanding emergent flood risk in cities based on urban big data and machine intelligence. It has the potential to be applied to other concepts, such as resilience and reliability through selecting suitable measurement variables and modeling their complex and non-linear interactions. One unique aspect of FloodRisk-Net is the capture of complex interactions among heterogeneous flood risk-related variables, such as flood hazards, exposure, and social and physical vulnerabilities, into latent representations through unsupervised deep graph neural network. Therefore, the method is flexible to change the input variables of the according to the measured concepts. Machine intelligence enables the use of urban big data to identify the complex interplay between urban features and flood hazards for better understanding and analysis of climate risk and resilience in cities. Developments in the urban big data arena will make the acquisition of large-scale high-resolution urban big data faster and more affordable in the future, thus providing greater potential for our data-driven and machine intelligence-based flood risk rating models by adding additional features based on better and more recent datasets. For example, if data related to future flood exposure under 14 climate change is available or if new flood events occur, the new data could be conveniently added as input features for enhanced characterization of flood risk profiles in cities over time. 15 15 Methods The graph structure generated by the graph learner is treated as learned graph view (𝑮𝐿= (𝑨̃∗, 𝑩𝑭)), and the graph structure generated by 𝐾𝑁𝑁 is treated as KNN graph view (𝑮𝐾= (𝑲, 𝑩𝑭)). (K is KNN graph structure, see S3 in Supplementary Notes for details of its construction.) These two views are regarded as being generated from the hidden actual graph structure. Two frequently used data augmentation methods are adopted—attribute masking and edge perturbation—to corrupt these two views. Details of the data augmentation are described in Section S4 in the Supplementary material. 𝑩𝑭 ̅̅̅̅, 𝑮𝐾 ̅̅̅̅, and 𝑮𝐿 ̅̅̅̅ are the augmented binary flood occurrence matrix, 𝐾𝑁𝑁 graph view, and learned graph view, respectively. A node-level contrastive learning framework applied to these two augmented graph views (𝑮𝐾 ̅̅̅̅ and 𝑮𝐿 ̅̅̅̅) to train unknown parameters in the graph learner. A neural network-based encoder (𝑓𝜃(∙)) is applied for two augmented graph views (𝑮𝐾 ̅̅̅̅ and 𝑮𝐿 ̅̅̅̅) to obtain representation: g p ( 𝐾 𝐿) 𝑯𝐾 = 𝑓𝜃(𝑮𝐾 ̅̅̅̅), 𝑯𝐿 = 𝑓𝜃(𝑮𝐿 ̅̅̅̅) g p ( 𝐾 𝐿) p 𝑯𝐾 = 𝑓𝜃(𝑮𝐾 ̅̅̅̅), 𝑯𝐿 = 𝑓𝜃(𝑮𝐿 ̅̅̅̅) (4) 𝑑 (4) where 𝜃 is the parameter of the encoder; it is the same for the two views. 𝑯𝐾 and 𝑯𝐿∈𝑅𝑚∗𝑑1 ( 𝑑1 is the encoded representation dimension) are the encoded node representations for augmented 𝐾𝑁𝑁 graph and learned graph views, respectively. We choose GCN as encoder network48. where 𝜃 is the parameter of the encoder; it is the same for the two views. 𝑯𝐾 and 𝑯𝐿∈𝑅𝑚∗𝑑1 ( 𝑑1 is the encoded representation dimension) are the encoded node representations for augmented 𝐾𝑁𝑁 graph and learned graph views, respectively. We choose GCN as encoder network48. An MLP-based projector (𝑔𝜉(∙)) is further applied on the encoded node representations to map them to the space where contrastive loss is applied. pp 𝒁𝐾 = 𝑔𝜉(𝑯𝐾) , 𝒁𝐿 = 𝑔𝜉(𝑯𝐿) (5) (5) 𝜉 𝜉 where 𝜉 is the parameter of the projector 𝑔𝜉(∙); it is the same for both views. 𝒁𝐾 and 𝒁𝐿∈ 𝑅𝑚∗𝑑2 (𝑑2 is the projected representation dimension) are the projected node representations for augmented 𝐾𝑁𝑁 graph and learned graph views, respectively. A symmetric normalized temperature-scaled cross-entropy loss (𝑁𝑇−𝑋𝑒𝑛𝑡)46,49,50 is adopted as the contrastive loss function. 𝑁𝑇−𝑋𝑒𝑛𝑡 aims to maximize the mutual information between 𝑧𝑘,𝑖 and 𝑧𝑙,𝑖 of node 𝑣𝑖 on two views. Methods Embeddings of node features could provide useful information for learning better graph structures, owing to parameters of node embedding learning could highlight different dimensions of raw node features43. 𝑨̃ = 𝜙(ℎ𝑤(𝑩𝑭)) = 𝜙(𝑬) (1) 𝑨̃ = 𝜙(ℎ𝑤(𝑩𝑭)) = 𝜙(𝑬) (1) where, ℎ𝑤(∙) is the embedding network which adopts neural network to conduct node embedding with the parameter 𝑤, 𝜙(∙) is cosine similarity function to calculate pairwise similarity, 𝑬∈𝑅𝑚∗𝑑𝐵𝐹 is the node embedding with respect to flood occurrence, and 𝑨̃ ∈𝑅𝑚∗𝑚 is the learned adjacency matrix of the spatial flood dependence graph with unknown parameters. where, ℎ𝑤(∙) is the embedding network which adopts neural network to conduct node embedding with the parameter 𝑤, 𝜙(∙) is cosine similarity function to calculate pairwise similarity, 𝑬∈𝑅𝑚∗𝑑𝐵𝐹 is the node embedding with respect to flood occurrence, and 𝑨̃ ∈𝑅𝑚∗𝑚 is the learned adjacency matrix of the spatial flood dependence graph with unknown parameters. Multi-Layer Perceptron (MLP)44 is chosen as embedding network because it could consider “the correlation and combination of input features,” which helps “generate more informative embeddings for similarity metric learning.”45 A single embedding layer can be written as: 𝑬(𝑙) = ℎ(𝑤) (𝑙) (𝑬(𝑙−1)) = 𝜎(𝑬(𝑙−1)𝜴(𝑙)) (2) (2) where, 𝑬(𝑙) ∈𝑅𝑚∗𝑑𝐵𝐹 is the output matrix of the 𝑙-th layer of the embedding model, 𝜴(𝑙) ∈ 𝑅𝑑𝐵𝐹∗𝑑𝐵𝐹 is the learnable parameter matrix of the 𝑙-th layer, 𝜎(∙) is a non-linear function to make the training more stable. The input in the first layer of 𝑬(0) is 𝑩𝑭, and the output of the last layer 𝑬(𝐿𝑒𝑛) is the obtained node embedding matrix (𝐿𝑒𝑛 is the number of layers of the graph leaner embedding network). The 𝐿2 normalization function is adopted as 𝜎(∙) to make training more stable, and the activation function of each layer in MLP is set as 𝑅𝑒𝐿𝑈 function. A i dj i 𝑨̃∗i f h d f 𝑨̃ h l i A non-negative adjacency matric 𝑨̃∗ is further extracted from 𝑨̃ to ensure the value in the matrix ranges from [0,1] as shown in Equation 3. 𝑨̃∗ = 𝑝𝑎𝑐𝑡(𝑨̃) = 𝑅𝑒𝐿𝑢(𝑨̃) (3) 𝑨̃∗ = 𝑝𝑎𝑐𝑡(𝑨̃) = 𝑅𝑒𝐿𝑢(𝑨̃) (3) 16 Multi-view graph contrastive learning module. A multi-view graph contrastive learning framework (Fig. 1 Layer 1) is developed, with experience from SUBLIME,45 SimCLR,46 and GRACE,47 to acquire supervision signal from data itself for training unknown parameters of graph structure learner. Two graph views are constructed to contrast. Methods 𝐿𝐺 = 1 2𝑛∑[𝑙(𝑧𝑘,𝑖, 𝑧𝑙,𝑖) + 𝑙(𝑧𝑙,𝑖, 𝑧𝑘,𝑖)] 𝑛 𝑖=1 (6) (6) 𝑙(𝑧𝑘,𝑖, 𝑧𝑙,𝑖) = −𝑙𝑜𝑔 𝑒𝜙(𝑧𝑘,𝑖,𝑧𝑙,𝑖)/𝑡 ∑ 1[𝑖≠𝑗]𝑒𝜙(𝑧𝑘,𝑖,𝑧𝑙,𝑗)/𝑡 𝑛 𝑖=1 (7) (7) where 𝑡 is the temperature parameter, 𝑙(𝑧𝑘,𝑖, 𝑧𝑙,𝑖) is the loss function for a positive sample (𝑧𝑘,𝑖, 𝑧𝑙,𝑖), and 1[𝑖≠𝑗] ∈{0,1} is an indicator function that equals to 1 iff 𝑖≠𝑗. The loss function for the positive sample (𝑧𝑙,𝑖, 𝑧𝑘,𝑖) is calculated similarly as 𝑙(𝑧𝑘,𝑖, 𝑧𝑙,𝑖). where 𝑡 is the temperature parameter, 𝑙(𝑧𝑘,𝑖, 𝑧𝑙,𝑖) is the loss function for a positive sample (𝑧𝑘,𝑖, 𝑧𝑙,𝑖), and 1[𝑖≠𝑗] ∈{0,1} is an indicator function that equals to 1 iff 𝑖≠𝑗. The loss function for the positive sample (𝑧𝑙,𝑖, 𝑧𝑘,𝑖) is calculated similarly as 𝑙(𝑧𝑘,𝑖, 𝑧𝑙,𝑖). The final loss 𝐿𝐺 is computed as the average value across all positive pairs, and it is minimized to maximize the agreement between two views. The final optimal adjacency matrix of spatial flood dependence network 𝐴∗ is obtained after minimizing 𝐿𝐺. Bootstrapping. To prevent the learned graph view from inheriting error information from and over-fitting the 𝐾𝑁𝑁 graph view, a bootstrapping mechanism45 is applied to the adjacency matrix of the 𝐾𝑁𝑁 graph view as follows: 𝐾←𝜏𝐾+ (1 −𝜏)𝑨̃∗ (8) (8) where 𝜏∈[0,1] is a coefficient controlling the update of 𝐾. The adjacency matrix of the 𝐾𝑁𝑁 graph view will be updated by the adjacency matrix of the learned graph view in a slow-moving step every 10 iterations instead of keeping unchanged. where 𝜏∈[0,1] is a coefficient controlling the update of 𝐾. The adjacency matrix of the 𝐾𝑁𝑁 graph view will be updated by the adjacency matrix of the learned graph view in a slow-moving step every 10 iterations instead of keeping unchanged. Incorporate spatial flood dependence relationships. Flood frequency in one grid cell will be directly impacted by other grid cells that have spatial flood dependence with it33. Hence, the ncorporate spatial flood dependence relationships. Flood frequency in one grid cell will be directly impacted by other grid cells that have spatial flood dependence with it33. Hence, the 17 flood exposure and flood vulnerability in this grid cell will be indirectly impacted by these spatially dependent grid cells. This role of spatial flood dependence relationship in flood risk rating is incorporated in node representation learning by aggregating node attributes (𝑭𝑹) from neighbors on the spatial flood dependence network (𝑨∗) using GCN. The representation learned by the 𝑙−𝑡ℎ layer of GCN, 𝒁(𝑙), can be expressed by the following convolutional operation: 𝒁(𝑙) = 𝑅𝑒𝐿𝑢(𝑫̃−1 2𝑨∗ ̅̅̅𝑫̃−1 2𝒁(𝑙−1)𝑾(𝑙−1)) (9) 𝒁(𝑙) = 𝑅𝑒𝐿𝑢(𝑫̃−1 2𝑨∗ ̅̅̅𝑫̃−1 2𝒁(𝑙−1)𝑾(𝑙−1)) (9) where, 𝑨∗ ̅̅̅ = 𝑨∗+ 𝑰 and 𝑫𝑖𝑖 ̃ = ∑𝑨∗ ̅̅̅𝑖𝑗 𝑗 . 𝑰 is the identity diagonal matrix of the adjacency matrix 𝑨∗. 𝑾∈𝑅𝑑𝑖𝑛∗𝑑𝑜𝑢𝑡 is the learnable parameter matrix. 𝒁(0) is 𝑭𝑹 and 𝒁(𝐿𝐺𝐸) is the output data. where, 𝑨∗ ̅̅̅ = 𝑨∗+ 𝑰 and 𝑫𝑖𝑖 ̃ = ∑𝑨∗ ̅̅̅𝑖𝑗 𝑗 . 𝑰 is the identity diagonal matrix of the adjacency matrix 𝑨∗. 𝑾∈𝑅𝑑𝑖𝑛∗𝑑𝑜𝑢𝑡 is the learnable parameter matrix. 𝒁(0) is 𝑭𝑹 and 𝒁(𝐿𝐺𝐸) is the output data. Capture complex interactions among features. An autoencoder framework is adopted, where DNN is adopted as the encoding network to capture complex and nonlinear interactions among individual flood risk-related features. In the encoder part, node representation learned by the 𝑙−𝑡ℎ layer of DNN, 𝐻(𝑙), can be expressed as follows: 𝑯(𝑙) = 𝑅𝑒𝐿𝑢(𝑾𝑒 (𝑙)𝑯(𝑙−1) + 𝒃𝑒 (𝑙)) (10) ( ) (10) where 𝑾𝑒 (𝑙) and 𝒃𝑒 (𝑙) are the weight matrix and bias of the 𝑙-th layer in the encoding part. 𝑯(0) is the input data and 𝑯(𝐿𝐺𝐸) is the output data. The decoder aims to reconstruct the input data through several fully connected layers as follows: 𝑯𝒅 (𝑙) = 𝑅𝑒𝐿𝑢(𝑾𝑑 (𝑙)𝑯(𝑙−1) + 𝒃𝑑 (𝑙)) (11) (11) where 𝑾𝑑 (𝑙) and 𝒃𝑑 (𝑙) are the weight matrix and bias of the 𝑙-th layer in the encoding part respectively. The autoencoder aims to reconstruct the input data without missing any information, which leads to the following reconstruction loss function: 𝐿𝑟𝑒𝑠= 1 2𝑁∑|| 𝑁 𝑖=1 ℎ𝑖− ℎ̂𝑖||2 2 (12) (12) where ℎ𝑖 is the 𝑖 -the row data of input data 𝑭𝑹 and ℎ̂ is the 𝑖-th row data of 𝑯𝒅 (𝐿). Node representation integration. The most likely cluster 𝑘 that instance 𝑖 belongs (𝑠𝑖) to is. 𝑍= 𝑠𝑜𝑓𝑡𝑚𝑎𝑥 (𝑫̃−1 2𝑨∗ ̅̅̅𝑫̃−1 2𝒁(𝐿𝐺𝐸)𝑾(𝐿𝐺𝐸)) (15) Incorporate spatial flood dependence relationships. Flood frequency in one grid cell will be directly impacted by other grid cells that have spatial flood dependence with it33. Hence, the Node representation 𝑯(𝑙−1)learned from DNN preserves the latent information of complex and nonlinear interactions among individual risk-related features for each grid cell. Node representation 𝒁(𝑙−1) incorporates spatial flood dependence relationships among different grid cells. We thus try to combine these two node representations: 𝒁̃(𝑙−1) = (1 −𝜖)𝑯(𝑙−1) + 𝜖𝒁(𝑙−1) (13) ( ) where 𝜖 is the balance coefficient, we set 𝜖 as 0.5 to obtain an informative representation51. ̃(𝑙1) (𝑙) where 𝜖 is the balance coefficient, we set 𝜖 as 0.5 to obtain an informative representation51. Then 𝒁̃(𝑙−1) is adopted as the input of the 𝑙-layer in GCN to learn the representation 𝒁(𝑙): where 𝜖 is the balance coefficient, we set 𝜖 as 0.5 to obtain an informative representation51. Then, 𝒁̃(𝑙−1) is adopted as the input of the 𝑙-layer in GCN to learn the representation 𝒁(𝑙): 1 1 Then, 𝒁̃(𝑙−1) is adopted as the input of the 𝑙-layer in GCN to learn the representation 𝒁(𝑙) 𝒁(𝑙) = 𝑅𝑒𝐿𝑢(𝑫̃−1 2𝑨∗ ̅̅̅𝑫̃−1 2𝒁̃(𝑙−1)𝑾(𝑙−1)) (14) (𝑙 ) (14) The representation 𝑯(𝑙−1) learned from each DNN layer is integrated with the representation learned from the corresponding GCN layer using equation (13) as shown in layer 2 in Figure 1. 𝒁(𝐿𝐺𝐸) is the output node representation, where 𝐿𝐺𝐸 is the layer number of GCN or encoding network of DNN. Soft Assignment. Grid cells with similar flood risk are clustered into the same group based on node embeddings 𝒁(𝐿𝐺𝐸), as 𝒁(𝐿𝐺𝐸) capture both nonlinear interactions among flood risk- related features and integrate spatial flood dependence relationships. A SoftMax function is implemented on the output of the GCN module following the work of Bo et al51: 1 1 𝑍= 𝑠𝑜𝑓𝑡𝑚𝑎𝑥 (𝑫̃−1 2𝑨∗ ̅̅̅𝑫̃−1 2𝒁(𝐿𝐺𝐸)𝑾(𝐿𝐺𝐸)) (15) (15) 𝑍= 𝑠𝑜𝑓𝑡𝑚𝑎𝑥 (𝑫̃−1 2𝑨∗ ̅̅̅𝑫̃−1 2𝒁(𝐿𝐺𝐸)𝑾(𝐿𝐺𝐸) The most likely cluster 𝑘 that instance 𝑖 belongs (𝑠𝑖) to is. The most likely cluster 𝑘 that instance 𝑖 belongs (𝑠𝑖) to is. 18 𝑠𝑖= argmax 𝑘 (𝑧𝑖𝑘) (16) (16) where 𝑧𝑖𝑘∈𝑍 is the probability that sample 𝑖 belongs to the cluster 𝑘 and 𝑠𝑖 is the clustering that grid cell 𝑖 belongs to. “Self-training” strategy. A dual self-training strategy51 is adopted to train the clustering module. The KL-divergence loss between cluster assignment distribution 𝑧𝑖𝑘 and the target distribution 𝑝𝑖𝑘 is minimized to obtain the optimal cluster assignment52 as follows: 𝐿𝑐𝑙𝑢= 𝐾𝐿(𝑃||𝑍) = ∑∑𝑝𝑖𝑘𝑙𝑜𝑔𝑝𝑖𝑘 𝑧𝑖𝑘 𝑘 𝑖 (17) (17) where 𝑃 is the target distribution. Incorporate spatial flood dependence relationships. Flood frequency in one grid cell will be directly impacted by other grid cells that have spatial flood dependence with it33. Hence, the The target distribution 𝑃 works as the “ground-truth labels” in the training process53. Reasons for its eligibility for being selected as the training signal are summarized in Section S5 in Supplementary Notes and it is obtained as follows: where 𝑃 is the target distribution. The target distribution 𝑃 works as the “ground-truth labels” in the training process53. Reasons for its eligibility for being selected as the training signal are summarized in Section S5 in Supplementary Notes and it is obtained as follows: 1. The student’s 𝑡-distribution is firstly used to calculate the ancillary cluster assignmen distribution 𝑄 based on node embedding 𝑯(𝐿𝐺𝐸), following Maaten and Hinton’s54 work: g g 𝑞𝑖𝑘= (1 + ||ℎ𝑖−𝑢𝑘 ||2 2 2 ⁄ ) −(𝛼+1) 2 ∑(1 + ||ℎ𝑖−𝑢𝑗 ||2 2 2 ⁄ ) −(𝛼+1) 2 𝐾 𝑗 (18) (18) where, 𝑞𝑖𝑘 is the ancillary cluster assignment distribution that measures the probability of sample 𝑖 assigned to the cluster 𝑘, ℎ𝑖 is the i-th row of node embedding 𝑯(𝐿𝐺𝐸), 𝑢𝑘 is the cluster center which is initialized by 𝐾-means on node embedding 𝑯(𝐿𝐺𝐸) and 𝛼 is the freedom degree of the student’s 𝑡-distribution, and we set 𝛼= 1 following Xie et al52’s work. 2. The target distribution 𝑃 is set as the variant of the ancillary cluster assignment 𝑄 following Xie et al52’s work. It is computed by firstly raising 𝑞𝑖 to the second power and then normalizing it based on the frequency as the following: 𝑝𝑖𝑘= 𝑞𝑖𝑘2 𝑓𝑘 ⁄ ∑(𝑞𝑖𝑗2/𝑓𝑗) 𝑗 (19) (19) where, 𝑓𝑘= ∑𝑞𝑖𝑘 𝑖 is the soft cluster frequencies and can be viewed as “the sum of the probability that instance 𝑖 belongs to the 𝑘-th cluster”55. To obtain the initial target distribution 𝑃, the DNN module is first pre-trained to obtain an initial node embedding 𝑯(𝐿𝐺𝐸) by minimizing 𝐿𝑟𝑒𝑠, as shown in Equation (12). 𝐾-means clustering is conducted once to obtain the initial clustering centers 𝑢 on the node embedding 𝑯(𝐿𝐺𝐸) before training the whole clustering model. The initial target distribution 𝑃 is calculated using Equation (19) after ancillary distribution 𝑄 is calculated using Equation (18). Incorporate spatial flood dependence relationships. Flood frequency in one grid cell will be directly impacted by other grid cells that have spatial flood dependence with it33. Hence, the Then, in the main training process, clustering centers 𝑢 and node embedding ℎ are updated based on gradients of 𝐿𝑡𝑎 with respect to 𝑢 and ℎ, which could help node embedding closer to the clustering center 51–53: 𝐿𝑡𝑎= 𝐾𝐿(𝑃||𝑄) = ∑∑𝑝𝑖𝑘𝑙𝑜𝑔𝑝𝑖𝑘 𝑞𝑖𝑘 𝑘 𝑖 (20) (20) 𝑘 𝑖 Where, 𝐿𝑡𝑎 is the KL-divergence loss between ancillary cluster assignment distribution 𝑞𝑖𝑘 and the target distribution 𝑝𝑖𝑘. Distribution 𝑄 is updated in each iteration using Equation (18) with the updated ℎ and 𝑢. Target distribution 𝑃 is updated every 5 iterations with the updated 𝑄 to keep training process stable using Equation (19) following Wang et al.53’s work. 𝑘 𝑖 Where, 𝐿𝑡𝑎 is the KL-divergence loss between ancillary cluster assignment distribution 𝑞𝑖𝑘 and the target distribution 𝑝𝑖𝑘. Distribution 𝑄 is updated in each iteration using Equation (18) with the updated ℎ and 𝑢. Target distribution 𝑃 is updated every 5 iterations with the updated 𝑄 to keep training process stable using Equation (19) following Wang et al.53’s work. p g p g q g g The overall loss function is defined as follows: function is defined as follows: The overall loss function is defined as follows: 𝐿= 𝐿𝑟𝑒𝑠+ 𝛼𝐿𝑐𝑙𝑢+ 𝛽𝐿𝑡𝑎 b l h i i f h 𝐿= 𝐿𝑟𝑒𝑠+ 𝛼𝐿𝑐𝑙𝑢+ 𝛽𝐿𝑡𝑎 (21) b l h i i f h d l d h d f (21) where 𝛼 and 𝛽 are coefficients to balance the training of the GCN module and the update of 𝑯(𝐿𝐺𝐸) between reconstruction loss, respectively. Clustering results are obtained using Equation (16) after training until the maximum epochs. where 𝛼 and 𝛽 are coefficients to balance the training of the GCN module and the update of 𝑯(𝐿𝐺𝐸) between reconstruction loss, respectively. Clustering results are obtained using Equation (16) after training until the maximum epochs. Flood-risk-level calculation. Grid cells in the same group are assumed to have the same flood 19 risk level as the clustering module has clustered grid cells with similar flood risk into the same group and ensures that grid cells in different clusters are different from each other as much as possible. The flood-risk level of each cluster is calculated as the following steps: risk level as the clustering module has clustered grid cells with similar flood risk into the same group and ensures that grid cells in different clusters are different from each other as much as possible. Incorporate spatial flood dependence relationships. Flood frequency in one grid cell will be directly impacted by other grid cells that have spatial flood dependence with it33. Hence, the The flood-risk level of each cluster is calculated as the following steps: risk level as the clustering module has clustered grid cells with similar flood risk into the same group and ensures that grid cells in different clusters are different from each other as much as possible. The flood-risk level of each cluster is calculated as the following steps: p g p 1. Following Mojaddadi et al.56, Siam et al.57, and Xu et al.58,’s work, flood risk-related features are normalized to reduce the impact of the difference of unit using Min-Max scaler as the following equation: p g p 1. Following Mojaddadi et al.56, Siam et al.57, and Xu et al.58,’s work, flood risk-related features are normalized to reduce the impact of the difference of unit using Min-Max scaler as the following equation: 𝑥′ = 𝑥−𝑥𝑚𝑖𝑛 𝑥𝑚𝑎𝑥− 𝑥𝑚𝑖𝑛 (22) (22) 𝑚𝑎𝑥 𝑚𝑖𝑛 Where, 𝑥′ is the scaled value which is in the range of [0,1]. 2. Calculate each flood risk-related feature’s mean values for each cluster: 2. Calculate each flood risk-related feature’s mean values for each cluster 𝑭𝑖𝑘𝑢 ̅̅̅̅̅̅ = ∑ 𝐹𝑖𝑘𝑢_𝑙 𝑛 𝑙=1 𝑛 (23) (23) 𝑛 where, 𝑭𝑖𝑘𝑢 ̅̅̅̅̅̅ is the average value for feature 𝑖 in cluster 𝑘 in MSA 𝑢, and 𝐹𝑖𝑘𝑢_𝑙 is the 𝑙-th value of feature 𝑖 in cluster 𝑘 in MSA 𝑢. where, 𝑭𝑖𝑘𝑢 ̅̅̅̅̅̅ is the average value for feature 𝑖 in cluster 𝑘 in MSA 𝑢, and 𝐹𝑖𝑘𝑢_𝑙 is the 𝑙-th value of feature 𝑖 in cluster 𝑘 in MSA 𝑢. 3. Calculate the value of flood hazard, flood exposure, and flood vulnerability of each cluster 3. Calculate the value of flood hazard, flood exposure, and flood vulnerability of each cluster: 𝑭𝑯𝑘𝑢 = 𝑭𝑭𝑘𝑢 ̅̅̅̅̅̅̅ + 𝑭𝑰𝑘𝑢 ̅̅̅̅̅̅ (24) 𝑭𝑬𝑘𝑢 = 𝑷𝑵𝑘𝑢 ̅̅̅̅̅̅̅ + 𝑩𝑨𝑘𝑢 ̅̅̅̅̅̅̅ (25) (25) 𝑭𝑽𝑘𝑢 = 𝑺𝑵𝑘𝑢 ̅̅̅̅̅̅̅ + 𝑷𝑵𝑘𝑢 ̅̅̅̅̅̅̅ 𝑭𝑽𝑘𝑢 = 𝑺𝑵𝑘𝑢+ 𝑷𝑵𝑘𝑢 = 𝑷𝒐𝑹𝑘𝑢 ̅̅̅̅̅̅̅̅̅ + 𝑫𝒊𝑹𝑘𝑢 ̅̅̅̅̅̅̅̅̅ + 𝑳𝑬𝑹𝑘𝑢 ̅̅̅̅̅̅̅̅̅ +𝑷𝑶𝑰 ̅̅̅̅̅̅ + 𝑭𝒅𝑯 ̅̅̅̅̅̅ + 𝑩𝑨𝒈 ̅̅̅̅̅̅ (26) (26) 𝒈 where, 𝑭𝑯𝑘𝑢, 𝑭𝑬𝒌𝒖, 𝑭𝑽𝑘𝑢, 𝑭𝑭𝒌𝒖 ̅̅̅̅̅̅̅, 𝑭𝑰𝑘𝑢 ̅̅̅̅̅̅, 𝑷𝑵𝑘𝑢 ̅̅̅̅̅̅̅, 𝑩𝑨𝑘𝑢 ̅̅̅̅̅̅̅, 𝑺𝑵𝑘𝑢 ̅̅̅̅̅̅̅, and 𝑷𝑵𝒌𝒖 ̅̅̅̅̅̅̅ are the flood hazard, flood exposure, flood vulnerability, average flood frequency, average flood intensity, average population number, average building area, social vulnerability, physical vulnerability value of cluster 𝑘 in MSA 𝑢, respectively. Competing interests The authors declare no competing interests. Data Availability Data Availability The dataset used in this paper are publicly accessible and cited in this paper. The dataset used in this paper are publicly accessible and cited in this paper. Author contributions Kai Yin: Conceptualization, Methodology, Software, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Ali Mostafavi: Conceptualization, Methodology, Writing—Reviewing and Editing, Supervision, Funding acquisition. Code Availability The code that supports the findings of this study is available from the corresponding author upon request. Acknowledgements This work was supported by the National Science Foundation under CRISP 2.0 Type 2 No. 1832662 grant. Any opinions, findings, conclusions, or recommendations expressed in this research are those of the authors and do not necessarily reflect the view of the funding agencies. Incorporate spatial flood dependence relationships. Flood frequency in one grid cell will be directly impacted by other grid cells that have spatial flood dependence with it33. Hence, the where, 𝑭𝑯𝑘𝑢, 𝑭𝑬𝒌𝒖, 𝑭𝑽𝑘𝑢, 𝑭𝑭𝒌𝒖 ̅̅̅̅̅̅̅, 𝑭𝑰𝑘𝑢 ̅̅̅̅̅̅, 𝑷𝑵𝑘𝑢 ̅̅̅̅̅̅̅, 𝑩𝑨𝑘𝑢 ̅̅̅̅̅̅̅, 𝑺𝑵𝑘𝑢 ̅̅̅̅̅̅̅, and 𝑷𝑵𝒌𝒖 ̅̅̅̅̅̅̅ are the flood hazard, flood exposure, flood vulnerability, average flood frequency, average flood intensity, average population number, average building area, social vulnerability, physical vulnerability value of cluster 𝑘 in MSA 𝑢, respectively. where, 𝑭𝑯𝑘𝑢, 𝑭𝑬𝒌𝒖, 𝑭𝑽𝑘𝑢, 𝑭𝑭𝒌𝒖 ̅̅̅̅̅̅̅, 𝑭𝑰𝑘𝑢 ̅̅̅̅̅̅, 𝑷𝑵𝑘𝑢 ̅̅̅̅̅̅̅, 𝑩𝑨𝑘𝑢 ̅̅̅̅̅̅̅, 𝑺𝑵𝑘𝑢 ̅̅̅̅̅̅̅, and 𝑷𝑵𝒌𝒖 ̅̅̅̅̅̅̅ are the flood hazard, flood exposure, flood vulnerability, average flood frequency, average flood intensity, average population number, average building area, social vulnerability, physical vulnerability value of cluster 𝑘 in MSA 𝑢, respectively. p y 1. Following the work of Xu et al58, the aggregated flood risk value 𝑭𝑹𝑘𝑢 of each cluster is calculated by multiplying values of flood hazard, exposure and vulnerability of each cluster: 1. Following the work of Xu et al58, the aggregated flood risk value 𝑭𝑹𝑘𝑢 of each cluster i calculated by multiplying values of flood hazard, exposure and vulnerability of each cluster 𝑭𝑹𝑘𝑢= 𝑭𝑯𝑘𝑢∗𝑭𝑬𝑘𝑢∗𝑭𝑽𝑘𝑢 (27) (27) 2. Finally, calculate the flood-risk level value 𝑭𝑳𝑘𝑢 by ranking the value of 𝑭𝑹𝑘𝑢 in an ascending way: 2. Finally, calculate the flood-risk level value 𝑭𝑳𝑘𝑢 by ranking the value of 𝑭𝑹𝑘𝑢 in an ascending way: 𝑭𝑳𝑘𝑢 = 𝑆𝑜𝑟𝑡𝑒𝑑 (𝑭𝑹𝑘𝑢) (28) (28) Model training. Hyperparameters tuning, the algorithm of the urban flood risk rating model, and the determination of the optimal shared clustering number for all five MSAs are described in Section S6 in Supplementary Notes. Model training. Hyperparameters tuning, the algorithm of the urban flood risk rating model, and the determination of the optimal shared clustering number for all five MSAs are described in Section S6 in Supplementary Notes. 20 Reference 1. NASEM (National Academies of Sciences, Engineering, and M. 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Insight into the Role of the STriatal-Enriched Protein Tyrosine Phosphatase (STEP) in A2A Receptor-Mediated Effects in the Central Nervous System
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Insight into the Role of the STriatal-Enriched Protein Tyrosine Phosphatase (STEP) in A2A Receptor-Mediated Effects in the Central Nervous System Maria Rosaria Domenici 1*, Cinzia Mallozzi 2, Rita Pepponi 1, Ida Casella 1, Valentina Chiodi 1, Antonella Ferrante 1 and Patrizia Popoli 1 1National Centre for Drug Research and Evaluation, Istituto Superiore di Sanitá, Rome, Italy, 2Department of Neuroscience, Istituto Superiore di Sanità, Rome, Italy The STriatal-Enriched protein tyrosine phosphatase STEP is a brain-specific tyrosine phosphatase that plays a pivotal role in the mechanisms of learning and memory, and it has been demonstrated to be involved in several neuropsychiatric diseases. Recently, we found a functional interaction between STEP and adenosine A2A receptor (A2AR), a subtype of the adenosine receptor family widely expressed in the central nervous system, where it regulates motor behavior and cognition, and plays a role in cell survival and neurodegeneration. Specifically, we demonstrated the involvement of STEP in A2AR-mediated cocaine effects in the striatum and, more recently, we found that in the rat striatum and hippocampus, as well as in a neuroblastoma cell line, the overexpression of the A2AR, or its stimulation, results in an increase in STEP activity. In the present article we will discuss the functional implication of this interaction, trying to examine the possible mechanisms involved in this relation between STEP and A2ARs. Edited by: Edited by: Francisco Ciruela, University of Barcelona, Spain Reviewed by: Hyung Nam, Louisiana State University Health Shreveport, United States Marco Milanese, University of Genoa, Italy *Correspondence: Maria Rosaria Domenici mariarosaria.domenici@iss.it *Correspondence: Maria Rosaria Domenici mariarosaria.domenici@iss.it Keywords: adenosine A2A receptor, striatal-enriched protein tyrosine phosphatase, brain, SH-SY5Y neuroblastoma cell lines, functional interaction Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology INTRODUCTION P1 adenosine receptors are the most investigated purinergic receptors within the central nervous system (CNS). Since their identification in the late 70s, they have been the subject of numerous studies that established their widespread distribution in the brain and their pivotal role in the functioning of the CNS. The adenosine A2A receptor (A2AR) is one of the four G protein coupled receptor subtypes (A1, A2A, A2B, and A3), it is coupled with Gs protein and its stimulation activates adenylate cyclase causing an increase in intracellular cAMP levels (Borea et al., 2018). With the exception of the dorsal and ventral striatum, where A2AR is present at remarkably high levels, in the rest of the brain the expression of the receptor is quite low (Rosin et al., 2003). Despite this, the huge importance of A2AR in the CNS is witnessed by its role in the regulation of fundamental functions such as movement, cognition and emotions and, for this reason, it has attracted the interest of researchers as a potential therapeutic target (Borah et al., 2019). Indeed, the A2AR antagonist istradefylline (Nourianz®) has recently been approved in the United States, after its first registration in Japan, for the treatment of Parkinson’s disease, as an add-on to levodopa (Chen and Cunha, 2020). Received: 05 January 2021 Accepted: 26 February 2021 Published: 19 April 2021 PERSPECTIVE published: 19 April 2021 doi: 10.3389/fphar.2021.647742 PERSPECTIVE Citation: Domenici MR, Mallozzi C, Pepponi R, Casella I, Chiodi V, Ferrante A and Popoli P (2021) Insight into the Role of the STriatal-Enriched Protein Tyrosine Phosphatase (STEP) in A2A Receptor- Mediated Effects in the Central Nervous System. Front. Pharmacol. 12:647742. doi: 10.3389/fphar.2021.647742 April 2021 | Volume 12 | Article 647742 1 Frontiers in Pharmacology | www.frontiersin.org Modulation of STEP Activity by Adenosine A2AR Domenici et al. FIGURE 1 | Schematic representation of the possible mechanisms involved in the regulation of STEP activity by A2ARs. Activation of A2ARs, directly with the selective A2AR agonist CGS 21680 or indirectly with cocaine, increases STEP activity through a peculiar mechanism involving mGlu5R, intracellular Ca++ increase and calcineurin recruitment. On the contrary, activation of PKA by forkolin promotes STEP inactivation through phosphorylation of Ser residue. FIGURE 1 | Schematic representation of the possible mechanisms involved in the regulation of STEP activity by A2ARs. Activation of A2ARs, directly with the selective A2AR agonist CGS 21680 or indirectly with cocaine, increases STEP activity through a peculiar mechanism involving mGlu5R, intracellular Ca++ increase and calcineurin recruitment. On the contrary, activation of PKA by forkolin promotes STEP inactivation through phosphorylation of Ser residue. One of the peculiarities of A2AR is the ability to modulate the activation and function of several other receptors, such as dopamine D2, cannabinoid CB1, metabotropic glutamate 5 receptor (mGlu5R), as well as adenosine A1 receptors, by forming heteroreceptor complexes (Cabello et al., 2009; Tebano et al., 2012; Moreno et al., 2018; Ferré and Ciruela, 2019). Recently, we identified a novel role of A2ARs in the rodent brain and in neuronal cells. Specifically, we demonstrated that the stimulation of A2ARs results in the activation of the STriatal-Enriched protein tyrosine phosphatase STEP, a brain-specific tyrosine phosphatase involved in several functions, including learning and memory (Goebel-Goody et al., 2012; Chiodi et al., 2014; Mallozzi et al., 2020). the substrates. When phosphorylated at the specific Ser residues (221 for STEP61 and 49 for STEP46) within the KIM domains, STEP61 and STEP46 become inactive since they lose their ability to bind to the substrates (Bult et al., 1996; Pulido et al., 1998; Kamceva et al., 2016). STEP activity is regulated by quite complex phosphorylation/dephosphorylation mechanisms, in which calcineurin (a calcium/calmodulin-activated serine/threonine phosphatase, also known as PP2B) and protein kinase A (PKA) play a major role (Figure 1). Citation: Calcineurin activates STEP through protein phosphatase 1 (PP1), which dephosphorylates the regulatory serine residue and activates STEP (Paul et al., 2000). The activation of PKA results in the inhibition of STEP activity either through the direct phosphorylation of STEP61 and STEP46 at the specific serine residues and, indirectly, through the phosphorylation of DARPP- 32 and the inhibition of PP1(Paul et al., 2000; Valjent et al., 2005; Giralt et al., 2011). Several neurotransmitter receptors, such as dopamine D1 receptor and nicotinic α7 nAChR, are able to modulate STEP activity (Paul et al., 2000; Zhang et al., 2013). Moreover, mGlu5R has been shown to increase STEP translation at dendritic levels that mediates AMPA receptor endocytosis, a mechanism that could be involved in DHPG-induced LTD. (Moult et al., 2002; Zhang et al., 2008; Goebel-Goody et al., 2012; Chen et al., 2013). As already mentioned, and as we will discuss later, STEP activity is also modulated by A2AR (Chiodi et al., 2014; Mallozzi et al., 2020). In this article we will present some recent results on the A2ARs/ STEP interaction and on the possible mechanisms involved. The physiological implication of this new receptor function will be discussed. STRIATAL-ENRICHED PROTEIN TYROSINE PHOSPHATASE In the early 90s, Paul J. Lombroso and collaborators isolated a new protein tyrosine phosphatase in the brain, particularly enriched in the striatum, that strongly colocalized with DARPP32 and tyrosine hydroxylase-positive neurons, which was denominated STEP (Lombroso et al., 1991, 1993). STEP exists in several isoforms that differ in intracellular localization and functions, and all originate by alternative splicing of a single Ptpn5 gene (Boulanger et al., 1995). The two major isoforms are STEP61, associated with membrane compartments, and the cytosolic protein STEP46, and both carry the consensus sequence required for the phosphatase catalytic activity and a kinase-interacting motif (KIM), that allows the interaction with Several substrates of STEP have been identified. The glutamate receptor subunits GluN2B and GluA1/GluA2 of the NMDA and AMPA receptors, respectively, are important STEP substrates whose dephosphorylation at specific tyrosine residues promotes receptor internalization and reduces NMDA- and AMPA- mediated synaptic transmission, having a strong impact on synaptic plasticity (Won and Roche, 2021). Pyk2 and Fyn, two other STEP substrates, are also involved in the modulation of synaptic transmission and synaptic plasticity by influencing April 2021 | Volume 12 | Article 647742 Frontiers in Pharmacology | www.frontiersin.org 2 Modulation of STEP Activity by Adenosine A2AR Domenici et al. respectively, and promoting cell survival or death (Xu et al., 2009). several mechanisms, including the direct or indirect phosphorylation of glutamate receptor subunits (Huang et al., 2001; Giralt et al., 2017; Matrone et al., 2020). Moreover, STEP shows a role at presynaptic level, modulating glutamate neurotransmitter release (Bosco et al., 2018). Additional STEP substrates are the extracellular signal-regulated kinases 1 and 2 (ERK1/2), involved in memory processes and in synaptic plasticity, and p38, implicated in cell death and survival, and both are inactivated by dephosphorylation of specific tyrosine residues upon STEP activation (Goebel-Goody et al., 2012). Another well identified role for STEP is the modulation of the effects of psychostimulant drugs such as cocaine and amphetamine (Valjent et al., 2005; Hopf and Bonci, 2009; Sun et al., 2013; Siemsen et al., 2018). As for cocaine effects, initial studies demonstrated that following acute cocaine treatment in mice, the increase in ERK1/2 phosphorylation (pERK1/2) in a subpopulation of dopamine D1R-containing striatal neurons was mediated, at least in part, by D1R-mediated STEP inactivation (Valjent et al., 2005). STRIATAL-ENRICHED PROTEIN TYROSINE PHOSPHATASE These effects were due to the blockade of neuroprotective responses initiated by the ERK/MAPK signaling pathway. On the other hand, in an in vivo model of cerebral ischemia, where excitotoxic cell death plays a major role, STEP exerts a neuroprotective effect by inhibiting the p38 MAPK signaling pathway. In fact, administration of the STEP-derived peptide prevents p38 MAPK activation and reduces ischemic brain damage in STEP KO mice (Deb et al., 2013). In order to reconcile those apparently conflicting results, it should be considered that, depending on the level of calcium increase following NMDA receptors stimulation, STEP activity can be increased and promote neuroprotection by reducing p38 activation or, in case of a prolonged insult, the resulting STEP degradation will facilitate cell death pathways by increasing the phosphorylation of p38 MAPK (Poddar et al., 2010). In addition, the stimulation of synaptic or extrasynaptic NMDA receptors differently impacts on STEP expression, resulting in the activation of ERK1/2 or p38 MAPK, Indeed, in the last years a dysregulation of STEP has been found in several neuropsychiatric diseases and its modulation, by genetic or pharmacological tools, was accompanied by the attenuation of the symptoms in animal models of diseases (Karasawa and Lombroso, 2014; Kulikova and Kulikov, 2017). The general idea is that elevated STEP levels or activity have detrimental effects on cognition by negatively influencing synaptic strengthening through the dephosphorylation of substrates regulating synaptic plasticity (Pelkey et al., 2002; Fitzpatrick and Lombroso, 2011). Indeed, high levels of STEP expression have been found in animal models of Alzheimer’s and Parkinson’s diseases (neurodegenerative diseases characterized by cognitive impairment), and in the hippocampus of aged mice, rats and rhesus monkeys and in the brain of individuals with mild cognitive impairment (Xu et al., 2012; Kurup et al., 2015; Castonguay et al., 2018). Furthermore, STEP over-expression induced memory deficits in mice, and its inhibition ameliorates memory performances in aged rats and in animal models of neuropsychiatric diseases (Castonguay et al., 2018). However, during aging reduced STEP activity and expression have also been reported (Rajagopal et al., 2016; Cases et al., 2018). EVIDENCE OF A FUNCTIONAL INTERACTION BETWEEN A2AR AND STRIATAL-ENRICHED PROTEIN TYROSINE PHOSPHATASE The first evidence of an involvement of STEP in A2AR-mediated effects came from our study investigating the synaptic effects of cocaine in the striatum (Chiodi et al., 2014). We found that cocaine reduced striatal synaptic transmission, evaluated by recording extracellular field potentials and AMPA- and NMDA-mediated currents in whole cell patch-clamp experiments in corticostriatal slices. Cocaine effects were reduced by A2AR antagonist, by inhibitor of protein tyrosine phosphatases, by a calcineurin inhibitor and by TAT-STEP, a substrate trapping mutant peptide that makes STEP enzymatically inactive. In addition, the effect of cocaine was strongly reduced in A2AR knock-out mice. In order to understand the relationship among cocaine, A2ARs and tyrosine phosphatases (and STEP in particular), we evaluated the enzimatic activity of the total tyrosine phosphatases, and of STEP in particular, in mice striatal tissue after cocaine stimulation. We could show that cocaine increased tyrosine phosphatase activity, and in particular STEP activity, in A2AR-dependent manner. In fact, cocaine failed to activate STEP in the presence of the A2AR antagonist or in A2AR knock-out mice. These results suggested that a possible mechanism through which cocaine reduced synaptic transmission is the recruitment of A2AR and STEP activation. Indeed, STEP activation results in the dephosphorylation and p ( j g p , ; , ) Beside its role in modulating synaptic plasticity and cognition, STEP is emerging as a key regulator of neuronal survival and death. As demonstrated by Choi et al. (2007), STEP increases neuronal vulnerability to excitotoxic cell death in primary hippocampal cultures and the sensitivity of neurons to excitotoxicity induced by Status Epilepticus in mice. These effects were due to the blockade of neuroprotective responses initiated by the ERK/MAPK signaling pathway. On the other hand, in an in vivo model of cerebral ischemia, where excitotoxic cell death plays a major role, STEP exerts a neuroprotective effect by inhibiting the p38 MAPK signaling pathway. In fact, administration of the STEP-derived peptide prevents p38 MAPK activation and reduces ischemic brain damage in STEP KO mice (Deb et al., 2013). In order to reconcile those apparently conflicting results, it should be considered that, depending on the level of calcium increase following NMDA receptors stimulation, STEP activity can be increased and promote neuroprotection by reducing p38 activation or, in case of a prolonged insult, the resulting STEP degradation will facilitate cell death pathways by increasing the phosphorylation of p38 MAPK (Poddar et al., 2010). STRIATAL-ENRICHED PROTEIN TYROSINE PHOSPHATASE However, in condition of chronic cocaine consumption, such as in models of cocaine self-administration, a decrease in STEP phosphorylation and pERK1/2 are observed in the rat prefrontal cortex, that could represent early events in withdrawal mechanisms (Sun et al., 2013). More recently, cocaine-induced STEP activation has been demonstrated in the early phase of abstinence, which mediates the decrease in p-ERK observed in the pre-limbic cortex of cocaine-seeking rats (Siemsen et al., 2018). These studies demonstrate an active role of STEP in cocaine-mediated effects. In line with this, as we will describe below, we found that the synaptic depression exerted by cocaine in the striatum involved STEP activation through the stimulation of A2ARs (Chiodi et al., 2014), suggesting an interaction between the receptor and the phosphatase. STEP appears to be strongly involved in neurological disorders where synaptic dysfunctions have been identified, as well as in diseases where excitotoxicity play a major role. diseases where excitotoxicity play a major role. Indeed, in the last years a dysregulation of STEP has been found in several neuropsychiatric diseases and its modulation, by genetic or pharmacological tools, was accompanied by the attenuation of the symptoms in animal models of diseases (Karasawa and Lombroso, 2014; Kulikova and Kulikov, 2017). The general idea is that elevated STEP levels or activity have detrimental effects on cognition by negatively influencing synaptic strengthening through the dephosphorylation of substrates regulating synaptic plasticity (Pelkey et al., 2002; Fitzpatrick and Lombroso, 2011). Indeed, high levels of STEP expression have been found in animal models of Alzheimer’s and Parkinson’s diseases (neurodegenerative diseases characterized by cognitive impairment), and in the hippocampus of aged mice, rats and rhesus monkeys and in the brain of individuals with mild cognitive impairment (Xu et al., 2012; Kurup et al., 2015; Castonguay et al., 2018). Furthermore, STEP over-expression induced memory deficits in mice, and its inhibition ameliorates memory performances in aged rats and in animal models of neuropsychiatric diseases (Castonguay et al., 2018). However, during aging reduced STEP activity and expression have also been reported (Rajagopal et al., 2016; Cases et al., 2018). Beside its role in modulating synaptic plasticity and cognition, STEP is emerging as a key regulator of neuronal survival and death. As demonstrated by Choi et al. (2007), STEP increases neuronal vulnerability to excitotoxic cell death in primary hippocampal cultures and the sensitivity of neurons to excitotoxicity induced by Status Epilepticus in mice. EVIDENCE OF A FUNCTIONAL INTERACTION BETWEEN A2AR AND STRIATAL-ENRICHED PROTEIN TYROSINE PHOSPHATASE In addition, the stimulation of synaptic or extrasynaptic NMDA receptors differently impacts on STEP expression, resulting in the activation of ERK1/2 or p38 MAPK, April 2021 | Volume 12 | Article 647742 Frontiers in Pharmacology | www.frontiersin.org 3 Modulation of STEP Activity by Adenosine A2AR Domenici et al. internalization of NMDA and AMPA receptor subunits causing depression of excitatory synaptic transmission (Zhang et al., 2008; Zhang et al., 2010; Zhang et al., 2011; Kurup et al., 2010). Moreover, the involvement of calcineurin suggests the need of intracellular calcium increase. These mechanisms have been very nicely examined and depicted by Robert Yasuda (Yasuda, 2020) who recapitulated the way through which A2AR modulates cocaine-induced synaptic depression and, possibly, cocaine self-administration, via STEP activation. (unpublished data) and that the signaling route of A2AR to STEP61 probably does not depend on their direct interaction. However, to definitively exclude a direct interaction between A2AR and STEP, BRET experiments should be performed also by using other STEP isoforms (i.e., STEP46). In a recent paper Won and collaborators used mass spectrometry to study STEP binding proteins and identified 315 candidate proteins and, among them, the authors recognized mGlu5R as an interactor of STEP (Won et al., 2019). This finding is particularly interesting since it is well known that A2AR and mGlu5R physically and functionally interact in several brain areas, that activation of A2ARs exerts a permissive role on mGluR5R-mediated effects (Ferre et al., 2002; Domenici et al., 2004; Tebano et al., 2005; Krania et al., 2018) and, most importantly, that mGlu5R stimulation results in an increase in STEP translation and, presumably, activation (Zhang et al., 2008). Moreover, mGlu5R interacts with Gq proteins and its stimulation enables the activation of PLC signaling and intracellular calcium increase (Conn and Pin, 1997). Interestingly, in our recent paper we found that A2AR-induced STEP activation is calcium-dependent since in SH-SY5Y cells it is prevented by the calcium chelator BAPTA-AM and by the calcineurin inhibitor FK506 (Mallozzi et al., 2020). Thus, on the basis of this calcium dependence, the mGlu5R could be a good candidate to mediate A2AR effects on STEP activity. Therefore, in preliminary experiments we verified in the SH-SY5Y cell line the effect of the selective A2AR agonist CGS 21680 on STEP activity in the presence of the mGlu5R antagonist MPEP, and we found that by blocking mGlu5R, CGS 21680 was no longer able to increase STEP activity (unpublished results). EVIDENCE OF A FUNCTIONAL INTERACTION BETWEEN A2AR AND STRIATAL-ENRICHED PROTEIN TYROSINE PHOSPHATASE Even though additional experiments are needed, these results clearly suggest that A2ARs modulate STEP activity through the involvement of mGlu5R (Figure 1). In a recent paper, in order to confirm and further investigate the relationship between A2AR and STEP, we used cellular, genetic, and pharmacological approaches to evaluated STEP activity in different condition of A2AR stimulation and in different brain areas (Mallozzi et al., 2020). We took advantage of a transgenic rat strain overexpressing A2AR in the brain (Chiodi et al., 2016) in which we evaluated STEP activity in the striatum and hippocampus. In basal conditions, we found a significant increase in STEP activity in the striatum and hippocampus of A2AR overexpressing rats with respect to wild type. Moreover, in the striatum the selective A2AR agonist CGS21680 increased STEP activity in wild type but not in A2AR overexpressing rats (where STEP activity was already high), while ZM241385, the A2AR antagonist, reduced STEP activity in overexpressing rats (up to wild type levels), without any effects in wild type animals. In addition, in A2AR overexpressing rats we found a decrease in the phosphorylation levels of GluN2B and Pyk2, two well-known STEP substrates, consistent with an increased phosphatase activity (Mallozzi et al., 2020). Similar results have been obtained in the neuroblastoma cell line SH-SY5Y, which expresses both STEP and A2ARs, where we confirmed that the stimulation of A2AR with CGS21680 causes an increase in STEP activity, evaluated also by western blotting analysis as a decrease in STEP phosphorylation status. An interesting point to address is by which mechanism the stimulation of A2ARs results in STEP activation. It is demonstrated, in fact, that the activation of the cAMP/PKA pathway, as it occurs with the activation of Gs-coupled receptors (and the A2AR belongs indeed to the family of Gs-coupled receptors), rather results in the phosphorylation and inactivation of STEP (Paul et al., 2000). Actually, also in our hands the treatment of SH-SY5Y cells with forskolin (Mallozzi et al., 2020), which induces activation of the cAMP/ PKA pathway, causes an up-regulation of phosphoSTEP, consistent with the inactivation of the phosphatase. Thus, a different mechanism must be hypothesized to explain A2AR-mediated STEP activation. Frontiers in Pharmacology | www.frontiersin.org REFERENCES Albasanz, J. L., Perez, S., Barrachina, M., Ferrer, I., and and Martín, M. (2008). Research article: up-regulation of adenosine receptors in the frontal cortex in alzheimer’s disease. Brain Pathol. 18 (2), 211–219. doi:10.1111/j.1750-3639. 2007.00112.x Cases, S., Saavedra, A., Tyebji, S., Giralt, A., Alberch, J., and and Pérez-Navarro, E. (2018). Age-related changes in STriatal-Enriched protein tyrosine Phosphatase levels: regulation by BDNF. Mol. Cell Neurosci. 86, 41–49. doi:10.1016/j.mcn. 2017.11.003 Arendash, G. W., Schleif, W., Rezai-Zadeh, K., Jackson, E. K., Zacharia, L. C., Cracchiolo, J. R., et al. (2006). Caffeine protects Alzheimer’s mice against cognitive impairment and reduces brain β-amyloid production. Neuroscience 142 (2), 941–952. doi:10.1016/j.neuroscience.2006.07.021 Castonguay, D., Dufort-Gervais, J., Ménard, C., Chatterjee, M., Quirion, R., Bontempi, B., et al. (2018). The tyrosine phosphatase STEP is involved in age-related memory decline. Curr. Biol. 28 (7), 1079–1089.e4. doi:10.1016/j.cub.2018.02.047 Borah, P., Deka, S., Mailavaram, R. P., and and Deb, P. K. (2019). 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Enhancement of long-term depression by soluble amyloid β protein in rat hippocampus is mediated by metabotropic glutamate receptor and involves activation of p38MAPK, STEP and caspase-3. Neuroscience 253, 435–443. doi:10.1016/j. neuroscience.2013.08.054 Bosco, F., Valente, P., Milanese, M., Piccini, A., Messa, M., Bonanno, G., et al. (2018). Altered intracellular calcium homeostasis underlying enhanced glutamatergic transmission in striatal-enriched tyrosine phosphatase (STEP) knockout mice. Mol. Neurobiol. 55 (10), 8084–8102. doi:10.1007/s12035-018- 0980-5 Chiodi, V., Ferrante, A., Ferraro, L., Potenza, R. L., Armida, M., Beggiato, S., et al. (2016). Striatal adenosine-cannabinoid receptor interactions in rats over- expressing adenosine A2Areceptors. J. Neurochem. 136 (5), 907–917. doi:10. 1111/jnc.13421 Boulanger, L., Lombroso, P., Raghunathan, A., During, M., Wahle, P., and and Naegele, J. (1995). Cellular and molecular characterization of a brain-enriched protein tyrosine phosphatase. J. Neurosci. 15 (12), 1532–1544. DISCUSSION AND CONCLUSION Finally, STEP over-expression induced memory impairment in adult mice (Castonguay et al., 2018), and the same occurs in conditions of increased A2ARs activation (Gimenez-Llort et al., 2007; Li et al., 2015; Pagnussat et al., 2015). Accordingly, in A2AR overexpressing rats, in which we demonstrated an increased basal STEP activity in the striatum and hippocampus, working memory deficits have been reported (Gimenez-Llort et al., 2007; Mallozzi et al., 2020). Very recently, Ferrante et al. (2021) demonstrated that STEP protein expression and activity were increased in Fragile X mice and normalized by the A2AR antagonist KW6002 treatment, which improved the behavioral phenotype as well. aged brain, and inhibition of STEP activity or the blockade of A2ARs improved memory performances (Castonguay et al., 2018; Orr et al., 2018; Ferré and Ciruela, 2019; Temido- Ferreira et al., 2019; Temido-Ferreira et al., 2020). Finally, STEP over-expression induced memory impairment in adult mice (Castonguay et al., 2018), and the same occurs in conditions of increased A2ARs activation (Gimenez-Llort et al., 2007; Li et al., 2015; Pagnussat et al., 2015). Accordingly, in A2AR overexpressing rats, in which we demonstrated an increased basal STEP activity in the striatum and hippocampus, working memory deficits have been reported (Gimenez-Llort et al., 2007; Mallozzi et al., 2020). Very recently, Ferrante et al. (2021) demonstrated that STEP protein expression and activity were increased in Fragile X mice and normalized by the A2AR antagonist KW6002 treatment, which improved the behavioral phenotype as well. may contribute to the cognitive impairment that occurs in the disease (Kurup et al., 2015). In conclusion, the interaction between A2AR and STEP (possibly through the involvement of mGlu5R) could have clinical relevance and its possible consequences should be contemplated when proposing drugs targeting the A2ARs. Notably, particular attention should be payed when considering A2AR agonists as potential treatment for human pathologies (Borea et al., 2018; Borah et al., 2019), given their potential to impair cognitive performance by increasing STEP activity. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. Thus, one important conclusion is that the modulation of STEP activity could contribute to the effects of A2ARs on cognitive functions (Chen, 2014; Uchida et al., 2014; Temido- Ferreira et al., 2019). As for Parkinson’s disease, an interesting consideration is that long-term treatment of patients with istradefylline could result not only in the improvement of motor deficits but also in beneficial effects on cognitive dysfunction, and that the inhibition of STEP could play a major role in this effect. In fact, STEP levels are increased in human brains and in animal models of Parkinson’s disease, which AUTHOR CONTRIBUTIONS MRD and CM conceived the work; MRD drafted the manuscript; RP composed the figure; IC, CM, and RP, produced the original data, VC, AF, and PP contributed in writing and editing and all read and approved the present version of the manuscript for publication. Casella, I., Ambrosio, C., Grò, M. C., Molinari, P., and and Costa, T. (2011). Divergent agonist selectivity in activating β1- and β2-adrenoceptors for G-protein and arrestin coupling. Biochem. J. 438 (1), 191–202. doi:10.1042/ BJ20110374 DISCUSSION AND CONCLUSION The studies presented above provide a clear demonstration of a functional interaction between A2ARs and STEP in the striatum and hippocampus of the rat and mouse brain, which has been confirmed in the SH-SY5Y neuroblastoma cell line, suggesting that this interaction can occur in different cell types. The mechanism through which A2AR and STEP interact is still not clearly identified, but the calcium dependence and the involvement of mGlu5R are both very likely. Even though a strong evidence that this interaction occurs also in vivo is still lacking, a review of the scientific literature shows that in some neuropathologic conditions STEP and the A2AR are dysregulated in a similar way. For example, STEP levels are elevated in rodent models of Alzheimer’s disease, in postmortem brains of patients with Alzheimer’s disease and in the brain of individuals with mild cognitive impairment (Zhang et al., 2011; Xu et al., 2012; Castonguay et al., 2018). In the same way, A2ARs are upregulated in Alzheimer’s disease, both in animal models and in the brain of patients (Arendash et al., 2006; Albasanz et al., 2008; Orr et al., 2015; Temido-Ferreira et al., 2020). More interestingly, during aging both STEP and A2ARs are upregulated and show an enhanced activity in animal models and in the human To assess if a physical interaction between A2AR and STEP could be necessary, we performed Bioluminescence Resonance Energy Transfer (BRET) assays (Molinari et al., 2008; Casella et al., 2011) in SH-SY5Y cell populations co-expressing a green fluorescent version of STEP61 with either luminescent-A2AR (a kind gift from Francisco Ciruela) or luminescent-β-arrestin 2 protein (a well recognized G-protein independent signal transducer) (Sachs et al., 2005). In our experiments, exposure of these cells to the A2AR agonist CGS21680 failed to enhance the BRET signal over the level of unstimulated samples, suggesting that STEP61 is probably not an A2AR interacting partner April 2021 | Volume 12 | Article 647742 Frontiers in Pharmacology | www.frontiersin.org 4 Modulation of STEP Activity by Adenosine A2AR Domenici et al. aged brain, and inhibition of STEP activity or the blockade of A2ARs improved memory performances (Castonguay et al., 2018; Orr et al., 2018; Ferré and Ciruela, 2019; Temido- Ferreira et al., 2019; Temido-Ferreira et al., 2020). REFERENCES doi:10.1523/ jneurosci.15-02-01532.1995 Chiodi, V., Mallozzi, C., Ferrante, A., Chen, J. F., Lombroso, P. J., Di Stasi, A. M. M., et al. (2014). Cocaine-induced changes of synaptic transmission in the striatum are modulated by adenosine A2A receptors and involve the tyrosine phosphatase STEP. 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Neurobiol. 119, 169–189. doi:10.1016/B978-0-12-801022-8.00008-8 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Valjent, E., Pascoli, V., Svenningsson, P., Paul, S., Enslen, H., Corvol, J.-C., et al. (2005). From the Cover: regulation of a protein phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum. Proc. Natl. Acad. Sci. 102 (2), 491–496. doi:10.1073/pnas.0408305102 Natl. Acad. Sci. 102 (2), 491–496. doi:10.1073/pnas.0408305102 Copyright © 2021 Domenici, Mallozzi, Pepponi, Casella, Chiodi, Ferrante and Popoli. 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. Won, S., Incontro, S., Li, Y., Nicoll, R. A., and and Roche, K. W. (2019). The STEP61 interactome reveals subunit-specific AMPA receptor binding and synaptic regulation. Proc. Natl. Acad. Sci. U.S.A. 116 (16), 8028–8037. doi:10.1073/pnas.1900878116 Won, S., and and Roche, K. W. (2021). Regulation of glutamate receptors by striatal-enriched tyrosine phosphatase 61 (STEP 61 ). J. Physiol. 599 (2), 443–451. doi:10.1113/JP278703 April 2021 | Volume 12 | Article 647742 Frontiers in Pharmacology | www.frontiersin.org 7
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Arg-type dihydroflavonol 4-reductase genes from the fern Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins
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PLOS ONE PLOS ONE RESEARCH ARTICLE OPEN ACCESS Dihydroflavonol 4-reductase (DFR), a key enzyme involved in the biosynthesis of anthocya- nins, has been cloned from various species. However, little research has been conducted on this enzyme in ferns, which occupy a unique evolutionary position. In this study, we iso- lated two novel DFR genes from the fern Dryopteris erythrosora. In vitro enzymatic analysis revealed that DeDFR1 and DeDFR2 enzymes can catalyze dihydrokaempferol and dihydro- quercetin but cannot catalyze dihydromyricetin. Amino acid sequence analysis showed that DeDFR1 and DeDFR2 have an arginine at the same substrate-specificity-determining site as that in the ferns Salvinia cucullata and Azolla filiculoides. Thus, we speculate that the Arg-type DFR is a new DFR functional type. To further verify the substrate preferences of the Arg-type DFR, an amino acid substitution assay was conducted. When N133 was mutated to R133, Arabidopsis DFR protein completely lost its catalytic activity for dihydro- myricetin, as observed for DeDFR1 and DeDFR2. Additionally, heterologous expression of DeDFR2 in the Arabidopsis tt3-1 mutant resulted in increasing anthocyanin accumulation. In summary, DeDFR1 and DeDFR2 are considered to be a new type of DFR with unique structures and functions. The discovery of the Arg-type DFR provides new insights into the anthocyanin biosynthesis pathway in ferns. Citation: Chen X, Liu W, Huang X, Fu H, Wang Q, Wang Y, et al. (2020) Arg-type dihydroflavonol 4- reductase genes from the fern Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins. PLoS ONE 15(5): e0232090. https://doi.org/10.1371/journal. pone.0232090 Editor: Shaojun Dai, Northeast Forestry University, CHINA CHINA Received: March 14, 2020 Accepted: April 7, 2020 Published: May 1, 2020 Copyright: © 2020 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: March 14, 2020 Accepted: April 7, 2020 Published: May 1, 2020 Copyright: © 2020 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2020 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Arg-type dihydroflavonol 4-reductase genes from the fern Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins Xuefei Chen1,2, Wenli Liu2, Xianyan Huang2, Huanhuan Fu2, Quanxi Wang2,3, Youfang Wang1*, Jianguo CaoID2* Xuefei Chen1,2, Wenli Liu2, Xianyan Huang2, Huanhuan Fu2, Quanxi Wang2,3, Youfang Wang1*, Jianguo CaoID2* 1 College of Life Science, East China Normal University, Shanghai, China, 2 College of Life Science, 1 College of Life Science, East China Normal University, Shanghai, China, 2 College of Life Science, Shanghai Normal University, Shanghai, China, 3 Shanghai Key Laboratory of Plant Functional Genomics and Resource, Shanghai Chenshan Botanical Garden, Shanghai, China 1 College of Life Science, East China Normal University, Shanghai, China, 2 College of Life Science, Shanghai Normal University, Shanghai, China, 3 Shanghai Key Laboratory of Plant Functional Genomics and Resource, Shanghai Chenshan Botanical Garden, Shanghai, China a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * yfwang@bio.ecnu.cn (YW); cao101@shnu.edu.cn (JC) OPEN ACCESS Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Introduction Funding: This research was supported by the Natural Science Foundation of Shanghai (13ZR1429700 to J.C.). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Anthocyanins represent an important subgroup of flavonoids, which are important secondary metabolites produced by plants to adapt to the terrestrial environment. Anthocyanins, serving as pigments, signaling molecules, photoprotectants, and protectants against biotic and abiotic stress, have a broad spectrum of functions in plant development [1–3]. Anthocyanins in food plants are potentially valuable components of the human diet. In addition, they have Competing interests: The authors have declared that no competing interests exist. 1 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins antioxidant, anticancer, and anti-inflammatory activities and may contribute to the prevention and treatment of metabolic disorders, cancer, eye diseases and cardiovascular diseases [4–6]. Dihydroflavonol 4-reductase (DFR; EC1.1.1.219) is a pivotal enzyme in anthocyanin bio- synthesis [7]. DFR can use NADPH as a cofactor to reduce dihydroflavonols, including dihy- drokaempferol (DHK), dihydroquercetin (DHQ), and dihydromyricetin (DHM), to the corresponding flavan-3,4-diols (leucoanthocyanidins), which are the precursors of anthocya- nidins and proanthocyanidins [8–11]. DFR genes have been cloned from various species, such as Arabidopsis thaliana, Brassica rapa, Ginkgo biloba, Lotus japonicus and Zea mays [12–17]. The DFR mutation (tt3) in A. thaliana has been demonstrated to cause anthocyanin loss, and the color of the seed coat changes to yellow [18–20]. Targeted mutagenesis of the InDFR-B gene by CRISPR/Cas9 technology leads to the production of anthocyanin-free white flowers in Petunia [21]. In a study of transgenic sweet potato, inhibition of IbDFR expression signifi- cantly reduces anthocyanin accumulation and antioxidant capacity [22]. In addition, overex- pression of DFR can increase anthocyanin production in tobacco and apple [23, 24]. DFRs not only regulate the total anthocyanin content but also have substrate preferences that affect the accumulation of different types of anthocyanins. Crystal structure analysis of the Vitis vinifera DFR shows that this protein belongs to the short-chain dehydrogenase/reductase family, and the 131–156 region is the substrate-binding site [25]. Some studies have indicated that amino acid residue 133 plays an important role in substrate specificity and classified DFRs into differ- ent types based on this site. DFRs in most plants are Asn-type DFRs, which can catalyze three types of substrates (DHK, DHQ, DHM). Phylogenetic analysis Multisequence alignment of amino acid sequences of DFRs from other plants and deduced amino acid sequences of DeDFR1 and DeDFR2 was performed using DNAMAN. The phyloge- netic tree was constructed using the neighbor-joining method by using MEGA 5.0 software. The GenBank accession numbers for the DFR sequences from these plants are as follows: A. thaliana (AED94866.1), Bromheadia finlaysoniana (AAB62873.1), Callistephus chinensis (CAA91922.1), Daucus carota (AAD56578.1), Ginkgo biloba (AGR34043.1), Ipomoea purpurea (BAA74700.1), Iris x hollandica (BAF93896.1), Lilium hybrid (BAB40789.1), Lotus japonicus1 (BAE19948.1), L. japonicus2 (AAV71171.1), L. japonicus3 (BAE19950.1), L. japonicus4 (BAE19951.1), L. japonicus5 (BAE19953.1), Medicago truncatula1 (XP_013466134.1), M. trun- catula2 (XP_013466133.1), Nicotiana tabacum (BAF96936.1), Oryza sativa (BAA36183.1), Petunia hybrid (AGI96402.1), Phoenix dactylifera (XP_008797532.1), Selaginella moellendorf- fii1 (XP_002984902.1), S. moellendorffii2 (XP_002985959.1), Vitis vinifera (NP_001268144.1), and Zea mays (NP_001152467.2). The DFR sequences from Azolla filiculoides and Salvinia cucullata were found from the CoGe (https://genomevolution.org/coge/), and the GenBank accession numbers are A. filiculoides1-6 (MN939152-MN939157) and S. cucullata1-5 (MN939158-MN939162). Isolation and cloning of DeDFR1 and DeDFR2 Total RNA was extracted from 0.5 g of rolled immature fronds of D. erythrosora using the RNApure Plant Kit (Cwbiotech, China). First-strand cDNA was subsequently synthesized using the Superscript III First-Strand Synthesis Kit (Invitrogen, USA). All steps were per- formed by following the manufacturer’s instructions supplied with the kits. The transcriptome was sequenced on the Illumina HiSeq2000 platform (Illumina, USA) at BGI (Shenzhen, China). Based on the assembled transcriptomic sequences of rolled immature fronds of D. erythro- sora (TSA number: GHEW00000000), we designed pairs of specific primers (S1 Table) for DeDFR1 and DeDFR2. TaKaRa LA Taq (Takara, Japan) was used for PCR. The thermal cycling conditions were as follows: 94˚C for 1 min, followed by 30 cycles of 94˚C for 30 s, 55˚C for 30 s, and 72˚C for 60 s, and a final extension step at 72˚C for 5 min. The PCR products were ligated to the pEASY-T5 Zero vector (Transgen Biology, China), and single colonies were selected for sequencing. Plant materials Dryopteris erythrosora was grown in a field or in pots in a growth chamber at the College of Life Science, Shanghai Normal University, China. For gene expression analysis, we sampled rolled immature fronds, young fronds and mature fronds from plants grown in the field. All fronds were frozen in liquid nitrogen and stored at -80˚C. The Arabidopsis thaliana ecotype Columbia (Col) was a gift from Prof. Zhongnan Yang (Shanghai Normal University). The Ara- bidopsis tt3-1 mutant was purchased from SALK (SALK ID: CS2114). Introduction Some species, such as Petunia and Cymbidium spe- cies, have Asp-type DFRs, which do not have the ability to catalyze DHK. Therefore, these spe- cies do not produce brick red/orange flowers with pelargonidin-based anthocyanins [26, 27]. Thus far, most genes have been isolated from seed plants, and few studies on the function of DFR genes in spore plants. Ferns are members of vascular plants that reproduce via spores and have neither seeds nor flowers. Therefore, ferns have a unique evolutionary position between bryophytes and seed plants. Dryopteris erythrosora O. Kuntze is a common garden ornamental fern with young fronds that appear red to bronze throughout the spring. Recent studies have revealed that D. erythrosora is a flavonoid-rich plant with a total flavonoid content of more than 7%, containing at least 8 flavonols, 4 flavones, 3 chalcones, 2 flavanols, 2 flavanones, 1 homoisoflavone, 1 iso- flavone, and 1 isoflavanone [28, 29]. Additionally, the flavonoid extract of D. erythrosora has antioxidant, anticancer and acetylcholinesterase inhibitor activities [30]. In addition, D. ery- throsora was one of the first ferns to be reported to contain anthocyanins, which mainly con- tain cyanidin and delphinidin [31]. However, only a few studies have examined anthocyanin biosynthesis in D. erythrosora, and the pathways in anthocyanin biosynthesis of ferns are still unclear. Previous studies have identified two upstream genes in D. fragrans, namely, 4-couma- rate:coenzyme A ligase (4CL) and chalcone synthase (CHS) [32, 33]. Campanella, et al. ana- lyzed the phylogenetic relationship of the DFR gene from the lycophyte Selaginella moellendorffii, fern Adiantum capillus-veneris and other plants, but the function of DFR enzymes in ferns have not been characterized [34]. In this paper, by analyzing transcriptome data, we isolated two new DFRs from D. erythrosora, designated DeDFR1 and DeDFR2. DeDFRs have unique residues in the region that determines substrate specificity, and these two DFRs can catalyze DHK and DHQ in vitro. Using site-directed mutagenesis, we analyzed the relationship between the region that determines substrate specificity and the substrate prefer- ences of DFRs. In addition, we investigated the potential roles of DeDFRs in plants by intro- ducing DeDFRs into Arabidopsis DFR (tt3-1) mutant plants. The results suggest that DeDFR1 and DeDFR2 are associated with color development and are helpful for understanding antho- cyanin biosynthesis in ferns. Introduction 2 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins Expression analysis of DeDFR1 and DeDFR2 The expression levels of DeDFR1 and DeDFR2 in D. erythrosora were analyzed using qRT-PCR. The cDNA used for detection of expression was synthesized by using PrimeScript™ RT Master Mix (Takara). The PCR primers were designed using Primer Premier 5.0 and are listed in S1 Table. All reactions were carried out with an ABI 7500 real-time PCR system 3 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins (Applied Biosystems, USA) with a 20-μL reaction system consisting of 10 μL of SYBR Green™ Premix Ex Taq™(Takara), 0.4 μL of ROX Reference Dye II, 0.2 μM each specific primer, and 10 ng of template cDNA. The thermal cycling conditions were as follows: an initial step of 95˚C for 30 s, followed by 40 cycles of 95˚C for 5 s and 60˚C for 34 s. A melting curve was gen- erated for each sample at the end of each run to verify the purity of the amplified products, and at least three replicates of each sample were analyzed. The transcript levels were normal- ized using DeGAPDH (GenBank: MK920229) as the internal control and calculated using the 2(−ΔΔCt) analysis method. Determination of anthocyanidin content Fern leaf (0.2 g dry weight) extracts were prepared with 4 mL of extraction solution (ethanol: water:hydrochloric acid = 2:1:1), ultrasonic extraction for 30 min, and hydrolysis in boiling water for 1 h. The supernatant was then filtered through a 0.22-μm reinforced nylon mem- brane filter (Millipore, USA). Samples were detected using a Shimadzu LC-20AD instrument equipped with an SPD-M20A photodiode array detector (Shimadzu, Japan). Twenty microli- ters of the extract was separated with a Symmetry column (C18, 250 × 4.6 mm, 5 μm, Waters, USA) and detected at 530 nm. The column oven temperature was set at 35˚C. The mobile phase consisted of 1% formic acid (A) and acetonitrile (B) with the following gradient pro- gram: 0–2 min, 8–12% B; 2–5 min, 12–18% B; 5–10 min, 18–20% B; 10–12 min, 20–25% B; 12–15 min, 25–30% B; 15–18 min, 30–45% B; 18–20 min, 45–80% B; 20–22 min, 80–8% B; 22– 30 min, 8% B. The flow rate was 0.8 mL/min. Cyanidin chloride, delphinidin chloride and pelargonidin chloride (Sigma-Aldrich, USA) were used as standards. The process was repeated at least three times for each sample. Production of recombinant DFR proteins in Escherichia coli DeDFR1 and DeDFR2 were cloned into the expression vector pET28a (+) (Novagen, USA) by using the In-Fusion PCR Cloning Kit (Takara). The PCR primers were designed using In- Fusion Cloning tools (https://www.takarabio.com). Bam HI and Sac I were used as the two restriction sites. The list of primers is provided in S1 Table. In-Fusion cloning was performed according to the manufacturer’s instructions at 50˚C for 15 min with a 5-μL reaction system consisting of 1 μL of 5× In-Fusion HD Enzyme Premix, 50 ng of linearized vector and 25 ng of purified PCR fragment. Ligated products were introduced into competent Trans5α cells (Transgen Biology) for sequencing. After confirmation of the sequences, the subcloned vectors were used to transform E. coli strain BL21 (DE3) (Transgen Biology). The empty vector and pET28a-AtDFR were also introduced into E. coli as controls. The expression of each recombinant DFR protein was induced by 0.2 mM isopropyl-thio- β-D-galactoside (IPTG) in LB culture for 20 h at 20˚C. The E. coli cells were harvested by cen- trifugation and resuspended in extraction buffer (25 mM Tris-HCl, pH 8.0). The cells were lysed by sonication, and the debris was removed by centrifugation at 8,000 rpm for 5 min at 4˚C. The protein concentration was estimated by using the Easy Protein Quantitative Kit (Transgen Biology), and the protein quality was examined using 10% SDS-PAGE. Enzyme assay Measurements of the activity of the recombinant DeDFR1 and DeDFR2 were performed according to the methods of Petit et al. and Katsu et al. with minor modifications [25, 35]. A 500-μL reaction mixture consisted of 1.6 mM NADPH, 50 mM Tris-HCl (pH 7.0), 0.8 mM substrate, and 5 mg of total protein. Three dihydroflavonols were selected as experimental sub- strates: dihydrokaempferol (DHK), dihydroquercetin (DHQ) and dihydromyricetin (DHM) 4 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins (Sigma-Aldrich). Each reaction was terminated after 30 min at 30˚C by extraction with ethyl acetate. Because the obtained leucoanthocyanidins were unstable in solution, the residues were dissolved after evaporation in 200 μL of n-BuOH-HCl (95:5) and incubated at 95˚C for 30 min to form anthocyanidins. The HPLC analysis of the DFR products was similar to the anthocya- nidin detection described above. The process was repeated at least three times for each sample. Amino acid point mutant construction The 133rd amino acid residue of AtDFR protein (Asn) was modified to Asp or Arg by overlap PCR. The principle of overlap PCR is to design two pairs of primers to amplify sequences with overlapping modified sites and then extend the overlap to obtain the complete sequence. The list of primers is provided in S1 Table. The modified AtDFRN133D and AtDFRN133R genes were ligated into the vector pET28a (+), transformed into E. coli strain BL21 (DE3), and subjected to the above enzymatic assay. Plant transformation To further characterize the function of DeDFRs, we cloned DeDFR1 and DeDFR2 into pBI121 vector harboring the CaMV 35S constitutive promoter by using the In-Fusion PCR Cloning Kit. Xba I and Sma I were used as the two restriction sites. The list of primers is provided in S1 Table. The recombinant vector was transformed into Agrobacterium tumefaciens strain GV3101 using a freeze-thaw method. Seed germination and seeding growth followed the stan- dard Arabidopsis growth protocol. Genetic transformation followed the floral dipping trans- formation protocol [36]. The growth of infected plants and seed harvesting followed the same protocol as previously reported [9, 37]. T1 seeds were selected on PNS medium with 50 mg L−1 kanamycin to obtain T2 plants. Planting of T2 seeds provided more homozygous T3 plants by DNA identification and antibiotic screening. Wild type (Col) and tt3-1 mutant plants were grown as controls. The Arabidopsis tubulin gene was used as an internal control gene to detect the DeDFRs expression level. Isolation of DeDFRs and phylogenetic analysis PhDFR: Petunia hybrid; NtDFR: Nicotiana tabacum; VvDFR: Vitis vinifera; AtDFR: Arabidopsis thaliana; ZmDFR: Zea mays; GbDFR: Ginkgo biloba; DeDFR1-2: Dryopteris erythrosora; AfDFR1-6: Azolla filiculoides; ScDFR1- 4: Salvinia cucullata. Fig 1. Alignment of the deduced amino acid sequences of DFRs. Identical residues are highlighted in black, and similar residues are highlighted in gray. A putative NAD(P)-binding domain and a putative substrate binding domain are underlined. The red asterisk indicates the 133rd amino acid residue, which is particularly important for substrate specificity. PhDFR: Petunia hybrid; NtDFR: Nicotiana tabacum; VvDFR: Vitis vinifera; AtDFR: Arabidopsis thaliana; ZmDFR: Zea mays; GbDFR: Ginkgo biloba; DeDFR1-2: Dryopteris erythrosora; AfDFR1-6: Azolla filiculoides; ScDFR1- 4: Salvinia cucullata. https://doi.org/10.1371/journal.pone.0232090.g001 five Salvinia cucullata DFRs (ScDFR1-5) and six Azolla filiculoides DFRs (AfDFR1-6) based on genomic data. The DFR sequences of the lycophyte S. moellendorffii were used as the root of the tree. This phylogenetic tree indicated that DFRs of ferns and seed plants were clearly classi- fied into different branches. Spermatophyte DFRs further diverged into different groups of gymnosperms, monocots, and eudicots. The DFR sequences of ferns were located at the base of the phylogenetic tree. The figure shows that AfDFR1-3, ScDFR1-2 and DeDFR1-2 proteins clustered within a branch, which is more similar to seed plants. It is worth noting that all the DFRs we found from fern S. cucullata, A. filiculoides and D. erythrosora belonged to neither Asn- nor Asp-type DFR. The results indicated that the sequences of DFRs of ferns and seed plants were different; thus, their protein functions may also be different. Isolation of DeDFRs and phylogenetic analysis Based on the assembled transcriptomic sequences of D. erythrosora, we screened and obtained two DeDFR genes (CL10493.Contig3 and CL16087.Contig1) and designated them DeDFR1 (GenBank: MK920230) and DeDFR2 (GenBank: MK920231). The open reading frame (ORF) of each DeDFR was analyzed, and a pair of specific primers that contained the start and stop codons was designed. The full-length cDNA of each gene was obtained by PCR. The ORF of DeDFR1 was a 987-bp segment encoding 328 amino acids (Fig 1), and the theoretical molecu- lar weight and isoelectric point were 36.3 kD and 5.41, respectively (calculated using the ExPASy website https://web.expasy.org/compute_pi/, S2 Table). DeDFR2 was a 1026-bp seg- ment encoding 341 amino acids, and the theoretical molecular weight and isoelectric point were 37.8 kD and 5.54. The identity of the deduced amino acid sequences between two DeDFRs and GbDFR were 45.94% and 47.20%, respectively. These percentages were higher than the identity between the two DeDFRs and AtDFR of 43.48% and 46.34%. In addition, the alignment showed that DeDFR1 and DeDFR2 belonged to the SDR superfamily, including a conserved NAD(P)-binding domain and a substrate-binding domain. Phylogenetic analysis of the deduced amino acid sequences of DeDFR1 and DeDFR2 was performed, and the sequences were compared to those of homologous DFRs from different species (Fig 2). To further investigate the characteristics of DFR proteins in ferns, we found PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 5 / 19 PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins Fig 1. Alignment of the deduced amino acid sequences of DFRs. Identical residues are highlighted in black, and similar residues are highlighted in gray. A putative NAD(P)-binding domain and a putative substrate binding domain are underlined. The red asterisk indicates the 133rd amino acid residue, which is particularly important for substrate specificity. PhDFR: Petunia hybrid; NtDFR: Nicotiana tabacum; VvDFR: Vitis vinifera; AtDFR: Arabidopsis thaliana; ZmDFR: Zea mays; GbDFR: Ginkgo biloba; DeDFR1-2: Dryopteris erythrosora; AfDFR1-6: Azolla filiculoides; ScDFR1- 4: Salvinia cucullata. Fig 1. Alignment of the deduced amino acid sequences of DFRs. Identical residues are highlighted in black, and similar residues are highlighted in gray. A putative NAD(P)-binding domain and a putative substrate binding domain are underlined. The red asterisk indicates the 133rd amino acid residue, which is particularly important for substrate specificity. Expression of DeDFRs and anthocyanidin accumulation during frond development For the cyanidin content, A’ and B’ above the bars indicate a significant difference between the samples at p<0.01. (e) Relative gene expression levels of DeDFRs normalized to the level of DeGAPDH. Three replicates were used for each sample. Bars represent the mean ± SD. Different lowercase letters above the bars indicate a significant difference between the samples at p<0.05. Fig 3. Expression of DeDFRs and anthocyanidin accumulation during frond development in D. erythrosora. (a) Frond development in D. erythrosora: rolled immature fronds (RF), young fronds (YF) and mature fronds (MF). (b) HPLC chromatogram of anthocyanidin standards. Peak 1, delphinidin; Peak 2, cyanidin; Peak 3, pelargonidin. (c) The HPLC profile of anthocyanidin levels in young fronds of D. erythrosora shows the elution times for delphinidin and cyanidin. (d) Accumulation of delphinidin and cyanidin in fronds. Bars represent the mean ± SD. For the delphinidin content, A, B, and C above the bars indicate a significant difference between the samples at p<0.01. For the cyanidin content, A’ and B’ above the bars indicate a significant difference between the samples at p<0.01. (e) Relative gene expression levels of DeDFRs normalized to the level of DeGAPDH. Three replicates were used for each sample. Bars represent the mean ± SD. Different lowercase letters above the bars indicate a significant difference between the samples at p<0.05. https://doi.org/10.1371/journal.pone.0232090.g003 respectively (Fig 3B). The detection results showed that cyanidin and delphinidin, but almost no pelargonidin, were present in these fern fronds (Fig 3C). The contents of both cyanidin and delphinidin were enriched in young fronds (Fig 3D). Cyanidin could be detected in all stages, and delphinidin could hardly be detected in rolled immature fronds. Transcriptomic analysis revealed that DeDFR2 was present at high levels in raw fragments of the rolled immature fronds, indicating that DeDFR2 might be closely associated with frond development (Table 1). qRT-PCR was used to examine the expression patterns of DeDFR1 and DeDFR2 (Fig 3D). The transcripts of DeDFR1 and DeDFR2 were detected in all fronds tested. The expression of DeDFR1 and DeDFR2 showed an increasing trend at first, followed by a decreasing trend, with frond development. The highest expression was observed in young fronds, followed by rolled immature fronds, and relatively low expression was observed in mature fronds, which is consistent with the change trend observed for cyanidin and delphinidin. Expression of DeDFRs and anthocyanidin accumulation during frond development Based on the accumu- lation of anthocyanins and the expression of DeDFRs, we speculated that DeDFR1 and DeDFR2 might be involved in anthocyanin synthesis. Expression of DeDFRs and anthocyanidin accumulation during frond development In D. erythrosora, the fronds showed a significant color change during the growth process (Fig 3A). The anthocyanidin component in different stages of frond development was detect by HPLC. At 520 nm, the chromatograms showed that the standard peaks of cyanidin, delphini- din and pelargonidin appeared at approximately 11.09 min, 9.16 min, and 13.22 min, 6 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 PLOS ONE PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins Fig 2. Alignment of the deduced amino acid sequences of DFRs. A phylogenetic tree of DFRs constructed by the neighbor-joining method with 1000 bootstrap replicates. Asn-type (no label), Asp-type (labeled with a filled triangle), Arg-type (labeled with a filled circle) and non-Asn/ Asp/Arg-type (labeled with a square) DFRs are labeled with different shapes in front of the species. For accession numbers of the DFR sequences, see Materials and Methods. https://doi.org/10.1371/journal.pone.0232090.g002 Fig 2. Alignment of the deduced amino acid sequences of DFRs. A phylogenetic tree of DFRs constructed by the neighbor-joining method with 1000 bootstrap replicates. Asn-type (no label), Asp-type (labeled with a filled triangle), Arg-type (labeled with a filled circle) and non-Asn/ Asp/Arg-type (labeled with a square) DFRs are labeled with different shapes in front of the species. For accession numbers of the DFR sequences, see Materials and Methods. PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 7 / 19 7 / 19 Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins PLOS ONE .org/10.1371/journal.pone.0232090 May 1, 2020 8 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 8 / 19 PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins Fig 3. Expression of DeDFRs and anthocyanidin accumulation during frond development in D. erythrosora. (a) Frond development in D. erythrosora: rolled immature fronds (RF), young fronds (YF) and mature fronds (MF). (b) HPLC chromatogram of anthocyanidin standards. Peak 1, delphinidin; Peak 2, cyanidin; Peak 3, pelargonidin. (c) The HPLC profile of anthocyanidin levels in young fronds of D. erythrosora shows the elution times for delphinidin and cyanidin. (d) Accumulation of delphinidin and cyanidin in fronds. Bars represent the mean ± SD. For the delphinidin content, A, B, and C above the bars indicate a significant difference between the samples at p<0.01. Heterologous expression and catalytic activity of DeDFRs To identify the catalytic function of DeDFR enzymes on dihydroflavonols, prokaryotic expres- sion was implemented. Using the In-Fusion PCR cloning method, the ORFs of DeDFR1 and DeDFR2 were inserted into the expression vector pET28a (+) to obtain recombinant proteins. The SDS-PAGE results showed that these recombinant proteins were similar in total protein content (S1 Fig). The cumbersome purification process tended to inactivate the recombinant protein, and the impure protein obtained after purification affected the accuracy of the protein content determination. Therefore, our activity analysis was based directly on the total protein content. Crude extracts of Escherichia coli expressing DFRs were subjected to enzyme assays using DHK, DHQ, and DHM as substrates in the presence of NADPH. The reaction product with an empty vector was colorless, and the HPLC chromatogram showed no significant prod- uct peak at 520 nm. Additionally, the results for the positive control AtDFR, an Asn-type DFR, showed that all three dihydroflavonols could be converted to pink anthocyanins, which could be detected by HPLC at 520 nm (S1 Fig). DeDFR1 and DeDFR2 had the ability to catalyze the DHK and DHQ reaction to produce the desired products. However, they could not catalyze DHM to produce the products of AtDFR (S2 Fig). The product peak area in HPLC is shown in Fig 4. The results showed that the catalytic products of DeDFR1 and DeDFR2 were lower than those of AtDFR regardless of the use of DHK or DHQ as a substrate. The amount of product of DeDFR1 and DeDFR2 was different. When using DHK as a substrate, the target product of Table 1. The transcripts of DeDFR1 and DeDFR2 in rolled immature fronds in D. erythrosora. Given name Unigene Length ORF Length Raw fragments FPKM DeDFR1 1914 987 1147 13.6767 DeDFR2 1219 1026 25925 485.3722 https://doi.org/10.1371/journal.pone.0232090.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 9 / 19 Table 1. The transcripts of DeDFR1 and DeDFR2 in rolled immature fronds in D. erythrosora. PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 9 / 19 PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins DeDFR1 was comparable to that of DeDFR2. However, when using DHQ as a substrate, the target product of DeDFR2 was lower than DeDFR1 by approximately 24%. The above data provide evidence that the DeDFR1 and DeDFR2 in D erythrosora had DFR activity In addi Fig 4. Heterologous expression and catalytic activity of DeDFRs https://doi.org/10.1371/journal.pone.0232090.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 10 / 19 PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins Heterologous expression and catalytic activity of DeDFRs The product peak area of HPLC for DeDFR1, DeDFR2 and AtDFR proteins with DHK, DHQ, and DHM as substrates. Bars represent the mean ± SD. Statistically significant difference compared with AtDFR at 0.01<p<0.05. Statistically significant difference compared with AtDFR at p<0.01. https://doi.org/10.1371/journal.pone.0232090.g004 Fig 4. The product peak area of HPLC for DeDFR1, DeDFR2 and AtDFR proteins with DHK, DHQ, and DHM as substrates. Bars represent the mean ± SD. Statistically significant difference compared with AtDFR at 0.01<p<0.05. Statistically significant difference compared with AtDFR at p<0.01. Fig 4. The product peak area of HPLC for DeDFR1, DeDFR2 and AtDFR proteins with DHK, DHQ, and DHM as substrates. Bars represent the mean ± SD. Statistically significant difference compared with AtDFR at 0.01<p<0.05. Statistically significant difference compared with AtDFR at p<0.01. https://doi.org/10.1371/journal.pone.0232090.g004 https://doi.org/10.1371/journal.pone.0232090.g004 DeDFR1 was comparable to that of DeDFR2. However, when using DHQ as a substrate, the target product of DeDFR2 was lower than DeDFR1 by approximately 24%. The above data provide evidence that the DeDFR1 and DeDFR2 in D. erythrosora had DFR activity. In addi- tion, the substrate specificity of these enzymes differed from that of AtDFR, which could cata- lyze DHM. DeDFR1 was comparable to that of DeDFR2. However, when using DHQ as a substrate, the target product of DeDFR2 was lower than DeDFR1 by approximately 24%. The above data provide evidence that the DeDFR1 and DeDFR2 in D. erythrosora had DFR activity. In addi- tion, the substrate specificity of these enzymes differed from that of AtDFR, which could cata- lyze DHM. Fig 5. The product peak area of HPLC for modified AtDFR proteins with DHK, DHQ, and DHM as substrates. Bars represent the mean ± SD. Statistically significant difference compared with AtDFR at 0.01<p<0.05. Statistically significant difference compared with AtDFR at p<0.01. https://doi.org/10.1371/journal.pone.0232090.g005 Fig 5. The product peak area of HPLC for modified AtDFR proteins with DHK, DHQ, and DHM as substrates. Bars represent the mean ± SD. Statistically significant difference compared with AtDFR at 0.01<p<0.05. Statistically significant difference compared with AtDFR at p<0.01. Fig 5. The product peak area of HPLC for modified AtDFR proteins with DHK, DHQ, and DHM as substrates. Bars represent the mean ± SD. Statistically significant difference compared with AtDFR at 0.01<p<0.05. Statistically significant difference compared with AtDFR at p<0.01. Site-directed mutagenesis of the substrate-specificity-determining site We found that both DeDFR1 and DeDFR2 had an Arg at the substrate-specificity-determining site, and they did not have the same ability to catalyze DHM like the Asn-type DFR. To verify the substrate preferences of the Arg-type DFR, the overlap PCR method was used to modify AtDFR to AtDFRN133D and AtDFRN133R (S3 Fig). Because modification of the 133rd residue hardly affected the solubility of AtDFR, we analyzed the activity of the modified recombinant proteins by quantifying the total protein content. Crude extracts of E. coli expressing modified AtDFRs were subjected to enzyme assays using DHK, DHQ, and DHM as substrates in the presence of NADPH (S2 Fig). The results showed that the enzyme activity was changed after the site was modified (Fig 5). The catalytic capabilities of AtDFR, AtDFRN133D and AtDFRN133R were quite different. When DHK was used as a substrate, the AtDFRN133D activi- ties decreased significantly. However, when DHQ was used as a substrate, the target product of AtDFRN133R yielded approximately half of that obtained with AtDFR. Notably, when DHM was used as a substrate, products were not detected with AtDFRN133R, as observed for DeDFR1 and DeDFR2. The site-directed mutagenesis results showed that the 133rd amino acid residue was indeed an important site affecting substrate specificity. They also showed that Arg-type DFRs could hardly catalyze DHM. Overexpression of DeDFRs in Arabidopsis tt3-1 Mutant The Arabidopsis tt3-1 mutant is characterized by the knockout of DFR, resulting in a lack of anthocyanins, so the seed coat is yellow due to absence of pigment. To investigate the function of DeDFRs in plants, they were ectopically expressed in Arabidopsis tt3-1 mutant. Using the flower dip transformation method, transgenic DeDFR1 and DeDFR2 plants were obtained (Fig 6A). The results showed that the DeDFR1 gene could not be expressed in the transgenic plants, and the seeds of the transgenic plants were still yellow. By analyzing the codon preference, we found that unlike AtDFR or DeDFR2, multiple arginines were encoded by CGC in DeDFR1, which might have led to the lack of DeDFR1 expression in Arabidopsis (S4 Fig). Phenotypic observation showed that transgenic plants expressing DeDFR2 exhibited a restored brown color of the seed coat similar to wild type seeds. The anthocyanidin content (mainly cyanidin) in seeds of transgenic DeDFR2 Arabidopsis was higher than in the mutant and wild type (Fig 6B). The presence and expression of the foreign gene in the transgenic lines were further con- firmed by RT-PCR. The amplification of DeDFR2 was observed in transgenic lines, not in wild type or mutants plants (Fig 6C). These results indicated that the overexpression of DeDFR2 in the tt3-1 mutant reconstructed the mutated anthocyanin biosynthesis. PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 Discussion The Arabidopsis tt3-1 mutant is characterized by knockout of DFR, resulting in a lack of anthocyanins. Seed plant studies have shown that red/purple leaves or flowers are enriched in anthocyanins [38–41]. In Malus crabapple, the abundance of anthocyanins in red-leaf cultivars decreased with leaf maturation, but almost no anthocyanins were detected in the evergreen cultivar [42]. Similar to the above result, we found that the red young fronds had the highest anthocyanin content and that the greenish mature fronds had relatively low anthocyanin levels in D. erythrosora. Furthermore, researchers found that anthocyanin concentrations were posi- tively correlated with DFR expression in a variety of plants, such as B. oleracea, G. biloba and Pyrus communis [16, 43, 44]. By analyzing the transcripts, we found that the anthocyanin con- tent was highly consistent with the trend of changes in DeDFR1 and DeDFR2 expression. In D. erythrosora, DeDFR1 and DeDFR2 were initially expressed in rolled immature fronds to pro- duce anthocyanins. The expression levels of DeDFR1 and DeDFR2 and total anthocyanins PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 11 / 19 Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins PLOS ONE Fig 6. Overexpression of DeDFR1 and DeDFR2 in Arabidopsis tt3-1 mutant. (a) Phenotype of the wild type (WT, Col), tt3-1 mutant and transgenic lines seed coats. (b) HPLC analysis of cyanidin content in the seeds of wild-type, tt3-1 mutant and transgenic lines. Three replicates were used for each sample. Bars represent the mean ± SD. Different capital letters above the bars indicate a significant difference between the samples at p<0.01. (c) Expressional analysis of DeDFR1 and DeDFR2 by RT-PCR in the leaves of wild-type, tt3-1 mutant and transgenic lines. https://doi org/10 1371/journal pone 0232090 g006 Fi 6 O i f D DFR1 d D DFR2 i A bid i tt3 1 ( ) Ph f h ild (WT C l) 3 1 d i li Fig 6. Overexpression of DeDFR1 and DeDFR2 in Arabidopsis tt3-1 mutant. (a) Phenotype of the wild type (WT, Col), tt3-1 mutant and transgenic lines seed coats. (b) HPLC analysis of cyanidin content in the seeds of wild-type, tt3-1 mutant and transgenic lines. Three replicates were used for each sample. Bars represent the mean ± SD. Different capital letters above the bars indicate a significant difference between the samples at p<0.01. PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 Discussion DeDFR1, AfDFR1 and ScDFR1 clustered into one branch, while DeDFR2, AfDFR2, AfDFR3, and ScDFR2 clustered into another branch. Thus, it can be deduced that DeDFR1 and DeDFR2 are independently evolved and may have different functions in D. erythrosora. and ScDFR2 clustered into another branch. Thus, it can be deduced that DeDFR1 and DeDFR2 are independently evolved and may have different functions in D. erythrosora. Previous studies have indicated that amino acid residue 133 plays an important role in sub- strate specificity and classified DFRs into different types based on this site, i.e., Asn-type DFRs, Asp-type DFRs, and non-Asn/Asp-type DFRs, with the amino acid residue at the 133 position being asparagine (Asn), aspartic acid (Asp) and neither Asn nor Asp, respectively [37, 47]. Most previous reports have suggested that Asn-type DFRs and Asp-type DFRs have the func- tion of catalyzing dihydroflavonol [13, 47, 48]. However, we did not find an Asn-type or Asp- type DFR in D. erythrosora, S. cucullata and A. filiculoides. Unlike seed plants, most of these DFR proteins in the three ferns have an arginine at the substrate-specificity-determining site. On the other hand, unlike Asn-type or Asp-type DFRs, DeDFR1 and DeDFR2 proteins cannot catalyze DHM. Thus, we speculated that there is a new DFR functional type in ferns, and we named it Arg-type DFR. The Arg-type DFR was not reported in the current research on seed plants, and it might be unique to ferns. Site-directed mutagenesis is a good way to verify the contribution of a specialized amino acid residue to the activity and substrate preference of DFRs. Based on the experiments, we found that modification of the 133rd amino acid residue of AtDFR could change the enzy- matic activity efficiency. The present investigation showed that the modified AtDFRN133D exhibited a reduced ability to catalyze the conversion of DHK to leucopelargonidins, which is consistent with studies of M. truncatula and L. japonicus [12, 13]. Although Asp-type DFRs exhibit a certain catalytic ability toward DHK in vitro, pelargonidin-based anthocyanins are not detected in plants with Asp-type DFRs [26, 47]. We speculate that in plants with Asp-type DFRs, leucopelargonidins are converted to the highly stable cyanidin/delphinidin-based anthocyanins by F3’H or F3’5’H, making it difficult for the plants to produce pelargonidin- based anthocyanins. Discussion (c) Expressional analysis of DeDFR1 and DeDFR2 by RT-PCR in the leaves of wild-type, tt3-1 mutant and transgenic lines. https://doi.org/10.1371/journal.pone.0232090.g006 peaked in young fronds. After the fronds matured, the expression of DeDFR1 and DeDFR2 decreased, the anthocyanin content was diluted, and the color of the mature fronds gradually deceased in intensity. Similar phenomena also occur in M. crabapple; McDFR1 is mainly expressed in the early stage of red leaf growth [42]. Thus, DeDFR1 and DeDFR2 may be associ- ated with anthocyanin coloration in fronds of D. erythrosora. Multiple DFR genes are present in plants. At least five different DFRs exist in L. japonicus [13]. The P. hybrida genome contains three DFR genes located on chromosomes 2, 4 and 6 [45]. Both Medicago truncatula and Fagopyrum esculentum contain two different DFRs [12, 35]. The Arabidopsis genome contains only one DFR gene [19]. However, there have been few reports of DFRs in ferns, mainly due to the substantial differences between the genomes of ferns and seed plants, making it almost impossible to obtain functional genes by homologous cloning. Conversely, there is a lack of genomic data for ferns—only the genomes of A. filicu- loides and S. cucullata have been reported [46]—so there are few molecular studies on ferns. Transcriptome sequencing technology provides a wealth of data for functional genetic studies of species without genomic data. In the present study, we found two DFRs from the transcrip- tomic data of D. erythrosora, five DFRs from the genomic data of S. cucullata and six DFRs from the genomic data of A. filiculoides. All of these genes belong to the SDR superfamily, with a conserved NAD(P)-binding domain and a substrate-binding domain. DFR evolution can be 12 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins clearly understood by generating a phylogenic tree with a broad range of plant species. As clearly understood by generating a phylogenic tree with a broad range of plant species. As shown in Fig 2, DFRs of ferns and seed plants were clearly classified into different branches. DeDFR1, AfDFR1 and ScDFR1 clustered into one branch, while DeDFR2, AfDFR2, AfDFR3, and ScDFR2 clustered into another branch. Thus, it can be deduced that DeDFR1 and DeDFR2 are independently evolved and may have different functions in D. erythrosora. PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 Discussion In summary, the 133rd amino acid residue of DFRs is an PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 13 / 19 PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins Arg type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 14 / 19 PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins Fig 7. Proposed pathway for anthocyanin biosynthesis in D. erythrosora. The enzymes studied herein are represented in red font. Abbreviations: CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; F30H, flavonoid 30-hydroxylase; F3050H, flavonoid 3050-hydroxylase; FLS, flavonol synthase; DHK, dihydrokaempferol; DHQ, dihydroquercetin; DHM, dihydromyricetin. https://doi.org/10.1371/journal.pone.0232090.g007 Fig 7. Proposed pathway for anthocyanin biosynthesis in D. erythrosora. The enzymes studied herein are represented in red font. Abbreviations: CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; F30H, flavonoid 30-hydroxylase; F3050H, flavonoid 3050-hydroxylase; FLS, flavonol synthase; DHK, dihydrokaempferol; DHQ, dihydroquercetin; DHM, dihydromyricetin. https://doi.org/10.1371/journal.pone.0232090.g007 Fig 7. Proposed pathway for anthocyanin biosynthesis in D. erythrosora. The enzymes studied herein are represented in red font. Abbreviations: CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; F30H, flavonoid 30-hydroxylase; F3050H, flavonoid 3050-hydroxylase; FLS, flavonol synthase; DHK, dihydrokaempferol; DHQ, dihydroquercetin; DHM, dihydromyricetin. https://doi.org/10.1371/journal.pone.0232090.g007 https://doi.org/10.1371/journal.pone.0232090.g007 important substrate recognition site, and different amino acid residues at this site directly affect the substrate binding ability. Catalytic activity analysis showed that the function of DeDFR1 and DeDFR2 is mainly to catalyze DHK and DHQ. Therefore, the expected products are pelargonidin-based and cyani- din-based anthocyanins, but we only detected cyanidin-based and delphinidin-based anthocy- anins in D. erythrosora. The lack of pelargonidin-based anthocyanins might be due to the influence of downstream genes, such as F3’H and F3’5’H, which convert the product of DeDFR1 and DeDFR2 to a more stable structure with two or three hydroxyl groups on ring B (Fig 7). In previous studies, a few glycosylation compounds of quercetin, kaempferol, and myr- icetin were detected in D. erythrosora [29, 30], indicating that ferns have enzymes similar to FLS, which can compete with DFRs for substrates. Interestingly, Zhang et al. detected DHM in D. erythrosora [49], indicating that the functional enzymes acting on DHM are not sufficient for complete digestion of this substrate. Conclusions The newly identified DeDFR1 and DeDFR2 genes were associated with the accumulation of anthocyanins in D. erythrosora. Moreover, both DeDFR1 and DeDFR2 enzyme proteins can catalyze DHK and DHQ but do not catalyze DHM. DeDFR1 and DeDFR2 are Arg-type DFRs, a previously unreported DFR type. This finding might provide new insights into the anthocya- nin biosynthesis pathway in ferns. Discussion This finding is consistent with the results showing that DeDFR cannot catalyze DHM. Whether the synthetic process of cyanidin-based and delphini- din-based anthocyanins is consistent with our hypothesis is worth exploring in the future. The development of plants is a synergistic process, and the synthesis of anthocyanins is not con- trolled by a single gene. The mechanism by which DeDFR and F3’H/F3’5’H synergistically con- trol the anthocyanin type, the competition mechanism between DeDFR and FLS, and whether the process of downstream anthocyanin synthesis in ferns is the same as in seed plants remain to be further studied. The discovery of the new functional Arg-type DFR also provides a new direction for flower cultivation. Supporting information S1 Fig. SDS-PAGE analysis of DFRs and catalytic activity of AtDFR. (a) Coomassie brilliant blue-stained polyacrylamide gel. M, protein marker. Lane 1, soluble lysate fraction from E. coli BL21 (DE3) harboring pET28a-AtDFR induced by IPTG. Lane 2, soluble lysate fraction from E. coli BL21 (DE3) harboring pET28a-DeDFR1 induced by IPTG. Lane 3, soluble lysate frac- tion from E. coli BL21 (DE3) harboring pET28a-DeDFR2 induced by IPTG. (b) DHK, DHQ, and DHM can be converted to pink compounds by AtDFR (1–3). DHK, DHQ, and DHM can- not be converted to colored compounds by the empty pET-28a (4–6). (c) Schematic HPLC chromatograms of the reaction products of the three substrates with AtDFR. (TIF) Discussion Conversely, we found that the modified AtDFRN133R exhibited a reduced ability to catalyze DHQ, and we did not detect the product of DHM, which is similar to the cat- alytic effects observed for DeDFR1 and DeDFR2. This finding demonstrates that Arg-type DFRs are significantly different from the Asp and Asn types in terms of function. Notably, the product yields of DeDFR1 and DeDFR2 were significantly lower than AtDFRN133R. This find- ing indicates that in Arabidopsis, the structure of DFR is favorable for substrate binding, so the modified AtDFRN133R is highly efficient. It is possible that other sites can affect binding effi- ciency via the spatial configuration. In the present study, the 133rd amino acids of the functional DFRs were Arg, Asn and Asp, all of which are polar amino acids [35]. Studies on the crystal structure of DFRs from V. vinif- era have shown that the side chains of N133 interact with both hydroxyl groups of ring B of DHQ. Moreover, the carboxyl and amino groups of N133 can make contacts with the ligand at C3’ and C4’ of DHQ, respectively [25]. Based on the docking model [35], we analyzed the dif- ferences in binding among proteins with different 133rd amino acids and for different ligands. The Asp-type DFR is able to catalyze DHQ via the carboxyl group on the side chain of D133; however, it is difficult to catalyze DHK due to the lack of an amino group in contact with the hydroxyl group on C4’. The Arg-type DFR catalyzes DHQ inefficiently, mainly due to the lack of a carboxyl group on the side chain of R133. The Arg-type DFR can catalyze DHK as effi- ciently as the Asn-type DFR, mainly via the interaction of the amino group with the hydroxyl group on C4’. These findings explain why different types of DFRs have different catalytic capa- bilities for dihydroflavonols. S4 Fig. Codon usage analysis of AtDFR, DeDFR1 and DeDFR2. (TIF) S4 Fig. Codon usage analysis of AtDFR, DeDFR1 and DeDFR2. (TIF) g g y , (TIF) S1 Table. List of primers used in this study. (DOCX) S2 Table. The theoretical molecular weight and isoelectric point for DFRs. (DOCX) S1 Raw Images. Original gel photos. (PDF) S2 Table. The theoretical molecular weight and isoelectric point for DFRs. (DOCX) Acknowledgments We thank Prof. Zhongnan Yang for supplying the seeds of Arabidopsis (Col). We thank Prof. Zhongnan Yang for supplying the seeds of Arabidopsis (Col). S2 Fig. HPLC chromatograms of the reaction products of three substrates with DeDFRs and modified AtDFR. (TIF) 15 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 PLOS ONE Arg-type DFRs from Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins S3 Fig. SDS-PAGE analysis of the site-directed mutagenesis of AtDFR protein. (a, b)The cDNA and deduced amino acid sequence of modified AtDFR. (c) Amplification of the AtDFRN133R gene. M, DL2000 DNA marker. Lane 1, amplification of the 5 ’end sequence. Lane 2, amplification of the 3 ’end sequence. Lane 3, amplification of the full-length AtDFRN133R. (d) Amplification of the full-length modified AtDFR. M, DL2000 DNA marker. Lane 1, ampli- fication of the full-length AtDFR. Lane 2, amplification of the full-length AtDFRN133R. Lane 3, amplification of the full-length AtDFRN133D. (e) Coomassie brilliant blue-stained polyacryl- amide gel of modified AtDFR. M, protein marker. Lane 1, soluble lysate fraction from E. coli BL21 (DE3) harboring pET28a-AtDFR induced by IPTG. Lane 2, soluble lysate fraction from E. coli BL21 (DE3) harboring pET28a- AtDFRN133R induced by IPTG. Lane 3, soluble lysate fraction from E. coli BL21 (DE3) harboring pET28a- AtDFRN133D induced by IPTG. (TIF) S3 Fig. SDS-PAGE analysis of the site-directed mutagenesis of AtDFR protein. (a, b)The cDNA and deduced amino acid sequence of modified AtDFR. (c) Amplification of the AtDFRN133R gene. M, DL2000 DNA marker. Lane 1, amplification of the 5 ’end sequence. Lane 2, amplification of the 3 ’end sequence. Lane 3, amplification of the full-length AtDFRN133R. (d) Amplification of the full-length modified AtDFR. M, DL2000 DNA marker. Lane 1, ampli- fication of the full-length AtDFR. Lane 2, amplification of the full-length AtDFRN133R. Lane 3, amplification of the full-length AtDFRN133D. (e) Coomassie brilliant blue-stained polyacryl- amide gel of modified AtDFR. M, protein marker. Lane 1, soluble lysate fraction from E. coli BL21 (DE3) harboring pET28a-AtDFR induced by IPTG. Lane 2, soluble lysate fraction from E. coli BL21 (DE3) harboring pET28a- AtDFRN133R induced by IPTG. Lane 3, soluble lysate fraction from E. coli BL21 (DE3) harboring pET28a- AtDFRN133D induced by IPTG. (TIF) S3 Fig. SDS-PAGE analysis of the site-directed mutagenesis of AtDFR protein. (a, b)T cDNA and deduced amino acid sequence of modified AtDFR. (c) Amplification of the AtDFRN133R gene. M, DL2000 DNA marker. Lane 1, amplification of the 5 ’end sequence 2, amplification of the 3 ’end sequence. Lane 3, amplification of the full-length AtDFRN13 (d) Amplification of the full-length modified AtDFR. M, DL2000 DNA marker. Lane 1, fication of the full-length AtDFR. S2 Fig. HPLC chromatograms of the reaction products of three substrates with DeDFRs and modified AtDFR. (TIF) Lane 2, amplification of the full-length AtDFRN133R. La amplification of the full-length AtDFRN133D. (e) Coomassie brilliant blue-stained polyacr amide gel of modified AtDFR. M, protein marker. Lane 1, soluble lysate fraction from E. BL21 (DE3) harboring pET28a-AtDFR induced by IPTG. Lane 2, soluble lysate fraction E. coli BL21 (DE3) harboring pET28a- AtDFRN133R induced by IPTG. Lane 3, soluble lys fraction from E. coli BL21 (DE3) harboring pET28a- AtDFRN133D induced by IPTG. (TIF) S4 Fig. Codon usage analysis of AtDFR, DeDFR1 and DeDFR2. (TIF) S1 Table. List of primers used in this study. (DOCX) S2 Table. The theoretical molecular weight and isoelectric point for DFRs. (DOCX) S1 Raw Images. Original gel photos. (PDF) PLOS ONE | https://doi.org/10.1371/journal.pone.0232090 May 1, 2020 References 1. 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Graves’ disease after exposure to the SARS-CoV-2 vaccine: a case report and review of the literature
BMC endocrine disorders
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(2023) 23:132 Takedani et al. BMC Endocrine Disorders https://doi.org/10.1186/s12902-023-01387-2 BMC Endocrine Disorders Open Access CASE REPORT Graves’ disease after exposure to the SARS‑CoV‑2 vaccine: a case report and review of the literature Kai Takedani1,2, Masakazu Notsu2* , Naoto Ishiai1, Yu Asami1, Kazuhiko Uchida3 and Keizo Kanasaki2 Abstract Background Autoimmune/inflammatory syndrome induced by adjuvants (ASIA) is characterized by immune system dysregulation after exposure to adjuvants, such as aluminum. Although cases of autoimmune thyroid diseases caused by ASIA have been reported, Graves’ disease is one of the rarer diseases. There are some reports that vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause ASIA. Here, we describe a case of Graves’ disease following SARS-CoV-2 vaccination and a review of the literature. Case presentation A 41-year-old woman was admitted to our hospital because of palpitations and fatigue. Two weeks after receiving the second SARS-CoV-2 vaccine (BNT162b2, Coronavirus Modified Uridine messenger RNA (mRNA) Vaccine, Pfizer), she developed fatigue and gradually worsened. On admission, she exhibited thyrotoxicosis (thyroid-stimulating hormone (TSH) < 0.01 mIU/L (0.08–0.54), free triiodothyronine (FT3) 33.2 pmol/L (3.8–6.3), and free thyroxine (FT4) 72.1 pmol/L (11.6–19.3)) and palpitations associated with atrial fibrillation. TSH receptor antibody (TRAb) was positive (TRAb 5.0 IU/L (< 2.0)), and 99mTc scintigraphy showed diffuse uptake in the thyroid gland, suggesting that the thyrotoxicosis in this case was caused by Graves’ disease. Thiamazole was prescribed to correct her condition, and soon after this treatment was initiated, her symptoms and thyroid hormone levels were significantly reduced. Conclusions This case report reinforces the potential correlation between ASIA affecting the thyroid and SARS-CoV-2 mRNA vaccines. The clinical course suggests that it is essential to consider the possibility of developing ASIA, such as Graves’ disease, after exposure to the SARS-CoV-2 vaccine. Keywords Graves’ disease, COVID-19, Vaccine, Autoimmune/inflammatory syndrome induced by adjuvants *Correspondence: Masakazu Notsu mnotsu25@med.shimane-u.ac.jp 1 Department of Internal Medicine, Masuda Red Cross Hospital, Masuda, Shimane, Japan 2 Department of Internal Medicine 1, Faculty of Medicine, Shimane University, 89‑1 Enya‑Cho, Izumo, Shimane 693‑8501, Japan 3 Department of Cardiology, Masuda Red Cross Hospital, Masuda, Shimane, Japan Background Graves’ disease is an autoimmune thyroid disease (AITD) and is recognized by the presence of thyroid-stimulating hormone (TSH) receptor antibody (TRAb) [1, 2]. That constitutively activates the thyroid gland, leading to a state of diffuse hyperthyroidism. Graves’ disease is the most common cause of hyperthyroidism. The underlying factors involved in the development of Graves’ disease remain uncertain. A few genetic loci have been suggested, as well as smoking, stress, and exposure to great amounts of iodine, despite a lack of conclusive causal relationships [3]. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Takedani et al. BMC Endocrine Disorders (2023) 23:132 Adjuvants encompass several substances commonly used in vaccines to boost immune reactivity toward antigens, and autoimmune/inflammatory syndrome induced by adjuvants (ASIA) is characterized by innate and adaptive immune system dysregulation after exposure to adjuvants, such as aluminum [4, 5]. Although cases of AITD caused by ASIA have been reported, most of them were destructive thyroiditis, and Graves’ disease is a rare occurrence [6–8]. COVID-19 poses a significant threat to the entire world. Vaccines of different technologies and types have been developed to reduce COVID-19 infection and severity [9]. However, there are some reports that vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause ASIA, including cases of Graves’ disease [10, 11]. Here, we present a case of Graves’ disease following SARS-CoV-2 vaccination and describe other cases found through a literature review. Case presentation A 41-year-old woman was admitted to our hospital because of palpitations and fatigue. Three months prior to hospitalization, she received the first vaccine for SARS-CoV-2 (BNT162b2, Coronavirus Modified Uridine messenger RNA (mRNA) Vaccine, Pfizer), and only mild pain at the vaccination site was observed. Three weeks later, she received the second dose of the vaccine. The next day, she had a fever and took acetaminophen orally, and the fever disappeared in one day. Two weeks later, she developed fatigue and gradually worsened. A week before admission, she went to the emergency department because of palpitations associated with atrial fibrillation and demonstrated thyrotoxicosis (TSH < 0.01 mIU/L (0.08–0.54, CLEIA method), free triiodothyronine (FT3) 28.3 pmol/L (3.8–6.3, CLEIA method), and free thyroxine (FT4) 61.8 pmol/L (11.6–19.3, CLEIA method)). At that time, she went home having received only symptomatic treatment. At the follow-up visit, her general status was not improved, and she was hospitalized to treat thyrotoxicosis. She had no family history of endocrine diseases, including thyroid disease. She was a nonsmoker and nondrinker. She was not exposed to intense stress or excess iodine. She had no past history and did not have a preceding cold. She did not have a prior COVID-19 infection. Two months before the appearance of her symptoms, the electrocardiogram showed sinus rhythm, and 18 months before, her thyroid hormone levels were normal. Her body weight had decreased by 3 kg in the three months before admission. Her menstruation was regular before hospitalization, and she was not pregnant. She had irregularly taken iron supplements due to iron deficiency anemia. Page 2 of 7 Her height was 163 cm, her body weight was 39 kg, and her body mass index (BMI) was 14.7 kg/m2. Her consciousness was clear. Her blood pressure was 107/52 mmHg, her pulse rate was 100 beats/min and irregular, her body temperature was 36.7 °C, and her oxygen saturation (­SpO2) was 100%. Her anterior neck was not swollen and without pain. She had a tremor. She had diarrhea without stomach pain. The other physical findings were unremarkable. She did not have eye symptoms. The laboratory findings on admission are shown in Table 1. She had severe thyrotoxicosis (TSH < 0.01 mIU/L, FT3 33.2 pmol/L, FT4 72.1 pmol/L). The presence of only gastrointestinal manifestation (diarrhea) suggested that she did not have a thyroid crisis. TRAb and thyroid-stimulating antibody (TSAb) were positive (TRAb 5.0 IU/L (< 2.0, CLEIA method) and TSAb 262% (< 120, EIA method)). Inflammatory markers were not elevated. Thyroglobulin antibody (Tg-Ab) and thyroid peroxidase antibody (TPO-Ab) were negative. Antinuclear antibody, which was screened to check for other autoimmune diseases, was also negative. Ultrasound revealed heterogeneous tissue and increased blood flow throughout the thyroid gland without diffuse goiter (Fig. 1). 99mTc pertechnetate scintigraphy showed diffuse uptake in the thyroid gland, and 99mTc uptake was 11.0% (0.5–3.5) (Fig. 2). These findings suggested that thyrotoxicosis in this case was caused by Graves’ disease, not destructive thyroiditis. An electrocardiogram showed slight tachycardia due to atrial fibrillation. Echocardiography revealed no abnormalities. Thiamazole and bisoprolol were prescribed to correct her condition (Fig. 3). Soon after this treatment was initiated, her palpitations and fatigue were significantly relieved, and she was discharged from the hospital. After Table 1 Baseline laboratory data Parameter Observed Reference range Serum characteristics CRP, mg/dL 0.1 < 0.3 ESR (1 h), mm 13 3–15 FT3, pmol/L 33.2 3.8–6.3 FT4, pmol/L 72.1 11.6–19.3 TSH, mIU/L < 0.01 0.08–0.54 TRAb, IU/L 5.0 < 2.0 TSAb, % 262 < 120 Tg-Ab, IU/mL 0.9 < 4.1 TPO-Ab, IU/mL 2.2 < 5.6 Tg, ng/mL 121 < 34 CRP C-reactive protein, ESR erythrocyte sedimentation rate, FT3 free triiodothyronine, FT4 free thyroxine, TSH thyroid-stimulating hormone, TRAb TSH receptor antibody, TSAb thyroid stimulating antibody, Tg-Ab thyroglobulin antibody, TPO-Ab thyroid peroxidase antibody, Tg thyroglobulin Takedani et al. BMC Endocrine Disorders (2023) 23:132 Page 3 of 7 Fig. 1 Thyroid ultrasonography. Right lobe: 15 × 50 × 13 mm. Left lobe: 13 × 50 × 11 mm. Increased blood flow throughout the thyroid gland without diffuse goiter was revealed that, her thyroid hormone levels improved, and the bisoprolol dose was reduced. There were no side effects associated with the treatment. Fig. 2 99mTc scintigraphy. Diffuse uptake in the thyroid gland was revealed Discussion and conclusions We reported a case of Graves’ disease following SARSCoV-2 vaccination. Treatment with thiamazole was significantly effective for her condition. Graves’ disease is an AITD marked by the presence of TRAb, with manifestations of diffuse goiter, thyrotoxicosis, and ophthalmopathy [1, 2]. The most common cause of hyperthyroidism is Graves’ disease. The causes of Graves’ disease are a loss of immunotolerance and the development of autoantibodies that stimulate thyroid follicular cells by binding to the TSH receptor. Our patient displayed symptoms due to excess thyroid hormones, such as palpitations due to atrial fibrillation, weight loss, fatigue, tremor, diarrhea, and menstrual disturbances. In addition, she demonstrated thyrotoxicosis with TSH levels under the detection limit and elevated levels of thyroid hormone, TRAb and TSAb positivity, and diffuse uptake in the thyroid gland on 99mTc scintigraphy, so her symptoms were consistent with a diagnosis of Graves’ Takedani et al. BMC Endocrine Disorders (2023) 23:132 Page 4 of 7 Fig. 3 Clinical course. When the patient went to the emergency department because of palpitations associated with atrial fibrillation, her TSH was under the detection limit, and her thyroid hormone levels were elevated. After admission, she was absolutely rested and treatment with bisoprolol was initiated. After diagnosis with Graves’ disease, treatment with thiamazole was initiated and her symptoms and thyroid hormones improved dramatically disease. She did not display diffuse goiter or ophthalmopathy. The low BMI found in our case was already present prior to the vaccination and is not part of the present discussion. COVID-19 is caused by the novel beta-coronavirus SARS-CoV-2 [12, 13]. There are some reports of cases with Graves’ disease after COVID-19. The spike proteins covering SARS-CoV-2 bind to angiotensin-converting enzyme 2 (ACE2) receptors for its initial entry [14]. ACE2 expression levels are higher in the small intestine, testis, kidneys, heart, lung, adipose tissue, and thyroid [15]. SARS-CoV-2 may affect the thyroid via ACE2, the viral receptor, resulting in the onset of AITD, such as Graves’ disease [12, 13]. The cytokine storm associated with COVID-19 infection may also induce thyroid dysfunction. An immunologic adjuvant is a substance that enhances the antigen-specific immune response without triggering one on its own [4, 5]. Adjuvants are commonly used to boost an immune response to treatments such as vaccination. ASIA was first coined in 2011 with the aim of codifying disorders characterized by innate and adaptive immune system dysregulation after adjuvant exposure [4]. Although the diagnostic criteria of ASIA are not clearly determined, the major signs of exposure to a preceding external stimulus (infection, vaccine, silicone, and adjuvant), ‘typical’ clinical manifestations, and improvement induced by removal of the inciting agent suggest a diagnosis of ASIA. In addition, the appearance of autoantibodies or antibodies directed at the suspected adjuvant is a minor diagnostic sign. Genetic predispositions for vaccine reactions such as ASIA remain uncertain [4]. ASIA includes various autoimmune diseases, such as collagen disease, blood disease, hepatic disease, neurological disease, and endocrine disease [5]. Most cases of AITD caused by ASIA are autoimmune subacute thyroiditis, which appear most often after exposure to human papillomavirus (HPV) vaccine, followed by influenza vaccine [8]. There are some reports of autoimmune Hashimoto’s thyroiditis as an AITD; however, there are few reports of Graves’ disease [6–8]. Our patient was exposed to the SARS-CoV-2 vaccine prior to clinical manifestations due to excess thyroid hormone and the appearance of thyroid antibodies of Graves’ disease. These findings suggested that the thyrotoxicosis in this case was due to Graves’ disease caused by ASIA. To date, 11 different vaccines have been granted an emergency use listing by the World Health Organization and used worldwide, including mRNA, protein subunit, viral vector, and inactivated vaccines [9]. Similar to other vaccines, there are some reports of the development of autoimmune diseases after exposure to the SARS-CoV-2 vaccine, such as collagen disease, heart disease, blood disease, hepatic disease, and endocrine disease [10, 16, 17]. No predictive factors have been associated with SARS-CoV-2 vaccine-associated Takedani et al. BMC Endocrine Disorders (2023) 23:132 Page 5 of 7 ASIA. Side effects after exposure to the SARS-CoV-2 vaccine were reported to each appropriate authority worldwide, and reports of Graves’ disease caused by ASIA were included in these reports. The cases that had been published in journals at the time of submission are shown in Table 2. We identified a total of 62 cases of Graves’ disease following SARS-CoV-2 vaccination, excluding our case. All patients showed positive TRAb or thyroid-stimulating immunoglobulin, and TRAb-positive cases without thyrotoxicosis were excluded. The table was categorized according to the sales company of the vaccines. We identified 32 patients who received the Pfizer mRNA vaccine; [11, 18–36] six patients who received the Moderna mRNA vaccine; [20, 28, 31, 37–39] 12 patients who received mRNA vaccines from unknown companies; [40] ten patients who received the AstraZeneca vaccine, including those from the Serum Institute of India; [37, 41–44] and one patient each who received Johnson & Johnson and Sinovac vaccines [27, 38]. Characteristically, across all companies, most patients with Graves’ disease are relatively young women, which is consistent with Graves’ disease in general. In most cases, symptoms developed within several days after vaccination. Inconsistent with the common clinical signs of Graves’ disease, there were some patients without goiter. Almost all confirmed cases showed good treatment responsiveness. According to previous reports, patients and their immunological background were not related. There were few descriptions of ophthalmopathy and past history of COVID-19 infection, and we were not able to confirm these data. Comparing patients who received mRNA vaccines with those from other companies, we recognized frequent onset of symptoms after not only the first but also the second and later mRNA vaccinations. The mRNA-based SARS-CoV-2 vaccine (Pfizer and Moderna) uses lipid nanoparticles to facilitate the transport of mRNA into cells and is widely used worldwide, including in our patient [45, 46]. The vaccine contains a number of excipients and lipids, one of which is based on polyethylene glycol (PEG), which may induce an immune response in specific individuals in rare cases [46]. In a viral vector vaccine (AstraZeneca and Johnson & Johnson), this role could be played by polysorbate 80; in an inactivated vaccine (Sinovac), this role could be played by aluminum salts [47, 48]. On the other hand, Vojdani et al. showed that many thyroid peroxidase (TPO) peptide sequences shared homology or similarity with sequences in various SARS-CoV-2 proteins. Furthermore, the SARS-CoV-2 spike protein, nucleoprotein, and membrane protein all cross-reacted with TPO. This suggests that cross-recognition between the modified SARSCoV-2 spike protein encoded in the mRNA vaccine and thyroid target proteins may promote AITD [49, 50]. In summary, we reported a case of Graves’ disease after exposure to the SARS-CoV-2 vaccine. Treatment with thiamazole was significantly effective for her condition. This case report reinforces the potential correlation between ASIA affecting the thyroid and SARS-CoV-2 vaccination. The clinical course suggests that it is essential to consider the possibility of developing ASIA, such as Graves’ disease, after exposure to the SARS-CoV-2 vaccine. Table 2 Reported cases of Graves’ disease after exposure to SARS-CoV-2 vaccination Our case Pfizer (n = 32) Moderna (n = 6) Pfizer and Moderna (n = 50) AstraZeneca (n = 10) Johnson & Johnson (n = 1) Sinovac (n = 1) Vaccine Type mRNA (Pfizer) mRNA mRNA mRNA Viral vector Viral vector Inactivated Age, year 41 44.8 (22–74) 45.8 (36–63) 43.4 (22–74) 43.3 (19–70) 68 44 Male, n (%) Female 9 (28) 2 (33) 12 (24) 3 (30) Female Female Time of symptom onset after vaccination, day 14 18.5 (1–120) 16.3 (2–46) 18.7 (1–120) 13.2 (2–31) 32 7 Dose, n (%) Second First 21 (66) Second 10 (31) Third 1 (3) First 3 (50) First 29 (58) Second 3 (50) Second 20 (40) Third 1 (2) First 9 (90) Second 1 (10) First First Enlarged thyroid, n (%) No 18/24 (75) 2/4 (50) 32/40 (80) 1/8 (13) NA NA Relapse, n (%) No 4 (13) 1/5 (20) 11/49 (22) 2 (20) No Yes FT3, pmol/L 33.2 19.8 (7.8–44.1) 27.5 (n = 1) 21.3 (6.3–44.1) 30.7 (n = 1) 21.2 14.8 FT4, pmol/L 72.1 48.4 (21–108) 50.4 (20–77) 48.2 (14–108) 43.7 (29–61) 46.3 34.4 Positive Tg-Ab, n (%) Yes 14/22 (64) 3/4 (75) 17/26 (65) 3/3 (100) NA Yes Positive TPO-Ab, n (%) Yes 17/26 (65) 5/5 (100) 22/31 (71) 7/8 (83) Yes Yes mRNA messenger RNA, FT3 free triiodothyronine, FT4 free thyroxine, Tg-Ab thyroglobulin antibody, TPO-Ab thyroid peroxidase antibody, NA not available Takedani et al. BMC Endocrine Disorders (2023) 23:132 Abbreviations AITD Autoimmune thyroid disease TSH Thyroid stimulating hormone TRAb TSH receptor antibody ASIA Autoimmune/inflammatory syndrome induced by adjuvants SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 mRNA Messenger RNA FT3 Free triiodothyronine FT4 Free thyroxine BMI Body mass index SpO2 Oxygen saturation TSAb Thyroid stimulating antibody Tg-Ab Thyroglobulin antibody TPO-Ab Thyroid peroxidase antibody ACE2 Angiotensin-converting enzyme 2 HPV Human papillomavirus PEG Polyethylene glycol TPO Thyroid peroxidase Page 6 of 7 7. 8. 9. 10. 11. 12. 13. Acknowledgements Not applicable. 14. Authors’ contributions KT, NI, YA, and KU managed the patient as the attending physicians. MN and KK evaluated the medical management strategies. KT, MN, and KK wrote the paper. All authors read and approved the final manuscript. 15. Funding Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 16. 17. 18. 19. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Consent for publication was obtained from all individual participants included in the study. Competing interests The authors declare that they have no competing interests in this paper. 20. 21. 22. 23. Received: 19 October 2021 Accepted: 12 June 2023 24. 25. References 1. De Leo S, Lee SY, Braverman LE. Hyperthyroidism Lancet. 2016;388:906–18. 2. Subekti I, Pramono LA. Current Diagnosis and Management of Graves’ Disease. Acta Med Indones. 2018;50:177–82. 3. Effraimidis G, Wiersinga WM. Mechanisms in endocrinology: autoimmune thyroid disease: old and new players. Eur J Endocrinol. 2014;170:R241–52. 4. Shoenfeld Y, Agmon-Levin N. “ASIA” - autoimmune/inflammatory syndrome induced by adjuvants. 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Shall We Focus on the Eosinophil to Guide Treatment with Systemic Corticosteroids during Acute Exacerbations of COPD?: PRO
Medical sciences
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Received: 12 July 2018; Accepted: 4 September 2018; Published: 11 September 2018 Abstract: In an era of precision medicine, it seems regressive that we do not use stratified approaches to direct treatment of oral corticosteroids during an exacerbation of chronic obstructive pulmonary disease (COPD). This is despite evidence suggesting that 40% of COPD patients have eosinophilic inflammation and this is an indicator of corticosteroid response. Treatments with oral corticosteroids are not always effective and not without harm, with significant and increased risk of hyperglycemia, sepsis, and fractures. Eosinophils are innate immune cells with an incompletely understood role in the pathology of airway disease. They are detected at increased levels in some patients and can be measured using non-invasive methods during states of exacerbation and stable periods. Despite the eosinophil having an unknown mechanism in COPD, it has been shown to be a marker of length of stay in severe hospitalized exacerbations, a predictor of risk of future exacerbation and exacerbation type. Although limited, promising data has come from one prospective clinical trial investigating the eosinophil as a biomarker to direct systemic corticosteroid treatment. This identified that there were statistically significant and clinically worsened symptoms in patients with low eosinophil levels who were prescribed prednisolone, demonstrating the potential utility of the eosinophil. In an era of precision medicine our patients’ needs are best served by accurate diagnosis, correct identification of maximal treatment response and the abolition of harm. The peripheral blood eosinophil count could be used towards reaching these aims. Keywords: COPD; Eosinophils; Inflammation Keywords: COPD; Eosinophils; Inflammation Shall We Focus on the Eosinophil to Guide T with Systemic Corticosteroids during Acute Exacerbations of COPD?: PRO James Camp 1, Jennifer L. Cane 1,2 and Mona Bafadhel 1,* James Camp 1, Jennifer L. Cane 1,2 and Mona Bafadhel 1,* 1 Respiratory Medicine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7FZ, UK; jamescamp438@gmail.com (J.C.); jennifer.cane@ndm.ox.ac.uk (J.L.C.) 2 NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford OX3 7FZ, UK * Correspondence: mona.bafadhel@ndm.ox.ac.uk James Camp 1, Jennifer L. Cane 1,2 and Mona Bafadhel 1,* 1 Respiratory Medicine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7FZ, UK; jamescamp438@gmail.com (J.C.); jennifer.cane@ndm.ox.ac.uk (J.L.C.) 2 NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford OX3 7FZ, UK * Correspondence: mona.bafadhel@ndm.ox.ac.uk Received: 12 July 2018; Accepted: 4 September 2018; Published: 11 September 2018 Received: 12 July 2018; Accepted: 4 September 2018; Published: 11 September 2018 medical sciences medical sciences 1. Eosinophil Cell Biology Eosinophils are inflammatory leukocytes comprising of bi-lobed nuclei and large acidophilic cytoplasm granules. The cationic protein granules bind to acid stains and in particular eosin which allowed them to be identified by Paul Ehrlich in 1879 [1]. Four proteins are found to make up the granules including major basic protein (MBP) located in the core and eosinophil basic proteins forming the matrix, consisting of eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN). These granules are toxic to various tissues and are capable of inducing damage and dysfunction upon their secretion [2]. Produced in healthy bone marrow derived from CD34+ myeloid progenitors, the number of eosinophils generated is typically low with circulating eosinophils range between 1–4% of the total white blood cell count. Once mature, eosinophils enter the systemic circulation where they can reside for 8–12 h. Unless stimulated, the cells then migrate to tissues [3]. Differentiation for this lineage is promoted by interleukin (IL)-3, granulocyte/macrophage-colony-stimulating factor (GM-CSF) and Med. Sci. 2018, 6, 74; doi:10.3390/medsci6030074 www.mdpi.com/journal/medsci www.mdpi.com/journal/medsci 2 of 7 Med. Sci. 2018, 6, 74 IL-5 cytokines from a hematopoietic stem cell into a mature eosinophil [4]. These same cytokines also act as priming agents, transforming the eosinophil from a quiescent cell into a hyper-responsive state. This includes an increased response to chemotaxis, degranulation, and cytokine production [5]. The mechanism of entry of the eosinophil from the blood stream into tissue involves crossing a microvascular wall. Eotaxin (CCL11) and IL-5 are the two main ligands that promote eosinophil migration into the tissue [6]. The Eotaxins, in addition to RANTES, produced from epithelial, mesenchymal, and endothelial cells are involved in migration and priming of eosinophils once in the airway [7,8]. Expression of C-C chemokine receptor type 3 (CCR3) and IL-5 receptor subunit alpha (IL5Rα) on the cell surface is integral to eosinophil recruitment to tissues. The role of the eosinophil in both innate and adaptive immunity is poorly defined, and their response varies depending on the environment and/or stimulus. Resident eosinophils are predominately found in the gastrointestinal tract, although other resident populations are also found in healthy individuals in physiological conditions in the thymus, spleen, lymph nodes, mammary glands and the uterus indicating other potential roles in homeostasis. Their accumulation at these sites has given rise to the eosinophil role extending to local immunity and/or remodeling and repair in health and disease (the so called “LIAR” hypothesis) [9]. Med. 1. Eosinophil Cell Biology Sci. 2018, 6, x FOR PEER REVIEW 2 of 7 act as priming agents, transforming the eosinophil from a quiescent cell into a hyper-responsive state. This includes an increased response to chemotaxis, degranulation, and cytokine production [5]. The mechanism of entry of the eosinophil from the blood stream into tissue involves crossing a microvascular wall. Eotaxin (CCL11) and IL-5 are the two main ligands that promote eosinophil migration into the tissue [6]. The Eotaxins, in addition to RANTES, produced from epithelial, mesenchymal, and endothelial cells are involved in migration and priming of eosinophils once in the airway [7,8]. Expression of C-C chemokine receptor type 3 (CCR3) and IL-5 receptor subunit alpha (IL5Rα) on the cell surface is integral to eosinophil recruitment to tissues. The role of the eosinophil in both innate and adaptive immunity is poorly defined, and their response varies depending on the environment and/or stimulus. Resident eosinophils are predominately found in the gastrointestinal tract, although other resident populations are also found in healthy individuals in physiological conditions in the thymus, spleen, lymph nodes, mammary glands and the uterus indicating other potential roles in homeostasis. Their accumulation at these sites has given rise to the eosinophil role extending to local immunity and/or remodeling and repair in health and disease (the so called ”LIAR” hypothesis) [9]. Eosinophils play an important role in immune-regulation by priming B cells and maintaining type-2 immunity [10]. In the airway, the eosinophil can act as an antigen-presenting cell while several stored and secreted mediators highlight their role as both an immunomodulatory and effector cell in the airway (Figure 1) [11]. Eosinophils are capable of disrupting the pulmonary epithelial barrier and causing alveolar epithelium cell lysis upon granular secretion, further exacerbating the inflammatory response [10]. LIAR hypothesis) [9]. Eosinophils play an important role in immune-regulation by priming B cells and maintaining type-2 immunity [10]. In the airway, the eosinophil can act as an antigen-presenting cell while several stored and secreted mediators highlight their role as both an immunomodulatory and effector cell in the airway (Figure 1) [11]. Eosinophils are capable of disrupting the pulmonary epithelial barrier and causing alveolar epithelium cell lysis upon granular secretion, further exacerbating the inflammatory response [10]. Figure 1. Sputum slide showing eosinophils. Figure 1. Sputum slide showing eosinophils. Figure 1. Sputum slide showing eosinophils. Figure 1. Sputum slide showing eosinophils. Figure 1. Sputum slide showing eosinophils. Figure 1. 1. Eosinophil Cell Biology Sputum slide showing eosinophils. 2. Eosinophils in Chronic Obstructive Pulmonary Disease 2. Eosinophils in Chronic Obstructive Pulmonary Disease In asthma, another common airway disease, the utility of the eosinophil to identify a corticosteroid response has been established [12], leading the way to the successful development of monoclonal antibodies to target severe eosinophilic asthma [13–17]. The lack of an underlying mechanism for the role of eosinophils in asthma has not diminished their use in clinical practice to identify the patient that requires treatment with anti-eosinophil depleting treatments, such as inhaled or oral corticosteroids, or monoclonal antibodies. In asthma, another common airway disease, the utility of the eosinophil to identify a corticosteroid response has been established [12], leading the way to the successful development of monoclonal antibodies to target severe eosinophilic asthma [13–17]. The lack of an underlying mechanism for the role of eosinophils in asthma has not diminished their use in clinical practice to identify the patient that requires treatment with anti-eosinophil depleting treatments, such as inhaled or oral corticosteroids, or monoclonal antibodies. or oral corticosteroids, or monoclonal antibodies. The involvement of the eosinophil in the pathogenesis of chronic obstructive pulmonary disease (COPD) has not been fully elucidated [18] and their role remains controversial. Up to 40% of patients with COPD have eosinophilic airway inflammation and both invasive and non-invasive methods have been used when measuring for this phenotype [19]. Historically, measurements of sputum eosinophils have been undertaken to categorize the degree of eosinophilic inflammation found in patients with COPD [20]. Although sputum induction is both a safe and repeatable procedure [20], it requires time, technical processing and expertise in slide preparation and counting [21]. Hence, sputum measurements in COPD (nor asthma) have not been adopted in routine clinical practice. The The involvement of the eosinophil in the pathogenesis of chronic obstructive pulmonary disease (COPD) has not been fully elucidated [18] and their role remains controversial. Up to 40% of patients with COPD have eosinophilic airway inflammation and both invasive and non-invasive methods have been used when measuring for this phenotype [19]. Historically, measurements of sputum eosinophils have been undertaken to categorize the degree of eosinophilic inflammation found in patients with COPD [20]. Although sputum induction is both a safe and repeatable procedure [20], it requires time, technical processing and expertise in slide preparation and counting [21]. Hence, sputum measurements in COPD (nor asthma) have not been adopted in routine clinical practice. The peripheral 3 of 7 Med. Sci. 2. Eosinophils in Chronic Obstructive Pulmonary Disease 2. Eosinophils in Chronic Obstructive Pulmonary Disease 2018, 6, 74 blood eosinophil count has emerged as an ideal surrogate for sputum eosinophilic inflammation [22]. Near-patient testing, validated against standard venipuncture laboratory analysis [23] allows for the rapid measurement of the peripheral blood eosinophil count in real-time and in the clinic room or surgery. In stable COPD, sputum eosinophils have been shown to identify both inhaled [24] and oral corticosteroid [25] response with respect to lung function improvements, quality of life and exercise capacity. Furthermore, in a direct replica of the seminal asthma study by Green et al. [12], reduction of sputum eosinophils in stable COPD has been shown to reduce exacerbations in the order of 65% [26]. Since the emergence of the peripheral blood eosinophil as a useful biomarker in COPD, several post-hoc analysis have explored its utility in directing inhaled corticosteroids to impact on exacerbation burden [27–30]. These studies have unequivocally found that in patients with COPD and a history of exacerbations, the peripheral blood eosinophil identifies patients with an increased risk of exacerbations and the best response to inhaled corticosteroids [27–29] or a worsened response to withdrawal of inhaled corticosteroids [31,32]. It is thus conceivable and arguably plausible that the measurement of eosinophilic inflammation is likely to be crucial in determining the phenotype of the disease and direction of therapy, making it critically useful as a biomarker to aid understanding in COPD pathogenesis and treatment response [18]. 3. Eosinophils in Exacerbations of COPD Exacerbations of COPD are heterogeneous and attempts to delineate the biological heterogeneity using plasma markers and symptoms have been made [33]. Unsupervised cluster analysis of sputum mediators has highlighted that there are independent biological clusters, which relate to unique inflammatory pathways and to underlying pathogenic etiology [22]. The biology of the exacerbation varies with the presence of bacteria, virus, eosinophils and in some a low inflammatory state (likely because of cardiac dysfunction or co-morbidity) [22]. The utility of the peripheral blood eosinophil as a suitable, sensitive and specific biomarker in COPD and in particular COPD exacerbations, identifying a type-2 eosinophilic phenotype was first reported in 2011 [22] and has been further validated by other groups [34]. Exacerbations of COPD that are associated with a type-2 inflammatory response have been shown to have increased concentrations of IL-5 and CCL11 and increased concentrations of sputum and blood eosinophils [22]. Furthermore, the clinical characteristics at the onset of an exacerbation cannot distinguish either type-1 or type-2 immune response [22,35,36]. Measurements of biological expression during an exacerbation of COPD could be used to stratify treatment. 5. Eosinophils at the Onset of an Exacerbation to Direct Prednisolone Treatment: Time to Move Towards Precision Clinical trials using biomarkers such as Procalcitonin [49] or C-reactive protein [50] have been successful in determining antibiotic prescription in exacerbations of COPD and go to some lengths to drive antibiotic stewardship [51,52]. In the only prospective study so far, the eosinophil has been successfully used to direct systemic corticosteroids at the time of a moderate exacerbation of COPD [53]. This single-center proof of concept study reached its primary outcome of non-inferiority in treatment failure rates in biomarker-directed treatment of systemic corticosteroids versus standard therapy with the additional signal of increased harm in patients who had low eosinophil levels (“biomarker low” peripheral blood eosinophil counts below 2%) prescribed prednisolone. Furthermore, the “biomarker low” patient population which received prednisolone therapy reported both statistically significant and clinically worsened symptoms and a slower rate of recovery than in “biomarker low” patient population receiving placebo. These findings were almost replicated in a pooled analysis of studies with available eosinophil results at the time of an exacerbation, with the worst outcomes (significantly higher treatment failure rates) in patients with eosinophilic exacerbations and treatment with placebo [54]. This has the implication that prednisolone is causing harm in some patients with COPD. At this current time there are a further two multi-center randomized trials seeking to validate this finding (NCT02857842 [55] and ISRCTN27510582), with planned read-outs by 2020. In an era of precision medicine [56], our patients’ needs are best served by accurate diagnosis, correct identification of maximal treatment response and the abolition of harm. The peripheral blood eosinophil count could be used towards reaching these aims. Funding: M.B. is funded by the NIHR on a personal fellowship award. J.L.C. is funded by the NIHR Oxford Respiratory Biomedical Research Centre. The work presents independent research funded by the NIHR, and the views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. Conflicts of Interest: M.B. has received honoraria and travel expenses for attendance at educational meetings from AstraZeneca, Chiesi, GlaxoSmithKline, Boehringer Ingelheim, Novartis and Pfizer. 4. Systemic Corticosteroids at the Onset of an Exacerbation: A Poorly Effective Treatment? Finally, the corroboration that eosinophils are an important biomarker in COPD exacerbations is demonstrated with the DECAF index, with eosinophils (and low levels, below 50 cells/mm3) being an independent predictor of mortality in severe hospitalized exacerbations of COPD [48]. In a post-hoc analysis from severe hospitalized exacerbations, length of hospital stay is significantly shorter in eosinophilic exacerbations treated with systemic corticosteroids [46] while retrospective analysis in the intensive care unit, demonstrates that eosinophilic exacerbations are associated with a lower mortality in COPD patients which are invasively ventilated [47]. Finally, the corroboration that eosinophils are an important biomarker in COPD exacerbations is demonstrated with the DECAF index, with eosinophils (and low levels, below 50 cells/mm3) being an independent predictor of mortality in severe hospitalized exacerbations of COPD [48]. 4. Systemic Corticosteroids at the Onset of an Exacerbation: A Poorly Effective Treatment? At present, there is no ideal method to direct treatment during an exacerbation of COPD. In the presence of dyspnea, exacerbations are usually always treated with systemic corticosteroids [37]. This is despite a small number of patients studied and heterogeneous evidence [37–39]. These treatments are routinely given in attempt to improve patient outcomes such as symptom recovery and prevent a treatment failure (defined as re-treatment, hospitalization or death), but have no effect on length of intensive treatment unit (ITU) stay or longer term lung function and are not without harm [40–43]. A Cochrane review for systemic corticosteroids in the management of an exacerbation of COPD demonstrates no effect on mortality and a small reduction in treatment failures, with a number-need-to-treat of 10, but a number-needed-to-harm of 6 [42]. Approximately 1 in 13 patients with an exacerbation of COPD treated with systemic corticosteroids will develop significant hyperglycemia [42]. A recent retrospective cohort case-control study in the emergency department demonstrates that even one short course of systemic corticosteroids are associated with an increase rate of sepsis (incidence rate ratio 5.3, 95% confidence interval (CI) 3.8–7.4), venous thromboembolism (incidence rate ratio 3.3, 95% CI 2.8–4.0) and fractures (incidence rate ratio 1.9, 95% CI 1.7–2.1) [40]. Corticosteroids however are an effective yet non-specific anti-inflammatory [44]. Approximately 30% of exacerbations of COPD are associated with eosinophilic airway inflammation [22]. Exacerbations of COPD with sputum eosinophilia have been shown to have the best forced expiratory volume (FEV1) Med. Sci. 2018, 6, 74 4 of 7 response to systemic corticosteroid therapy [45]. Despite their unknown mechanism in airways disease such as asthma and COPD, the eosinophil does inform of the likelihood of response to corticosteroids. response to systemic corticosteroid therapy [45]. Despite their unknown mechanism in airways disease such as asthma and COPD, the eosinophil does inform of the likelihood of response to corticosteroids. In a post-hoc analysis from severe hospitalized exacerbations, length of hospital stay is p p In a post-hoc analysis from severe hospitalized exacerbations, length of hospital stay is significantly shorter in eosinophilic exacerbations treated with systemic corticosteroids [46] while retrospective analysis in the intensive care unit, demonstrates that eosinophilic exacerbations are associated with a lower mortality in COPD patients which are invasively ventilated [47]. 5. Luijk, B.; Lindemans, C.A.; Kanters, D.; van der Heijde, R.; Bertics, P.; Lammers, J.W.; Bates, M.E.; Koenderman, L. Gradual increase in priming of human eosinophils during extravasation from peripheral blood to the airways in response to allergen challenge. J. Allergy Clin. Immunol. 2005, 115, 997–1003. [CrossRef] [PubMed] p g g gy 6. Matthews, A.N.; Friend, D.S.; Zimmermann, N.; Sarafi, M.N.; Luster, A.D.; Pearlman, E.; Wert, S.E.; Rothenberg, M.E. Eotaxin is required for the baseline level of tissue eosinophils. 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Van Velzen, P.; Ter Riet, G.; Bresser, P.; Baars, J.J.; van den Berg, B.T.J.; van den Berg, J.W.K.; Brinkman, P.; Dagelet, J.W.F.; Daniels, J.M.A.; Groeneveld-Tjiong, D.R.G.L.; et al. Doxycycline for outpatient-treated acute exacerbations of COPD: A randomised double-blind placebo-controlled trial. Lancet Respir Med. 2017, 5, 492–499. [CrossRef] 52. Rohde, G.G.; Koch, A.; Welte, T. Randomized double blind placebo-controlled study to demonstrate that antibiotics are not needed in moderate acute exacerbations of COPD—The ABACOPD study. BMC Pulm. Med. 2015, 15, 5. [CrossRef] [PubMed] 53. Bafadhel, M.; McKenna, S.; Terry, S.; Mistry, V.; Pancholi, M.; Venge, P.; Lomas, D.A.; Barer, M.R.; Johnston, S.L.; Pavord, I.D.; et al. Blood eosinophils to direct corticosteroid treatment of exacerbations of chronic obstructive pulmonary disease: A randomized placebo-controlled trial. Am. J. Respir. Crit. Care Med. 2012, 186, 48–55. [CrossRef] [PubMed] 54. Bafadhel, M.; Davies, L.; Calverley, P.M.; Aaron, S.D.; Brightling, C.E.; Pavord, I.D. Blood eosinophil guided prednisolone therapy for exacerbations of COPD: A further analysis. Eur. Respir. J. 2014, 44, 789–791. [CrossRef] [PubMed] 55. Sivapalan, P.; Moberg, M.; Eklof, J.; Janner, J.; Vestbo, J.; Laub, R.R.; Browatzki, A.; Armbruster, K.; Wilcke, J.T.; Seersholm, N.; et al. A multi-center randomized, controlled, open-label trial evaluating the effects of eosinophil-guided corticosteroid-sparing therapy in hospitalised patients with COPD exacerbations—The CORTICO steroid reduction in COPD (CORTICO-COP) study protocol. BMC Pulm. Med. 2017, 17, 114. [CrossRef] [PubMed] 56. Agusti, A.; Bafadhel, M.; Beasley, R.; Bel, E.H.; Faner, R.; Gibson, P.G.; Louis, R.; McDonald, V.M.; Sterk, P.J.; Thomas, M.; et al. Precision medicine in airway diseases: Moving to clinical practice. Eur. Respir. J. 2017, 50, 1701655. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4388200720
http://vca.univd.edu.ua/index.php/vca/article/download/64/40
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ТЕОРЕТИЧНІ ОСНОВИ ЗАСТОСУВАННЯ ЗАХОДІВ ЗАБЕЗПЕЧЕННЯ КРИМІНАЛЬНОГО ПРОВАДЖЕННЯ ПІД ЧАС ДОСУДОВОГО РОЗСЛІДУВАННЯ КОРУПЦІЙНИХ КРИМІНАЛЬНИХ ПРАВОПОРУШЕНЬ
Vìsnik Krimìnologìčnoï asocìacìï Ukraïni
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ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) 3:343.35.08 DOI: https://doi.org/10.32631/vca.2023.2.13 ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) 3:343.35.08 DOI: https://doi.org/10.32631/vca.2023.2.13 УДК 343.13:343.35.08 Роман Ілліч ЛОГІН (Харківський національний університет внутрішніх справ, м. Харків, Україна) Роман Ілліч ЛОГІН (Харківський національний університет внутрішніх справ, м. Харків, Україна) ТЕОРЕТИЧНІ ОСНОВИ ЗАСТОСУВАННЯ ЗАХОДІВ ЗАБЕЗПЕЧЕННЯ КРИМІНАЛЬНОГО ПРОВАДЖЕННЯ ПІД ЧАС ДОСУДОВОГО РОЗСЛІДУВАННЯ КОРУПЦІЙНИХ КРИМІНАЛЬНИХ ПРАВОПОРУШЕНЬ Проведено аналіз стану наукової розробленості теоретичних знань про заходи забезпечення кримінального провадження як основи досліджень застосування заходів забезпечення кримінального провадження під час досудового розслідування корупційних кримінальних правопорушень. Надано огляд існуючих у вітчизняній доктрині кримінального процесу сучасних підходів до визначення поняття заходів забезпечення кримінального провадження, його співвідношення із поняттям кримінального процесуального примусу, видової класифікації заходів забезпечення кримінального провадження та їх функціонального призначення. Виокремлено основні напрямки досліджень заходів забезпечення кримінального провадження на сучасному етапі розвитку науки кримінального процесу. Визначено діалектичний зв’язок із загальнотеоретичними положеннями щодо заходів забезпечення кримінального провадження та перспектив подальшого дослідження застосування заходів забезпечення кримінального провадження під час досудового розслідування корупційних кримінальних правопорушень. Ключові слова: процесуальний примус, заходи забезпечення кримінального провадження, досудове розслідування корупційних кримінальних правопорушень. Ключові слова: процесуальний примус, заходи забезпечення кримінального провадження, досудове розслідування корупційних кримінальних правопорушень. Постановка проблеми. Прикладні дослідження, до яких слід віднести й дослідження проблем застосування заходів забезпечення кримінального провадження під час досудового розслідування корупційних кримінальних правопорушень потребують належної теоретичної основи. Із набуттям чинності Кримінального процесуального кодексу України 19 листопада  180   180  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) 2012 року (далі – КПК України), у кримінальний процес було запроваджено ряд нових інститутів кримінального судочинства. Серед іншого, одним із таких нововведень постало запровадження інституту заходів забезпечення кримінального провадження: визначено мету їх застосування, надано їх вичерпний перелік, сформульовано порядок застосування кожного з їх різновидів. Окремо слід згадати, що було введено ряд абсолютно нових, у порівнянні із положеннями Кримінально-процесуального кодексу 1960 року, процесуальних заходів, які спрямовані на забезпечення дієвості кримінального провадження, а саме: накладення грошового стягнення, тимчасове обмеження у користуванні спеціальним правом, тимчасовий доступ до речей і документів, тимчасове вилучення майна, а також запроваджено новий запобіжний захід – домашній арешт. 2012 року (далі – КПК України), у кримінальний процес було запроваджено ряд нових інститутів кримінального судочинства. Серед іншого, одним із таких нововведень постало запровадження інституту заходів забезпечення кримінального провадження: визначено мету їх застосування, надано їх вичерпний перелік, сформульовано порядок застосування кожного з їх різновидів. Окремо слід згадати, що було введено ряд абсолютно нових, у порівнянні із положеннями Кримінально-процесуального кодексу 1960 року, процесуальних заходів, які спрямовані на забезпечення дієвості кримінального провадження, а саме: накладення грошового стягнення, тимчасове обмеження у користуванні спеціальним правом, тимчасовий доступ до речей і документів, тимчасове вилучення майна, а також запроваджено новий запобіжний захід – домашній арешт. 1 Авраменко О.В., Благута Р.І., Хитра А.Я. Заходи забезпечення кримінального провадження: навчальний посібник. Львів: ЛьвДУВС, 2014. С. 4. ТЕОРЕТИЧНІ ОСНОВИ ЗАСТОСУВАННЯ ЗАХОДІВ ЗАБЕЗПЕЧЕННЯ КРИМІНАЛЬНОГО ПРОВАДЖЕННЯ ПІД ЧАС ДОСУДОВОГО РОЗСЛІДУВАННЯ КОРУПЦІЙНИХ КРИМІНАЛЬНИХ ПРАВОПОРУШЕНЬ Метою цієї роботи є визначення теоретичної основи для подальшого дослідження проблем застосування заходів забезпечення кримінального провадження під час досудового розслідування корупційних кримінальних правопорушень. Під такою основою слід розуміти сучасний стан розвитку загальнотеоретичних положень про заходи забезпечення кримінального провадження у кримінальному процесі України. Виклад основного матеріалу. У науковій літературі вірно зазначається, що введення у слідчу та судову практику нових заходів забезпечення кримінального провадження, є спробою законодавця систематизувати заходи кримінального процесуального примусу, що можуть бути застосовані у кримінальному провадженні, визначити додаткові гарантії особи від незаконного втручання у її життя з боку державних органів і посадових осіб. Розширення видів заходів забезпечення кримінального провадження, що не пов’язані із позбавленням волі, є вимогою часу та світового співтовариства у напрямі наближення національного законодавства до європейських цінностей та стандартів1. Вагомих змін зазнав механізм застосування тримання під вартою як запобіжного заходу. Природнім наслідком суттєвої трансформації положень кримінального процесуального законодавства постала активізація наукових досліджень у цьому напрямку, зокрема, і з метою удосконалення нових законодавчих положень. Аналіз літературних джерел, присвячених дослідженню заходів забезпечення кримінального провадження, призводить до висновку, що вони здійснюються за трьома основними напрямками: 1) визначення теоретичних основ застосування заходів забезпечення кримінального провадження (визначення їх природи, поняття, ознак, мети, класифікації, співвідношення із іншими процесуальними діями у кримінальному провадженні); 2) вивчення підстав та порядку застосування окремих видів заходів забезпечення кримінального провадження, як то: виклик слідчим, 2) вивчення підстав та порядку застосування окремих видів заходів забезпечення кримінального провадження, як то: виклик слідчим,  181  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) прокурором, судовий виклик і привід; накладення грошового стягнення; тимчасове обмеження у користуванні спеціальним правом тощо; прокурором, судовий виклик і привід; накладення грошового стягнення тимчасове обмеження у користуванні спеціальним правом тощо; у р у р 3) дослідження особливостей застосування заходів забезпечення кримінального провадження щодо окремих категорій осіб (неповнолітніх, жінок тощо); 3) дослідження особливостей застосування заходів забезпечення кримінального провадження щодо окремих категорій осіб (неповнолітніх, жінок тощо); 4) наукова розвідка особливостей застосування заходів забезпечення кримінального провадження у кримінальних провадженнях щодо окремих видів кримінальних правопорушень. Дослідження проблем застосування заходів забезпечення кримінального провадження під час досудового розслідування корупційних кримінальних правопорушень можна віднести до останнього напрямку розвитку наукових уявлень. Проте, перед тим, ніж перейти до безпосереднього огляду стану досліджень за цим напрямком, вважаємо за необхідне зупинитись на аналізі робіт за іншими напрямками, і в першу чергу – теоретичного спрямування. Адже вони становлять наукову основу для подальшого розвитку досліджень проблем застосування заходів забезпечення кримінального провадження під час досудового розслідування корупційних кримінальних правопорушень. 1 Кожевніков Г. Заходи забезпечення кримінального провадження. Вісник Національної академії прокуратури України. 2012. № 3. С. 69 ТЕОРЕТИЧНІ ОСНОВИ ЗАСТОСУВАННЯ ЗАХОДІВ ЗАБЕЗПЕЧЕННЯ КРИМІНАЛЬНОГО ПРОВАДЖЕННЯ ПІД ЧАС ДОСУДОВОГО РОЗСЛІДУВАННЯ КОРУПЦІЙНИХ КРИМІНАЛЬНИХ ПРАВОПОРУШЕНЬ Одним із перших вітчизняних науковців піддав науковому аналізу поняття заходів забезпечення кримінального провадження та їх співвідношення із поняттям процесуального примусу у світлі Кримінального процесуального кодекс України 2012 року є Г. К. Кожевніков. Вчений дійшов висновку, що поняття заходів забезпечення кримінального провадження є тотожнім поняттю процесуального примусу. Нагадаємо, що під заходами процесуального примусу у літературі розглядають передбачені кримінальним процесуальним законом заходи примусового характеру, які застосовуються уповноваженими на те посадовими особами та державними органами до підозрюваного, обвинуваченого, потерпілого, свідка, а також до інших осіб, які приймають участь у кримінальному провадженні, з метою недопущення дійсних і можливих перешкод, які виникають та не дають можливості реалізувати завдання кримінального судочинства1. Зазначається, що виходячи із наведеного визначення завдань заходів кримінально процесуального примусу, перелічені у ст. 131 КПК України заходи забезпечення кримінального провадження виконують головну кримінально-процесуальну функцію – забезпечення належної поведінки суб'єктів кримінального провадження. Поряд із цим, науковцем запропоновано ряд положень щодо удосконалення чинного законодавства. Так, запропоновано включити до переліку, визначеного у ст. 131 КПК України такий захід забезпечення кримінального провадження як поміщення неповнолітнього, який досяг одинадцятирічного віку у приймальник-розподільник для дітей на строк до  182  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) тридцяти днів. Поряд із цим, спірним, на думку науковця, є включення до заходів забезпечення кримінального провадження такого різновиду процесуальних дій як виклик слідчим, прокурором та судовий виклик – з огляду на те, що ця дія, на відміну, від інших, зазначених у ст. 131 КПК України не містить у собі елемент примусу. Виклик не може бути заходом примусу, зазначає науковець, адже по суті передбачає добровільне його виконання учасником кримінального провадження. Примус же у цьому випадку настає тільки у разі неявки особи без поважних причин за викликом у вигляді застосування до такої особи приводу1. у у р у Оскільки в КПК України відсутнє нормативне визначення поняття заходів забезпечення кримінального провадження, у літературі пропонується доктринальне визначення цього поняття. Наприклад В. І. Фаринник вважає, що під ними слід розуміти «передбачені кримінальним процесуальним законом заходи державно-правового примусу, що застосовуються уповноваженими державними органами та особами, у встановленому законом порядку до підозрюваного, обвинуваченого, потерпілого, свідка та інших осіб, котрі залучаються до сфери кримінальної процесуальної діяльності, з метою запобігання та припинення їхніх неправомірних дій, забезпечення виявлення та закріплення доказів, досягнення завдань кримінального судочинства та дієвості провадження2». Існують й інші визначення цього поняття. 1 Кожевніков Г. Заходи забезпечення кримінального провадження. Вісник Національної академії прокуратури України. 2012. № 3. С. 69. 2 Фаринник В. І. Заходи забезпечення кримінального провадження у новому кримінальному процесуальному законодавстві: сутність та класифікація. Вісник кримінального судочинства. 2015. № 1. С. 135. 4 Бондаренко О. М. Загальні правила застосування заходів забезпечення кримінального провадження URL: http://www.lsej.org.ua/2_2014/29.pdf 1 Кожевніков Г. Заходи забезпечення кримінального провадження. Вісник Національної академії прокуратури України. 2012. № 3. С. 69. 2 Фаринник В. І. Заходи забезпечення кримінального провадження у новому кримінальному процесуальному законодавстві: сутність та класифікація. Вісник кримінального судочинства. 2015. № 1. С. 135. 3 Кримінальний процесуальний кодекс України: науково-практичний коментар / за заг. ред. В.Я. Тація, В.П. Пшонки, А.В. Портнова; Національний університет «Юридична академія України імені Ярослава Мудрого», Національна академія правових наук України. Харків: «Право», 2012. С. 292. 4 Бондаренко О. М. Загальні правила застосування заходів забезпечення кримінального провадження URL: http://www.lsej.org.ua/2_2014/29.pdf 135. 3 Кримінальний процесуальний кодекс України: науково-практичний коментар / за заг. ред. В.Я. Тація, В.П. Пшонки, А.В. Портнова; Національний університет «Юридична академія України імені Ярослава Мудрого», Національна академія правових наук України. Харків: «Право», 2012. С. 292. 4 Б О М З і і б і 1 Бобечко Н. Заходи забезпечення кримінального провадження: процесуальна природа, зміст та обсяг. Право України. 2017. № 12. С. 28. 2 Назаров В. В. Застосування заходів забезпечення кримінального провадження. Європейські перспективи. 2013. № 3. С. 103. 3 Миколенко О. М. Критерії класифікації заходів забезпечення кримінального провадження та їх співвідношення з заходами кримінального процесуального примусу. Правова держава. 2014. № 17. С. 83. ТЕОРЕТИЧНІ ОСНОВИ ЗАСТОСУВАННЯ ЗАХОДІВ ЗАБЕЗПЕЧЕННЯ КРИМІНАЛЬНОГО ПРОВАДЖЕННЯ ПІД ЧАС ДОСУДОВОГО РОЗСЛІДУВАННЯ КОРУПЦІЙНИХ КРИМІНАЛЬНИХ ПРАВОПОРУШЕНЬ Зокрема, у навчальній літературі це поняття подається як передбачені кримінально- процесуальним законом заходи примусового характеру, які застосовуються компетентними особами та органами за наявності підстав та в порядку, передбачених законом, з метою подолання негативних обставин, які перешкоджають вирішенню завдань кримінального судочинства, та забезпечення виконання рішень органів досудового розслідування та суду3. р р у р у у у О. М. Бондаренко, в свою чергу, пропонує визначати цю категорію як заходи примусового характеру, що полягають в обмеженні прав та законних інтересів підозрюваних, обвинувачених та інших учасників кримінального провадження, що застосовуються уповноваженими на те особами на підставі та у порядку, передбачених законом з метою досягнення дієвості кримінального провадження4. Дискусійним у науковій літературі залишається питання співвідношення понять кримінального процесуального примусу та заходів забезпечення кримінального провадження.  183   183  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) За результатами аналізу цієї проблематики, можна дійти висновку про існування двох підходів до вирішення цієї проблематики. Представники першого напрямку ототожнюють ці поняття, інші науковці говорять про заходи забезпечення кримінального провадження як групу заходів кримінального процесуального примусу, тобто їх різновид1. Наприклад, В. В. Назаров вважає, що ці поняття є тотожними, оскільки дають можливість виявляти, збирати та зберігати докази, попереджати можливу протиправну поведінку чи виключати можливість підозрюваного, обвинуваченого ухилятися від слідства та суду2. О. М. Миколенко наголошує на необхідності розмежування заходів забезпечення кримінального провадження і заходів кримінально- процесуального примусу. Вчений стверджує, що ці поняття співвідносяться як категорії загальне та приватне. Серед заходів забезпечення кримінального провадження існують й такі, що не носять примусовий характер та застосування яких не передбачає настання для особи певних обмежень. Як приклад наводяться судовий виклик, виклик слідчим чи прокурором, а також тимчасовий доступ до речей і документів. На цій підставі заходи забезпечення кримінального провадження слід поділяти на два види: примусові (заходи процесуального примусу) та непримусові3. Аналогічну позицію висловлює й Н. Р. Бобечко. Вчений стверджує, що поняття «заходи забезпечення кримінального провадження» є родовим по відношенню до заходів кримінального процесуального примусу. Поряд із тими, що відображені у розділі ІІ КПК України, на думку вченого, існують інші заходи забезпечення кримінального провадження. Цікавим є підхід автора до аналізу цього питання, виходячи із характеру кримінальних процесуальних норм. Зважаючи на те, що останні виконують не тільки охоронну, а й регулятивну функцію, то і заходи забезпечення кримінального провадження слід поділяти на дві групи: заходи примусового забезпечення кримінального провадження та заходи непримусового забезпечення кримінального провадження. Поряд із цим, непримусові заходи забезпечення кримінального провадження автор поділяє на: заходи інформаційного забезпечення (повідомлення, виклики); забезпечення неупередженості суб’єктів кримінального провадження (відвід, самовідвід, усунення присяжного); організаційно-технічного забезпечення (ексгумація трупа, розшук); забезпечення безпеки суб’єктів кримінального провадження (конфіденційність відомостей про особу, закритий судовий розгляд, прослуховування телефонних та інших переговорів); непримусового одержання і перевірки доказової інформації (допит, пред’явлення для впізнання, витребування речей, документів, висновків ревізій та актів  184  1 Бобечко Н. Заходи забезпечення кримінального провадження: процесуальна природа, зміст та обсяг. Право України. 2017. № 12. С. 34. 2 Рожнова В.В., Савицький Д.О., Конюшенко Я.Ю. та ін. Курс лекцій з кримінального процесу за новим Кримінальним процесуальним кодексом України (Загальна частина) / Національна академія внутрішніх справ, 2012. С. 334.. 1 Бобечко Н. Заходи забезпечення кримінального провадження: процесуальна природа, зміст Право України. 2017. № 12. С. 34. Рожнова В.В., Савицький Д.О., Конюшенко Я.Ю. та ін. Курс лекцій з кримінального процесу за римінальним процесуальним кодексом України (Загальна частина) / Національна академія ніх справ, 2012. С. 334.. ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) полягають у застосуванні відносно підозрюваного, обвинуваченого психічного впливу, що зовнішньо виражений у встановленні на таких осіб визначених обов’язків, метою яких є забезпечення їх належної поведінки (особисте зобов’язання, особиста порука, застава). полягають у застосуванні відносно підозрюваного, обвинуваченого психічного впливу, що зовнішньо виражений у встановленні на таких осіб визначених обов’язків, метою яких є забезпечення їх належної поведінки (особисте зобов’язання, особиста порука, застава). полягають у застосуванні відносно підозрюваного, обвинуваченого психічного впливу, що зовнішньо виражений у встановленні на таких осіб визначених обов’язків, метою яких є забезпечення їх належної поведінки (особисте зобов’язання, особиста порука, застава). 2. В залежності від форми досудового розслідування: 2. В залежності від форми досудового розслідування: 1) ті, що застосовуються під час досудового слідства (всі заходи забезпечення кримінального провадження, передбачені КПК України); 1) ті, що застосовуються під час досудового слідства (всі заходи забезпечення кримінального провадження, передбачені КПК України); 2) ті, що застосовуються під час дізнання (заборонено таких запобіжних заходів як домашній арешт, застава або тримання під вартою). 3. Залежно від кола осіб, відносно яких може бути застосовано запобіжні заходи: 1) загальні – ті, що можуть застосовуватись відносно всіх категорій підозрюваних, обвинувачених (особисте зобов’язання, застава, домашній арешт тощо); 2) спеціальні – що можуть застосовуватись відносно окремих категорій осіб: неповнолітніх (передання їх під нагляд батьків, опікунів чи піклувальників, адміністрації виховної дитячої установи), обмежено осудних осіб (поміщення до психіатричного закладу в умовах, що виключають небезпечну поведінку обмежено осудних осіб)1. Відома також класифікація, запропонована Л.М. Лобойко, коли заходи забезпечення кримінального провадження поділяються залежно від суворості наслідків, що настають для осіб, щодо яких вони застосовуються на: 1) запобіжні заходи та 2) інші заходи забезпечення кримінального процесу2. С. М. Смоков класифікує заходи забезпечення кримінального провадження виходячи з обмеження конституційних прав громадян на такі види: 1) заходи, які обмежують суспільне і приватне життя громадян (до них автором віднесено виклик слідчим, прокурором, судовий виклик, а також привід, тимчасове обмеження у користуванні спеціальним правом, відсторонення від посади, тимчасовий доступ до речей і документів; 2) заходи, які обмежують майнові права громадян (накладення грошового стягнення, тимчасове вилучення майна, арешт майна); 3) заходи, які обмежують свободу пересування громадян (особисте зобов’язання, домашній арешт, особиста порука, застава); 4) заходи, які обмежують особисту свободу громадян (затримання та тримання під вартою)3. Розроблена у науці й проблема функціонального призначення заходів забезпечення кримінального провадження. На думку В. І. Фаринника застосування заходів кримінального провадження у кримінальному процесі переслідує дві мети – забезпечувальну і превентивну. ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) перевірок, отримання пояснень)1. перевірок, отримання пояснень)1. Отже, важливе пізнавальне значення має й сформульована у літературі їх класифікація. Можна зустріти й інші підходи, коли їх класифікують за: Отже, важливе пізнавальне значення має й сформульована у літературі їх класифікація. Можна зустріти й інші підходи, коли їх класифікують за: 1) метою застосування: 1) метою застосування: – заходи, що забезпечують належну поведінку підозрюваного, обвинуваченого у кримінальному провадженні та виконання ними процесуальних обов’язків (запобіжні заходи); – заходи, що забезпечують отримання та збирання доказів (виклик слідчим, прокурором, судовий виклик, тимчасовий доступ до речей і документів тощо); – заходи, що спрямовані на забезпечення законного порядку під час провадження (привід, накладення грошового стягнення, відсторонення від посади); – заходи по забезпеченню цивільного позову та можливої конфіскації майна (тимчасове вилучення майна, арешт майна тощо); – заходи, що тривають протягом чітко визначеного періоду часу (запобіжні заходи, відсторонення від посади, арешт майна тощо); заходи, термін дії яких не визначено (привід тощо); – заходи, термін дії яких не визначено (привід тощо); 3) підставами застосування: ) підставами застосування: – заходи, що застосовуються у зв’язку з невиконанням (або можливим невизнанням) процесуальних обов’язків (накладення грошового стягнення, привід, запобіжні заходи тощо); – заходи, які застосовуються незалежно від процесуального правопорушення (тимчасовий доступ до речей і документів; тимчасове вилучення майна); 4) режимом обмеження прав і свобод людини: – заходи, що пов’язані із тимчасовою ізоляцією особи (тримання під вартою, домашній арешт, затримання особи); – заходи, які не пов’язані з тимчасовою ізоляцією особи (накладення грошового стягнення, тимчасове обмеження у користуванні спеціальним правом, особисте зобов’язання, особиста порука, застава тощо)2. Л. Д. Коваленко, в свою чергу, пропонує наступну класифікацію заходів забезпечення кримінального провадження: 1. За режимом обмеження прав і свобод: 1) ізоляційні - спрямовані на ізоляцію підозрюваного, обвинуваченого від суспільства та полягають у обмеженні особистої свободи таких осіб (тримання під вартою, затримання, домашній арешт); 2) неізоляційні (психолого-примусові запобіжні заходи) – ці засоби 2) неізоляційні (психолого-примусові запобіжні заходи) – ці засоби  185   185  1 Кримінальний процес: підручник / за заг. ред В.В. Коваленка, Л.Д. Удалова, Д.П. Письменного. Київ: ЦУЛ. 2013. С. 170. 2 Лобойко Л.М. Кримінальний процес: підручник. Київ: Істина. 2014. С. 161. 3 Смоков С.М. Види обмежень конституційних прав громадян у новому Кримінальному процесуальному кодексі України. Форум права. 2012. № 2. С. 629-631. Лобойко Л.М. Кримінальний процес: підручник. Київ: Істина. 2014. С. 161. Смоков С.М. Види обмежень конституційних прав громадян у новому Кримінальному льному кодексі України. Форум права. 2012. № 2. С. 629-631. Кримінальний процес: підручник / за заг. ред В.В. Коваленка, Л.Д. Удалова, Д.П. Письменного. Л. 2013. С. 170. Лобойко Л М Кримінальний процес: підручник Київ: Істина 2014 С 161 ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) Забезпечувальна мета полягає у створенні умов для виконання підозрюваним, обвинуваченим  186  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) покладених на нього процесуальних обов’язків, наприклад: прибути за викликом до слідчого, прокурора, слідчого судді, суду, заздалегідь повідомити про неможливість прибуття; виконання обов’язків, покладених на цих осіб рішенням про застосування запобіжних заходів; виконувати законні розпорядженням і вимогам слідчого, прокурора, слідчого судді, суду. Превентивна ж мета, на думку науковця, полягає у запобіганні спробам переховуватися від органів досудового розслідування та/або суду; знищенню, приховуванню чи спотворенню будь-яких речей чи документів, які мають істотне значення для встановлення обставин кримінального правопорушення; незаконного впливу на потерпілих, свідків, інших підозрюваних, обвинувачених, експертів, спеціалістів у кримінальному провадженні; вчиненню інших кримінальних правопорушень1. Висновки. Отже на сьогоднішній день слід вести мову про наявність у вітчизняній науці кримінального процесу належної теоретичної основи для подальшого розвитку уявлень про заходи забезпечення кримінального провадження та проведенні прикладних досліджень за зазначеними раніше напрямками, зокрема і щодо особливостей застосування заходів забезпечення кримінального провадження у кримінальних провадженнях щодо корупційних кримінальних правопорушень. Корупційні кримінальні правопорушення становлять досить велику групу, окремі складові якої різняться рівнем суспільної небезпечності, наявністю специфічних обставин по кожній окремій справі, які повинні враховуватись при обранні та застосуванні того чи іншого заходу забезпечення кримінального провадження. Саме тому дослідження існуючих проблем законодавчих прогалин у цій сфері, судово-слідчої практики застосування нормативно-правових положень повинно мати під собою належне наукове підґрунтя. 1 Фаринник В.І. Поняття та підстави застосування запобіжних заходів, не пов’язаних із тимчасовою ізоляцією особи. Бюлетень Міністерства юстиції України. 2015. № 5. С. 149. СПИСОК ВИКОРИСТАННИХ ДЖЕРЕЛ 1. Авраменко О.В., Благута Р.І., Хитра А.Я. Заходи забезпечення кримінального провадження: навчальний посібник. Львів: ЛьвДУВС, 2014. 1. Авраменко О.В., Благута Р.І., Хитра А.Я. Заходи забезпечення кримінального провадження: навчальний посібник. Львів: ЛьвДУВС, 2014. 2. Бобечко Н. Заходи забезпечення кримінального провадження: процесуальна природа, зміст та обсяг. Право України. 2017. № 12. С. 27–38. 2. Бобечко Н. Заходи забезпечення кримінального провадження: процесуальна природа, зміст та обсяг. Право України. 2017. № 12. С. 27–38. . Бондаренко О.М. Загальні правила застосування заходів ечення кримінального провадження. URL: 3. Бондаренко О.М. Загальні правила застосування заходів забезпечення кримінального провадження. URL: http://www.lsej.org.ua/2_2014/29.pdf 4. Кожевніков Г. Заходи забезпечення кримінального провадження. Вісник Національної академії прокуратури України. 2012. № 3. С. 68–70. 4. Кожевніков Г. Заходи забезпечення кримінального провадження. Вісник Національної академії прокуратури України. 2012. № 3. С. 68–70. 5. Кримінальний процесуальний кодекс України: науково-практичний коментар / за заг. ред. В.Я. Тація, В.П. Пшонки, А.В. Портнова; Національний 5. Кримінальний процесуальний кодекс України: науково-практичний коментар / за заг. ред. В.Я. Тація, В.П. Пшонки, А.В. Портнова; Національний  187  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 2 (29) університет «Юридична академія України імені Ярослава Мудрого», Національна академія правових наук України. Харків: «Право», 2012. університет «Юридична академія України імені Ярослава Мудрого», Національна академія правових наук України. Харків: «Право», 2012. університет «Юридична академія України імені Ярослава Мудрого», Національна академія правових наук України. Харків: «Право», 2012. університет «Юридична академія України імені Ярослава Мудрого», Національна академія правових наук України. Харків: «Право», 2012. ц д р у р р р 6. Кримінальний процес: підручник / за заг. ред В.В. Коваленка, Л.Д. Удалової, Д.П. Письменного. Київ: ЦУЛ. 2013. 6. Кримінальний процес: підручник / за заг. ред В.В. Коваленка, Л.Д. Удалової, Д.П. Письменного. Київ: ЦУЛ. 2013. 7. Лобойко Л.М. Кримінальний процес: підручник. Київ: Істина. 2014. 8. Миколенко О.М. Критерії класифікації заходів забезпечення кримінального провадження та їх співвідношення з заходами кримінального процесуального примусу. Правова держава. 2014. № 17. С. 81–84. 8. Миколенко О.М. Критерії класифікації заходів забезпечення кримінального провадження та їх співвідношення з заходами кримінального процесуального примусу. Правова держава. 2014. № 17. С. 81–84. 9. Назаров В.В. Застосування заходів забезпечення кримінального провадження. Європейські перспективи. 2013. № 3. С. 102–106. 9. Назаров В.В. Застосування заходів забезпечення кримінального провадження. Європейські перспективи. 2013. № 3. С. 102–106. 10. Рожнова В.В., Савицький Д.О., Конюшенко Я.Ю. та ін. Курс лекцій з кримінального процесу за новим Кримінальним процесуальним кодексом України (агальна частина) / Національна академія внутрішніх справ, 2012. 10. Рожнова В.В., Савицький Д.О., Конюшенко Я.Ю. СПИСОК ВИКОРИСТАННИХ ДЖЕРЕЛ та ін. Курс лекцій з кримінального процесу за новим Кримінальним процесуальним кодексом України (агальна частина) / Національна академія внутрішніх справ, 2012. 11. Смоков С.М. Види обмежень конституційних прав громадян у новому Кримінальному процесуальному кодексі України. Форум права. 2012. № 2. С. 629–631. 11. Смоков С.М. Види обмежень конституційних прав громадян у новому Кримінальному процесуальному кодексі України. Форум права. 2012. № 2. С. 629–631. 12. Фаринник В.І. Заходи забезпечення кримінального провадження у новому кримінальному процесуальному законодавстві: сутність та класифікація. Вісник кримінального судочинства. 2015. № 1. С. 133–142. 12. Фаринник В.І. Заходи забезпечення кримінального провадження у новому кримінальному процесуальному законодавстві: сутність та класифікація. Вісник кримінального судочинства. 2015. № 1. С. 133–142. Стаття надійшла до редакції 25.07.2023 Стаття надійшла до редакції 25.07.2023 Roman LOGIN, (Kharkiv National University of Internal Affairs, Kharkiv) THEORETICAL BASIS OF THE APPLICATION OF MEASURES TO ENSURE CRIMINAL PROCEEDINGS DURING THE PRE-JUDICIAL INVESTIGATION OF CORRUPTION CRIMINAL OFFENSES An analysis of the state of scientific development of theoretical knowledge about measures to ensure criminal proceedings as a basis for research into the use of measures to ensure criminal proceedings during pre-trial investigation of corruption criminal offenses was carried out. An overview of modern approaches to the definition of the concept of measures of criminal proceedings, its correlation with the concept of criminal procedural coercion, the type classification of measures of criminal proceedings and their functional purpose, existing in the domestic doctrine of criminal proceedings, was given. A dialectical connection with general theoretical provisions on measures to ensure criminal proceedings and further study of the use of measures to ensure criminal proceedings during pre-trial investigation of corruption criminal offenders is determined. Key words: procedural coercion, measures to ensure criminal proceedings, pre-trial investigation of corruption criminal offenders.  188   188 
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English
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Genotypic background of the recipient plant is crucial for conferring RB gene mediated late blight resistance in potato
BMC genomic data
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Shandil et al. BMC Genetics (2017) 18:22 DOI 10.1186/s12863-017-0490-x Shandil et al. BMC Genetics (2017) 18:22 DOI 10.1186/s12863-017-0490-x © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Genotypic background of the recipient plant is crucial for conferring RB gene mediated late blight resistance in potato Rajesh K. Shandil1, Swarup K. Chakrabarti1*, Bir Pal Singh1, Sanjeev Sharma1, S. Sundaresha1, Surinder K. Kaushik2, Arvind K. Bhatt3 and Nitya Nand Sharma4 Abstract Background: Late blight, caused by oomycetes pathogen Phytophthora infestans (Mont.) de Bary, is the most devastating potato disease in the world. RB gene from Solanum bulbocastanum has been shown to impart broad spectrum resistance against P. infestans races. In this study Katahdin transgenic event SP951 was used as male parent to cross with the popular Indian potato cultivars viz., Kufri Bahar (KB) and Kufri Jyoti (KJ) to enhance the late blight resistance. Results: Populations of 271 F1seedlings from the crosses KB × SP951 (87) and KJ × SP951 (184) were screened for inheritance of RB transgene through PCR and bioassay. Disease response based on AUDPC of different hybrid lines varied from immunity to complete susceptibility. High degree of resistance (<25% infection) was observed in KJ × SP951 derived seedlings (85.2%), whereas level of resistance in KB × SP951 (36.4% infection) derived seedlings was of low order. Conclusion: This study provides valuable genetic materials for development of potentially durable late blight resistant potato varieties. Besides, it also corroborates the fact that efficacy of R gene is not solely dependent on its presence in the variety but largely depends on the genetic background of the recipient genotype. Keywords: RB gene, Phytophthora infestans, Molecular breeding, Transgenic potato deployment of resistant varieties has been employed to manage this disease [2]. * Correspondence: Chakrabarti.sk@icar.gov.in 1ICAR-Central Potato Research Institute, Shimla 171001, Himachal Pradesh, India Full list of author information is available at the end of the article Background Potato (Solanum tuberosum L.) is an important horticul- tural food crop which has the potential to meet food demand of the fast growing human population across the world, including India. Late blight caused by the oomycetes pathogen Phytophthora infestans (Mont.) de Bary is the most devastating disease worldwide causing € 12 billion crop losses annually [1]. In India, late blight is more serious in temperate highlands and plateau region than in the Indo-Gangetic plains and average crop losses to the tune of 15% have been estimated which amounts to 6.7 million metric tonnes of potatoes. Integrated disease management strategy consisting of disease forecasting, fungicide spray schedules and Major R genes mainly derived from Solanum demissum have been exploited world over including India, to develop late blight resistant cultivars through classical breeding. The resistance conferred by these R genes, however, was short lived due to development of matching virulences of P. infestans in due course of time [3]. A wild diploid potato species S. bulbocastanum (2n = 2 × = 24) from Mexico and Guatemala, possessing very high degree of resistance to late blight controlled by R genes is an excep- tion. Classical breeding approach to transfer resistance from this species to cultivated potato is not possible because of differences in ploidy and Endosperm Balance Number [4]. To overcome this problem, the R gene responsible for broad-spectrum resistance in S. bulbocas- tanum was cloned by two independent groups in USA (RB, [5]) and The Netherlands (Rpi-blb1, [6]). The cloning * Correspondence: Chakrabarti.sk@icar.gov.in 1ICAR-Central Potato Research Institute, Shimla 171001, Himachal Pradesh, India Full list of author information is available at the end of the article l list of author information is available at the end of the article ull list of author information is available at the end of the article Shandil et al. BMC Genetics (2017) 18:22 Page 2 of 8 Page 2 of 8 of RB gene opened up the possibility of using recombinant DNA technology to transfer the gene to commercially im- portant susceptible potato varieties to diversify and strengthen late blight resistance in cultivated potatoes [7]. terminal portions of the gene were amplified by using the primer pairs 1–5/3–5 and cf1/cr1, respectively [8]. The allele specific primers MAMA2/INDEL-r were used to amplify the internal region of the RB gene [10]. The PCR reaction condition and temperature regimes as described by the respective authors were used for ampli- fication. Southern analysis Selected F1 lines that were PCR positive and showed late blight resistance were analyzed for the transgene integra- tion. For this, 15 μg of genomic DNA was digested with HindIII (New England Biolab) which cut once in the T- DNA. The digested DNA samples were electrophoressed on a 0.8% agarose gel for 16 h. The separated fragments were transferred onto a nylon membrane (Amersham, GE Healthcare, USA) and hybridization was performed using nptII gene fragment as the hybridized probes. Plant material The cultivar Katahdin and its transgenic event SP951 (RB-transgenic) used for introgression breeding was pro- vided by University of Wisconsin, Madison, USA under USAID-ABSP II project (Agricultural Biotechnology Support Project II). The potato cultivars Kufri Bahar and Kufri Jyoti were obtained from the Division of Seed Technology, CPRI, Shimla. The population of Kufri Jyoti × SP951 (184 F1 seedlings) and Kufri Bahar × SP951 (87 F1 seedlings) found positive for RB gene integration, were further evaluated for late blight resistance. All the plant materials including hybrid lines were clonally propagated inside transgenic containment facility, at ICAR-CPRI, Shimla. Kufri Bahar, Kufri Jyoti, Katahdin transgenic event SP951 (RB-transgenic line that contains one copy of the RB gene, [9]), and Katahdin (untrans- formed) were used as controls. Background The RP2-f1/r5 primer pairs (collectively referred to as “RP2 primers”) were used to amplify the RNA Polymerase II subunit 2 as internal check for all the samples [11]. g g p The RB gene isolated from S. bulbocastanum when transferred into Katahdin using Agrobacterium tumefa- ciens [5] imparted broad spectrum resistance to known races of P. infestans in selected transgenic events both in the greenhouse and in field experiments [7, 8]. In the present study, the RB-transgenic Katahdin event SP951 (obtained from University of Wisconsin, Madison, USA under USAID-ABSP II)was used as male parent in crosses with two popular Indian cultivars viz. Kufri Bahar (KB) and Kufri Jyoti (KJ) which occupy almost 400,000 hectares of potato area in India (CIP Social Sci- ence Working Paper 2005–06, page number 17). Kufri Jyoti is a day-neutral, widely adapted cultivar that was resistant to late blight and is grown throughout India including hills, plains and the plateau. Kufri Bahar on the other hand is susceptible to late blight but is the most preferred cultivar in west-central plains of India occupying about 60% area. In this study, introgression of RB gene in Indian popular potato cultivars was demon- strated for the enhancement of late blight resistance and generation of valuable genetic material for resistance breeding programme. It was also demonstrated that genotypes with variable level of late blight resistance can be developed by crossing a specific RB-transgenic event with well adapted Indian potato cultivars. DNA extraction and PCR reactions Genomic DNA was isolated from leaves of young shoots using GenElute™Plant Genomic DNA Mini Prep Kit (Sigma Aldrich). Template DNA from each young plant of 271 F1seedlings was extracted for PCR confirmation of RB gene. Multiplex PCR reaction (20 μL) was per- formed using 1 U of Taq DNA polymerase (Bangalore GeNei™, India), 100 ng of genomic DNA, 1X PCR Buffer (MgCl2 free), 2 mM MgCl2, 160 μM dNTP, 0.50 μM of each MAMA primers (MAMAF: CATCTTGAGAGAG TGAAGAATGATCT and MAMAR: CTAGTGCGCAA CACAATTGAA) and 0.10 μM of each RP2 primers (RP2F: TCGTGGATTTTTCCGATCTC and RP2R: ATC TCGCTCCATCTCTCCAA) with the following amplifi- cation conditions: 94 °C for 5 min; 35 cycles comprising of 94 °C for 1 min, 55 °C for 30 s, and 72 °C for 1 min; and final extension at 72 °C for 10 min. The RB gene in- sert was also analyzed using N-terminal and C-terminal primers. PCR reaction (20 μL) was performed using 1 U of Taq DNA polymerase (Bangalore GeNeiTM, India), 100 ng of genomic DNA, 1X PCR Buffer, 1.5 mM MgCl2, 160 μM dNTP, 0.20 μM of each N-terminal primers (RBNF: CTCATTTTACCCCTACAA and RBN R: GCGTTTTGGACCCTTTTA) and 0.20 μM of each C-terminal primers (RBCF: TAAGCATGAGTTGGAA TAACT and RBCR: GCCAGTCTTCTCCTATTCCCT) with amplification conditions as mentioned above. After completion of PCR, reaction products were loaded on 1% of agarose gel (containing ethidium bromide 0.5 μg/ mL) prepared in 1 X TAE buffer, and electrophoresed at 100 V for 2 h. Gel documentation was performed under UV light using a Fluor-STMMultiImager (BIO-RAD). Evaluation for foliar blight resistance g RB-positive F1seedlings were further tested for their effi- cacy in terms of resistance to foliar blight under P. infes- tans inoculation. The terminal disease severity was 45% (10 DAI)in transgenic Katahdin event SP951 as against 100% in the control cultivars thereby signifying the effectiveness of RB gene in providing resistance to late blight. The degree of late blight resistance in F1 progen- ies varied from immunity to complete susceptibility (Fig. 4). The distribution of the progeny (88 of KJ × SP951 clones)for late blight disease resistance scores are as follows: 1.1% (score 4), 3.4% (score 5), 10.2% (score 6), 15.9% (score 7), 65.9% (score 8) and 3.4% (score 9). All these clones had late bight infection <70% thereby demonstrating the effectiveness of RB gene in imparting resistance. Few seedlings like KJ/SP951-96, KJ/SP951-97 and KJ/SP951-104 showed very high degree of resistance (score 9, Fig. 4). Contrary to this, 12.7, 7.3, 12.7, 5.5, 16.4, 9.1, 20.0 and 16.4% seedlings of KB x SP951 were scored 1, 2, 3, 4, 5, 6, 7 and 8, respectively. In total 63.6% seedlings of the cross combination were suscep- tible (>25% infection) to late blight (Fig. 4). Disease severity was recorded at different time inter- vals (2, 6 & 10 days) after inoculation [13] i.e. 48, 52, 56 days after planting and populations of KB × SP951 and KJ × SP951 were grouped based on late blight resist- ance score (Table 1). Plants with score of > 7.0 (<25% infection) were scored as resistant whereas those with score of <7.0 (>25% infection) were scored as suscep- tible. Area Under the Disease Progress Curve (AUDPC) for some of the lines showing variation in degree of late blight resistance was also calculated [14]. Southern analysis The integration of the transgene was confirmed by Southern hybridization in the parental line SP951 as well as in the selected F1 genotypes (Fig. 3). The result showed that the transgene copy number was the same (single copy) in the parental transgenic Katahdin SP951 as well as in the progeny lines. This confirmed stable integration of the transgene in the genome of the RB- transgenic Katahdin line SP951 and segregation of the inserted locus in the progeny of crosses involving adapted Indian potato cultivars as female parent and SP951 as male parent. Late blight disease analysis of F1 seedlings Seedlings of Kufri Bahar x SP951 (55) and Kufri Jyoti x SP951(88) positive for RB gene, were evaluated for foliar blight resistance. Three tubers of each clone were planted in earthen pots under transgenic containment facility. The pots were shifted to green house at 40 days after planting (DAP) and were allowed to acclimatize for six days. Thereafter, the plants were shifted to late blight screening chamber where temperature (18 ± 1 °C) and relative humidity (≥90%) were maintained. P. infestans isolate belonging to A2 mating type and having all viru- lence genes was mass cultured on tuber slices of cv. Kufri Chandramukhi. The sporangia were washed from the tuber surface in sterilized distilled water and diluted to 40,000 sporangia/ml. The sporangial suspension was incubated at 4 °C for 30 min for release of zoospores. Plants were sprayed till run off with zoospore suspension using a hand atomizer. Molecular analysis of F1 seedlings Presence of RB gene in the plant samples was confirmed by amplification of both N- and C-terminal regions as well as internal region of the RB gene sequence using three different primer pairs. The N-terminal and C- Shandil et al. BMC Genetics (2017) 18:22 Page 3 of 8 Page 3 of 8 ratio of ~1:0.89 in the F1 which is expected in this experiment. Similarly, PCR amplification with N- and C- terminal region specific primers yielded fragment size of 619 bp (Fig. 2) and 840 bp (Fig. 2), respectively which confirmed the stability and inheritance of the transgene. DNA fragments were labelled with α[32P]- dCTP using a random primer DNA labelling kit (Amersham, GE Healthcare, USA). Hybridization was performed at 65 °C for 18 h. The filter was washed at room temperature with 2× SSC and 0.1% SDS, followed by 1× SSC and 0.1% SDS, for 10 min each [12] and the image analysed using a phosphorimager (BioRad, USA). DNA fragments were labelled with α[32P]- dCTP using a random primer DNA labelling kit (Amersham, GE Healthcare, USA). Hybridization was performed at 65 °C for 18 h. The filter was washed at room temperature with 2× SSC and 0.1% SDS, followed by 1× SSC and 0.1% SDS, for 10 min each [12] and the image analysed using a phosphorimager (BioRad, USA). Table 1 Scoring of late blight resistance based on the percentage of infected leaf tissue Late Blight Infection (%) >90 81–90 71–80 61–70 41–60 26–40 11–25 <10 0 Score 1 2 3 4 5 6 7 8 9 Molecular analysis of F1seedlings When Kufri Bahar and Kufri Jyoti cultivars of potato were crossed with Katahdin transgenic event SP951 (RB- transgenic line), 271 F1seedlings (87 of KB× SP951 and 184 of KJ × SP951) were obtained and were tested for RB gene integration. Stability and inheritance of RB gene in F1 seedlings were confirmed by PCR amplification that revealed a 712 bp fragment in 55 F1 seedlings of KB × SP951and 88 F1 seedlings of KJ × SP951 (Fig. 1) with mismatch amplification for mutation analysis (MAMA) primer. It showed that the RB gene segregated in the Late blight infection in KJ/SP951-4 at 52 DAP was 15% only which almost ceased thereafter. A very high level of late blight resistance was recorded in KJ/SP951- 96, KJ/SP951-97, KJ/SP951-104, KJ/SP951-8, KJ/SP951- 16 and KJ/SP951-19seedlings (Fig. 5). Whereas in KJ/ SP951-4, KJ/SP951-18, KJ/SP951-63, KJ/SP951-15, and KJ/SP951-54 seedlings it was 20 to 50% at 52 DAP, the disease progression almost ceased during 52–57 DAP. Table 1 Scoring of late blight resistance based on the percentage of infected leaf tissue Late Blight Infection (%) >90 81–90 71–80 61–70 41–60 26–40 11–25 <10 0 Score 1 2 3 4 5 6 7 8 9 Shandil et al. BMC Genetics (2017) 18:22 Page 4 of 8 Fig. 1 Multiplex PCR of F1seedlingsof the cross Kufri Jyoti x SP951 and Kufri Bahar x SP951using MAMA and RP2 primers paternal parent SP951 in terms of late blight resistance. This clearly indicates effectiveness of RB gene in inhibit- ing progression of late blight infection. On the other hand, very few seedlings of the cross KB × SP951 showed inhibitory effect on late blight progression (Fig. 5). In KB/SP951-4 (50% at 52 DAP), KB/SP951-13 (60% at 52 DAP) and KB/SP951-3 (70% at 52 DAP), late blight progression almost ceased even after very high infection percentage (50–70%) (Fig. 5). On the other hand, very few seedlings of the cross KB × SP951 showed inhibitory effect on late blight progression (Fig. 5). In KB/SP951-4 (50% at 52 DAP), KB/SP951-13 (60% at 52 DAP) and KB/SP951-3 (70% at 52 DAP), late blight progression almost ceased even after very high infection percentage (50–70%) (Fig. 5). Discussion Experiments to find out the best combination of RB gene with other gene(s) for obtaining best level of late blight resistance is underway in our laboratory. The correl- ation between the transcript abundance of RB gene and late blight resistance has been established recently [9, 15] in different transgenic events of the same variety. However, it may not be sufficient to explain the observed difference in the level of late blight resistance in the F1 progeny of a particular transgenic event as indicated in our study. Similarly, the Sgt1 gene has been reported essential for the RB-mediated late blight resistance and Sgt1- si- lenced transgenic lines of potato failed to exhibit RB- me- diated late blight resistance [16]. In some instances, pathogen induced up-regulation of R genes has also been noted previously in other pathosystems [17–20]. Fig. 3 Copy number analysis of parental line SP951 and F1 hybrid lines Fig. 3 Copy number analysis of parental line SP951 and F1 hybrid lines (2x) [1]. On the other hand, development of stable trans- genic potato plants with new R gene(s) derived from another potato species is relatively simple and has only a minor impact on the genetics of the recipient cultivated variety [8]. However, bio-efficacy of directly regenerated transgenic plants may differ because of differences in sites of gene integration, copy number variation of the inserted gene as well as genetic background of the recipient genotype(s). Therefore, the present attempt was made to develop RB-transgenic potato varieties by crossing well adapted indigenous potato cultivar(s) with well-characterized RB transgenic event of another exotic potato variety through conventional breeding. The Indian cultivar Kufri Jyoti was bred as a late blight resistant cultivar in 1968 using S. demissum as resistant parent. It possesses three major R genes (R3, R4, R7) but resistance in this cultivar was compromised due to development of matching virulences during 1980s that knocked down all the three R genes. However, the down- stream signal transduction cascade as well as PR pro- teins in Kufri Jyoti was presumably very effective since it showed high level of resistance to late blight before its A large variation in the level of late blight resistance in the F1population derived from KJ × SP951 and KB × SP951 was recorded. F1 seedlings derived from KJ × SP951 showed better resistance compared to those derived from Fig. Discussion Many tuber-bearing wild Solanum species are known to possess high degree of resistance to late blight. The RB/ Rpi-blb1 gene cloned from the S. bulbocastanum, when integrated into four popular potato cultivars susceptible to late blight, viz. Katahdin, Superior, Dark Red Norland, and Russet Burbank, conferred broad-spectrum late blight resistance consistently with little or no impact on total yield [5, 8]. The introgression of late blight resist- ance from wild Solanum species into currently cultivated potato through classical breeding is a long-term process. For instance, it took 46 years of breeding efforts to develop two varieties (Bionic and Toluca) from the first bridge cross between S. acaule (4x) × S. bulbocastanum Based on AUDPC, KB x SP951 seedlings were found more susceptible than those derived from KJ x SP951. Lowest AUDPC values (0–2) were recorded for 36 seed- lings of KJ × SP951 whereas only one hybrid of KB × SP951 fell in this category. AUDPC values for Kufri Jyoti seedlings were lower (139 out of 184 hybrid lines show- ing AUDPC values < 1) than Kufri Bahar seedlings (10 out of 87 hybrid lines showing AUDPC values < 1) indi- cating higher inherent late blight resistance in Kufri Jyoti seedlings. All the KJ × SP951 seedlings had better resist- ance than the maternal parent Kufri Jyoti whereas sev- enty six (out of 184 hybrid lines)were better than the Fig. 2 PCR confirmation of RB gene integration in F1seedlings using N-terminal (619 bp fragment size) and C-terminal (840 bp fragment size) primers mation of RB gene integration in F1seedlings using N-terminal (619 bp fragment size) and C-terminal (840 bp fragment Fig. 2 PCR confirmation of RB gene integration in F1seedlings using N-terminal (619 bp fragment size) and C-terminal (840 bp fragment size) primers Shandil et al. BMC Genetics (2017) 18:22 Page 5 of 8 Page 5 of 8 KB × SP951. High throughput gene expression analysis of the selected F1 seedlings showed conflicting results with respect to level of expression of R and R-like genes vis-à- vis late blight resistance level (unpublished data). It, there- fore, indicated that expression level of RB gene alone in particular F1 genotype was unable to explain its observed level of late blight resistance. Instead, it may be dependent on genotypic background, specifically combinations of pathogenesis related (PR) gene alleles with the RB allele in particular F1genotype derived from the introgression cross. Discussion 4 Distribution of RB positive population of Kufri Jyoti × SP951 and Kufri Bahar × SP951 (F1 seedlings) in the late blight resistance scale (Table 1) based on terminal disease severity at 10 DAI (days after inoculation) Fig. 4 Distribution of RB positive population of Kufri Jyoti × SP951 and Kufri Bahar × SP951 (F1 seedlings) in the late blight resistance scale (Table 1) based on terminal disease severity at 10 DAI (days after inoculation) Shandil et al. BMC Genetics (2017) 18:22 Page 6 of 8 Fig. 5 Progression of late blight infection in terms of Area Under the Disease Progress Curve (AUDPC) in Kufri Jyoti x SP951 and Kufri Bahar x SP951seedlings. The lines were grouped in the scale 1–9(DAP: Days after planting) breakdown due to evolution of matching races in the pathogen. It was evident from the high level of resist- ance observed in majority of F1 progeny derived from Kufri Jyoti where an effective R gene (RB) was intro- duced. Although, the stability and inheritance of the RB gene was confirmed through Southern analysis and late blight assay, variation in the resistance level of the F1 progeny having the RB gene was probably due to shuf- fling of other downstream genes during meiotic recom- bination. On the contrary, most of the progeny derived from the cross with the susceptible cultivar Kufri Bahar showed inferior level of resistance even after having RB gene, probably because of in-effective combination of downstream genes. This indicates that allelic combin- ation of downstream genes encoding signal transduction cascades as well as pathogenesis related proteins are very important for getting desired level of resistance in a par- ticular genotype. Besides, several other polygenic com- ponents may be responsible for the observed variation in late blight severity among the F1seedlings. For example, theSgt1 gene has been reported essential for the RB-me- diated late blight resistance and this may be another rea- son for the observed variation in R gene-mediated plant defense responses [16]. An understanding of the genetic relationship within potato hybrid lines is important to establish a broad genetic base for breeding purposes [21]. Potato germplasm with reported late blight resist- ance should be introgressed with RB for stronger and more durable resistance. Abbreviations AUDPC: Area under the disease progress curve; DAP: Days after planting; KB/ SP951: Lines obtained from Kufri Bahar crossed with SP951; KJ/SP951: Lines obtained from Kufri Jyoti crossed with SP951; MAMA: Mismatch amplification mutation assay; PR: Pathogenesis related; RB: R gene from Solanum bulbocastanum; SGT: Suppressor of G2 allele of S-phase Kinase Associated Protein 1 (SKP1) gene; SP951: RB transgenic Katahdin event (Male parent) Conclusions The study confirms that the classical R gene (RB) with NBS-LRR-CC motif from the wild potato species Solanum bulbocastanum confers broad spectrum resistance to po- tato late blight. It was also established that the RB trans- gene could be transferred to the F1 progeny by crossing non-transgenic locally adapted cultivar with the transgenic event of the cultivar Katahdin. In addition this study dem- onstrated that the level of late blight resistance varied greatly within the F1 progeny, i.e. a few F1 genotype pos- sessing the RB transgene showed immunity to late blight while some others were completely susceptible even after possessing the RB transgene. The differential late blight response could not be explained by the variation in level of RB gene expression in the F1 progeny. This study, therefore, clearly indicated that efficacy of R gene is not solely dependent on its presence in the particular geno- type but largely depends on the genetic background of the recipient genotype. It will be interesting to investigate role of other downstream genes in potato for achieving satis- factory level of resistance through the classical R gene. Funding Indian Council of Agricultural Research, New Delhi, India and Agricultural Biotechnology Support Project II, Cornell University, USA. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Ethics approval and consent to participate The RB-transgenic event SP951 of the potato cultivar Katahdin was imported from Wisconsin Alumni Research Foundation (WARF), University of Wisconsin, Madison, USA under the import permit No. 96/2005 and 116/2005 (IQ No. 156/05) dated 18.02.2005 of Department of Biotechnology, Government of India. The Indian Council of Agricultural Research signed a Material Transfer Agreement (MTA) with the Wisconsin Alumni Research Foundation (WARF) for utilizing the RB gene in development of late blight resistant variety. The efficacy trial of the material was done under confined condition as per the approval given by the Review Committee on Genetic Modification (RCGM), Department of Biotechnology, Government of India vide letter number BT/ BS/11/22/97-PID dated 12.06.2006 and BT/BS/11/22/97-PID dated 20.11.2009. The study did not involve human participants, human data or human tissue. Availability of data and materials The plant materials and other biological materials used and generated under this study are available at ICAR-Central Potato Research Institute, Shimla, India. Experimental data on late blight evaluation and molecular analysis of RB-transgenic materials are available with the corresponding author as digital data as well as in laboratory note book. Consent for publication Not applicable. Consent for publication Not applicable. Authors’ contributions RKS conducted all the experiments and collected data; SKC, BPS and AKB contributed in planning of experiments, data collection and analysis and interpretation of result; SKK contributed in making crosses and progeny development, SKC and RKS contributed in preparation of manuscript; SSh contributed in late blight screening; SSu and NNS contributed in molecular analysis of experimental materials. All the authors have read and approved the final manuscript. Therefore, RB transgene can be used effectively to reduce foliar late blight infection in cultivated potatoes. This enhanced resistance over the generation may be ascribed to the pooling of R gene activation or re- shuffling of R genes in the background of Kufri Jyoti that imparts resistance against Phytophthora infestans. This RB positive F1 genotypes showing different levels of late blight resistance will be useful for identifying promising downstream genes to build the future molecular breed- ing activities. Discussion The results corroborate the hypothesis that RB is an signal receptor protein that triggers a cache of defence proteins to induce localized hypersensitive response thereby halting the pathogenesis at the site of invasion [22]. Level of resistance depends on the combination of effective alleles of the defence proteins present in a par- ticular genotypic background. Our findings support the above hypothesis. The cessation of late blight progres- sion after 40–70% infection in the KB × SP951 and KJ × Page 7 of 8 Shandil et al. BMC Genetics (2017) 18:22 Page 7 of 8 Page 7 of 8 SP951 population, supports the results obtained for S. bulbocastanum clone PT29 derived somatic hybrids and a number of backcrossed progenies where the pathogen sometimes sporulated and the disease phenotype was general suppression but not elimination of symptom development [17, 23]. Promising lines were selected on the basis of per cent infection on the last day (57 day after planting, DAP) of scoring as well as AUDPC. Area under the disease progress curve is a measure of disease development over the whole course of the epidemic. The lines showing 40% or less late blight infection at 57 DAP and AUDPC values below that of control varieties Kufri Bahar and Kufri Jyoti were selected for further evaluation. Author details 1ICAR-Central Potato Research Institute, Shimla 171001, Himachal Pradesh, India. 2Division of Germplasm Evaluation, ICAR-National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi, India. 3Department of Biotechnology, Himachal Pradesh University, Shimla, H.P, India. 4Premas BiotechPvt. Ltd, Gurgaon, Haryana, India. Received: 2 September 2016 Accepted: 3 March 2017 Received: 2 September 2016 Accepted: 3 March 2017 References 1. Haverkort AJ, Struik PC, Visser RGF, Jacobsen E. Applied biotechnology to combat late blight in potato caused by Phytophthora infestans. Potato Res. 2009;52:249–64. 1. Haverkort AJ, Struik PC, Visser RGF, Jacobsen E. Applied biotechnology to combat late blight in potato caused by Phytophthora infestans. Potato Res. 2009;52:249–64. 2. Singh BP, Roy S, Bhattacharyya SK, Shekhawat GS. Schedule of metalaxyl-based fungicide and development of fungicide resistant strains.In: GS Shekhawat, SM Paul Khurana, SK Pandey, VK Chandla, editors. Potato, Present and Future. Shimla: Indian Potato Association, Central Potato Research Institute; 1994. pp. 179-183. Competing interests Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. 3. Zhu SX, Vossen JH, Bergervoet M, Nijenhuis M, Kodde L, Kessel GJT, Vleeshouwers VGAA, Visser RGF, Jacobsen E. An updated conventional and a novel GM potato late blight R gene differential set for virulence monitoring of Phytophthora infestans. Euphytica. 2015;202(2):219–34. Abbreviations AUDPC A 3. Zhu SX, Vossen JH, Bergervoet M, Nijenhuis M, Kodde L, Kessel GJT, Vleeshouwers VGAA, Visser RGF, Jacobsen E. An updated conventional and a novel GM potato late blight R gene differential set for virulence monitoring of Phytophthora infestans. Euphytica. 2015;202(2):219–34. 3. Zhu SX, Vossen JH, Bergervoet M, Nijenhuis M, Kodde L, Kessel GJT, Vleeshouwers VGAA, Visser RGF, Jacobsen E. 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Glutathione Deficiency in Cardiac Patients Is Related to the Functional Status and Structural Cardiac Abnormalities
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6,706
To cite this version: Thibaud Damy, Matthias Kirsch, Lara Khouzami, Philippe Caramelle, Philippe Le Corvoisier, et al.. Glutathione deficiency in cardiac patients is related to the functional status and structural cardiac abnormalities.. PLoS ONE, 2009, 4 (3), pp.e4871. ￿10.1371/journal.pone.0004871￿. ￿inserm-00448489￿ Glutathione deficiency in cardiac patients is related to the functional status and structural cardiac abnormalities. Thibaud Damy, Matthias Kirsch, Lara Khouzami, Philippe Caramelle, Philippe Le Corvoisier, Françoise Roudot-Thoraval, Jean-Luc Dubois-Randé, Luc Hittinger, Catherine Pavoine, Françoise Pecker Glutathione Deficiency in Cardiac Patients Is Related to the Functional Status and Structural Cardiac Abnormalities Thibaud Damy1,2,3., Matthias Kirsch1,3., Lara Khouzami2,3, Philippe Caramelle2,3, Philippe Le Corvoisier2,3,4,5, Franc¸oise Roudot-Thoraval3,6, Jean-Luc Dubois-Rande´ 1,2,3, Luc Hittinger1,2,3, Catherine Pavoine2,3, Franc¸oise Pecker1,2,3* 1 AP-HP, Groupe hospitalier Henri-Mondor Albert-Chenevier, Fe´de´ration de Cardiologie, De´partement de Chirurgie Cardiaque, Cre´teil, France, 2 INSERM, U955, Cre´teil, France, 3 Universite´ Paris12, Faculte´ de Me´decine, UMR-S955, Cre´teil, France, 4 INSERM, Centre d’Investigation Clinique 006, Cre´teil, France, 5 Platform of biological resources, Groupe hospitalier Henri-Mondor Albert-Chenevier, Cre´teil, France, 6 AP-HP, Groupe hospitalier Henri-Mondor Albert-Chenevier, De´partement de Recherche Clinique- Sante´ Publique, Cre´teil, France Abstract Funding: The funders for the present work were: Institut National de la Sante et de la Recherche Medicale, the Assistance Publique des Hopitaux de Paris (as a Contrat d’Interface to F.P.), the Universite Paris 12 and the Association Francaise contre les Myopathies (as an aide aux etudes to L.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: francoise.pecker@inserm.fr . These authors contributed equally to this work. Citation: Damy T, Kirsch M, Khouzami L, Caramelle P, Le Corvoisier P, et al. (2009) Glutathione Deficiency in Cardiac Patients Is Related to the Functional Status and Structural Cardiac Abnormalities. PLoS ONE 4(3): e4871. doi:10.1371/journal.pone.0004871 Editor: Alicia J. Kowaltowski, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Brazil Editor: Alicia J. Kowaltowski, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Brazil Received December 2, 2008; Accepted February 2, 2009; Published March 25, 2009 my et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. pyright:  2009 Damy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution L restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright:  2009 Damy et al. This is an open-access article distributed under the terms of the Creative Commons Attr unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The funders for the present work were: Institut National de la Sante et de la Recherche Medicale, the Assistance Publique des Hopitaux de Paris (as a Contrat d’Interface to F.P.), the Universite Paris 12 and the Association Francaise contre les Myopathies (as an aide aux etudes to L.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: francoise.pecker@inserm.fr . These authors contributed equally to this work. HAL Id: inserm-00448489 https://inserm.hal.science/inserm-00448489v1 Submitted on 19 Jan 2010 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. Abstract Background: The tripeptide glutathione (L-gamma-glutamyl-cysteinyl-glycine) is essential to cell survival, and deficiency in cardiac and systemic glutathione relates to heart failure progression and cardiac remodelling in animal models. Accordingly, we investigated cardiac and blood glutathione levels in patients of different functional classes and with different structural heart diseases. Methods: Glutathione was measured using standard enzymatic recycling method in venous blood samples obtained from 91 individuals, including 15 healthy volunteers and 76 patients of New York Heart Association (NYHA) functional class I to IV, undergoing cardiac surgery for coronary artery disease, aortic stenosis or terminal cardiomyopathy. Glutathione was also quantified in right atrial appendages obtained at the time of surgery. Results: In atrial tissue, glutathione was severely depleted (258%) in NYHA class IV patients compared to NYHA class I patients (P = 0.002). In patients with coronary artery disease, this depletion was related to the severity of left ventricular dysfunction (P = 0.006). Compared to healthy controls, blood glutathione was decreased by 21% in NYHA class I patients with structural cardiac disease (P,0.01), and by 40% in symptomatic patients of NYHA class II to IV (P,0.0001). According to the functional NYHA class, significant depletion in blood glutathione occurred before detectable elevation in blood sTNFR1, a marker of symptomatic heart failure severity, as shown by the exponential relationship between these two parameters in the whole cohort of patients (r = 0.88). Conclusions: This study provides evidence that cardiac and systemic glutathione deficiency is related to the functional status and structural cardiac abnormalities of patients with cardiac diseases. These data also suggest that blood glutathione test may be an interesting new biomarker to detect asymptomatic patients with structural cardiac abnormalities. Citation: Damy T, Kirsch M, Khouzami L, Caramelle P, Le Corvoisier P, et al. (2009) Glutathione Deficiency in Cardiac Patients Is Related to the Functional Status and Structural Cardiac Abnormalities. PLoS ONE 4(3): e4871. doi:10.1371/journal.pone.0004871 Editor: Alicia J. Kowaltowski, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Brazil Received December 2, 2008; Accepted February 2, 2009; Published March 25, 2009 Copyright:  2009 Damy 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. PLoS ONE | www.plosone.org Patients The study included 76 patients undergoing cardiac surgery (coronary artery bypass grafting, aortic valve replacement, ortho- topic heart transplantation and ventricular assist device implanta- tion) from 2004 to 2007. Clinical data and transthoracic echocardiographies (Vivid 7, GE, Norway), using american society of echocardiography recommendations [23], were obtained for all individuals. To distinguish patients with systolic LV dysfunction from those with preserved LV function, we used as cut-off value 45% LVEF, which is the mean of the 40–50% range proposed by the new ESC guidelines [24]. Permanent atrial fibrillation was defined as long standing atrial fibrillation in which cardioversion had failed or had been foregone, according to the ESC guidelines [25]. Venous blood samples and right atrial appendages were obtained from patients undergoing cardiac surgery for coronary artery bypass graft or aortic valve replacement with cardiopulmo- nary bypass. Blood samples only were obtained from patients undergoing left ventricular assist device implantation. Right atrial specimen and 2 venous blood samples were taken into cryotubes at initiation of cardiopulmonary bypass, immediately frozen in liquid nitrogen and stored at 280uC until use. Patients with sepsis, endocarditis, renal failure or impaired liver function were excluded. Introduction (BNP) and the amino-terminal fragment of its precursor hormone (NT-pro-BNP) secreted in response to myocardial stress have also received considerable attention as potential screening and prognos- tic tests for symptomatic, New York Heart Association (NYHA) class II to IV patients [12–16]. However, neither TNF nor sTNFR1 or BNP peptides do help to the screening of asymptomatic patients suspected of having a structural heart disease. Only very recently, circulating MMP-9 has been associated with cardiovascular risk factors in middle-aged normal population [17]. Despite considerable advances in treatment, heart failure remains associated with high morbidity and mortality worldwide [1–3]. Better identification of asymptomatic individuals with structural cardiac abnormalities would improve outcomes and reduce incidence of heart failure. The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF) and the cleaved extracellular domain of its type-1 receptor (sTNFR1) are recognized biomarkers of heart failure severity and adverse outcomes of the disease [4–11]. B-type natriuretic peptide Exacerbated TNF and sTNFR1 expression is related to systemic and cardiac glutathione deficiency in animal models of heart PLoS ONE | www.plosone.org March 2009 | Volume 4 | Issue 3 | e4871 1 Glutathione in Heart Diseases to GSH by glutathione reductase in the presence of NADPH. This method is simple, convenient, sensitive, accurate and rapid, and can assay glutathione in whole blood and tissues. In addition, it uses sulfosalicylic acid for sample preparation, which inhibits gamma- glutamyl transferase and limits glutathione loss [27]. sTNFR1 was quantified in whole blood with ELISA kits (Quantikine, R&D Systems). failure [18,19], and in advanced heart failing patients [19,20]. In fact, the antioxidant tripeptide glutathione (L-gamma-glutamyl– cysteinyl–glycine) is essential for vascular and cardiac function [19,20], and determines cell survival [21,22]. to GSH by glutathione reductase in the presence of NADPH. This method is simple, convenient, sensitive, accurate and rapid, and can assay glutathione in whole blood and tissues. In addition, it uses sulfosalicylic acid for sample preparation, which inhibits gamma- glutamyl transferase and limits glutathione loss [27]. We hypothesized that functional status and cardiac structural remodelling of patients were related to glutathione deficiency. The purpose of the present study was to explore the glutathione levels in cardiac tissue and blood of patients with cardiac structural abnormalities in relation to NYHA functional classification, left ventricular ejection fraction (LVEF) and blood sTNFR1. sTNFR1 was quantified in whole blood with ELISA kits (Quantikine, R&D Systems). Statistical analysis Results are given as means6sem. Continuous data were analyzed by Mann-Whitney test or Kruskal-Wallis test combined with Dunn post-test, as appropriate (Prism, GraphPad Software Inc). Discontinuous data were analyzed using a Chi square test. Differences were considered statistically significant at P,0.05 (two tailed). We determined the cut-off values of blood glutathione and blood sTNFR1 level to discriminate cardiac patients (NYHA class I to IV) from healthy controls by constructing receiver operating characteristics (ROC) curves relating each marker to NYHA class. Areas under the ROC curves (AUROCs) are given with their 95% confidence intervals. Cut-off values were chosen to optimize the couple of values sensitivity/specificity. Right atrial glutathione in patients with cardiac disease Right atrial glutathione in patients with cardiac disease Compared to patients of NYHA class I, patients of NYHA class II and III displayed quite preserved right atrial glutathione content (Fig. 2). In contrast, patients of NYHA class IV demonstrated a dramatic 58% depletion in atrial tissue glutathione, dropping to 1.060.2 nmol glutathione/mg tissue to be compared to 2.460.2 nmol glutathione/mg tissue in NYHA class I patients (P = 0.002) (Fig. 2). Of note, as previously pointed out by Carnes et al [28], patients with permanent atrial fibrillation displayed a significant decrease in atrial glutathione content compared to patients with sinus rhythm (1.160.3 vs 2.160.2 nmol/ mg tissue, respectively; P,0.05) (Table 1). PLoS ONE | www.plosone.org Clinical and biological characteristics of the patients The clinical and biological characteristics of the 76 patients undergoing surgery for dilated cardiomyopathy (transplantation or mechanical assist device implantation; n = 8), aortic valve stenosis (AS; n = 25) or coronary artery disease (CAD; n = 43) are reported in Table 1. Patients with CAD and patients with AS constituted the two principal groups of our cohort. The three groups had cardiac structural abnormalities. Patients with CAD and patients with dilated cardiomyopathy had reduced LVEF while AS patients displayed significant hypertrophy of septal (ST) and posterior (PWT) end-diastolic walls but preserved LVEF (Table 1). In the cohort, 22% of the patients were of functional NYHA class I, 31% of NYHA class II, 29% of NYHA class III and 18% of NYHA class IV. As compared to healthy controls (mean LVEF of 6261% and mean age of 5264 years), patients of NYHA class I had a preserved LVEF (Fig. 1A). In patients of NYHA class II to IV, LVEF declined progressively, but only patients of NYHA class III and IV had statistically depressed LVEF, with mean values approaching 4063% and 2564%, respectively (Fig. 1A), related to a 3- to 5-fold elevation in blood sTNFR1 level as compared to healthy controls (Fig. 1B). Of note, mean blood sTNFR1 level in NYHA class I patients was not statistically different from that of healthy controls (Fig. 1B). Fifteen healthy volunteers were recruited by the Centre d’Investigation Clinique of the Hoˆpital Henri Mondor. Blood samples obtained from each fasting volunteer, clinical data, and transthoracic echocardiographies were processed as described above for patients. Volunteers included 8 males and 7 females with mean age of 5264 years (range: 30–70 years), mean LVEF of 61.661.4% (range: 55–68%), mean blood glutathione level of 2.1360.4 (range: 1.84–2.36 mM) and mean blood sTNFR1 level of 0.2560.01 ng/ml (range: 0.20–0.33 ng/ml). All patients had given written informed consent before surgical procedures were performed. All studies are conformed to the Declaration of Helsinki and were approved by our institutional ethics committee (AP-HP, Groupe hospitalier Henri-Mondor Albert-Chenevier, Cre´teil, F-94010, France). Assays for glutathione and sTNFR1 Atrial tissue samples were cut into 20 mm sections. Homoge- nates were prepared from 5 frozen sections of each sample by homogenization at 4uC, in 200 ml of 50 mM Hepes, pH 7.4, containing protease inhibitors (1 mM PMSF, 2 mg/ ml leupeptin, 2 mg/ ml aprotinin), using a Qiagen TissueLyzer. Glutathione was measured in atrial homogenates or whole blood according to a modification of Tietze’s recycling assay [26] as previously used [18] and as thoroughly described by Rahman et al. [27]. In short, it is a spectrophotometric/microplate reader assay method, relying on oxidation of reduced glutathione (GSH) by the sulfhydryl reagent 5,59-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow derivative 59-thio-2-nitrobenzoic acid (TNB), measurable at 405 nm. Glutathione disulfide (GSSG) is recycled Next, we considered separately the two principal CAD and AS groups of patients, redistributed into 2 subgroups according to their LV function, after excluding patients with permanent atrial fibrillation. The subgroup of CAD patients with LVEF decline (#45%) displayed 40% deficiency in atrial glutathione content March 2009 | Volume 4 | Issue 3 | e4871 PLoS ONE | www.plosone.org 2 Glutathione in Heart Diseases Table 1. Baseline characteristics of patients undergoing cardiac surgery. Assays for glutathione and sTNFR1 p g g g y Parameters All patients CAD AS P-value Demographic Male/ female (n/ n) 60/ 16 37/ 6 15/ 10 0.01 Age (years) 6661 6262 7562 ,0.0001 NYHA (mean) 2.360.1 2.260.2 2.660.2 NS NYHA class I (%) 22 31 5 NYHA class II (%) 31 31 42 NYHA class III (%) 29 26 37 NYHA class IV (%) 18 12 16 Clinical Hypertension (%) 56 64 36 NS Hypercholesterolemia (%) 56 81 47 0.03 Diabetes mellitus (%) 35 45 17 NS Permanent atrial fibrillation (%) 12 5 24 0.01 Echocardiographic LVEF (%) 4562 4562 5364 0.06 LVEDD (mm) 5461 5562 5062 0.04 iLVEDD (mm.cm22) 3061 3061 2961 NS ST (mm) 11.660.4 10.360.4 13.360.7 0.0001 PWT (mm) 10.860.4 9.860.4 12.160.6 0.002 LA diameter (mm) 4162 4162 4263 NS Systolic PAP (mm Hg) 4462 4264 4363 NS Medication Beta-blockers (%) 51 71 22 0.0005 ACE inhibitors (%) 35 50 11 NS AT-II type 1 R antagonists (%) 21 20 22 NS Diuretics (%) 48 43 57 NS Aldosterone antagonists (%) 16 20 9 NS Statin (%) 60 77 35 0.001 Surgical Not urgent surgery, n(%) 83 81 100 0.02 Biochemical CRP (mg/ l) 1162 1163 1166 NS Haemoglobin (g/ dl) 13.160.2 13.360.3 12.860.5 NS Total bilirubin (mg/ dl) 1762 1863 1763 NS Creatinine (mmol/ l) 10665 10968 10066 NS CAD: patients with coronary artery diseases; AS: patients with aortic valve stenosis; LVEF: left ventricular ejection fraction; LVEDD: left ventricular end diastolic diameter; iLVEDD: indexed LVEDD; ST: end-diastolic septal wall thickness; PWT: end-diastolic posterior wall thickness; LAD: left atrial diameter; PAP: pulmonary artery pressure; CRP: C-reactive protein. In CAD and AS, blood glutathione was not correlated with the age (p = 0.46). Data are given as mean or percentage 6sem. P-values refer to the comparisons between CAD and AS patients. doi:10.1371/journal.pone.0004871.t001 CAD: patients with coronary artery diseases; AS: patients with aortic valve stenosis; LVEF: left ventricular ejection fraction; LVEDD: left ventricular end diastolic diameter; iLVEDD: indexed LVEDD; ST: end-diastolic septal wall thickness; PWT: end-diastolic posterior wall thickness; LAD: left atrial diameter; PAP: pulmonary artery pressure; CRP: C-reactive protein. In CAD and AS, blood glutathione was not correlated with the age (p = 0.46). Data are given as mean or percentage 6sem. P-values refer to the comparisons between CAD and AS patients. Discussion The new findings of our study are twofold. Firstly, asymptom- atic patients of NYHA I class display significantly deficiency in blood glutathione level compared to healthy controls. Blood glutathione deficiency worsens in patients of NYHA class II to IV in relation to blood sTNFR1 elevation, a marker of heart failure. Secondly, glutathione content in the atrial tissue is diminished by 58% in NYHA class IV patients compared to NYHA class I patients, and the degree of its decrease in CAD patients correlates with LVEF decline. Figure 1. Relations between LVEF or blood sTNFR1 level and NYHA class in cardiac patients and healthy controls. The cohort displayed standard relations between functional NYHA class and LVEF (A) or blood sTNFR1 level (B). LVEF: left ventricular ejection fraction. sTNFR1: cleaved extracellular domain of TNFR1. Linear trends P,0.0001. *P,0.05 vs healthy controls (C). {P,0.05 vs NYHA class I; {P,0.05 vs NYHA class II; $P,0.05 vs NYHA class III. doi:10.1371/journal.pone.0004871.g001 To our knowledge, our study is the first to show that blood glutathione deficiency correlates with the severity of heart failure symptoms in patients. Oxidative stress is a recognized contributor to heart failure progression [29,30], and previous studies have pointed out changes in the redox status of glutathione in the failing heart. However, they have overlooked a possible deficiency in total glutathione content [31]. The originality of the present study was to investigate this issue in a context of identifying a possible marker of cardiac disease severity with possible implication in clinical stratification. level of blood glutathione was not related with the age of the patients, approaching 1.561 mM in younger patients (#65 years, mean age of 5561 years) and 1.460.1 mM in older patients (.65 years, mean age of 7461 years). The relation between blood glutathione and sTNFR1 persisted whatever the age of the patients was (Fig. 5). sTNF and its receptors sTNFR1 and sTNFR2 are pro- inflammatory molecules, the blood levels of which are associated with oxidative stress and are predictive of heart failure adverse outcomes [4,6,9,11]. In keeping with previous reports, we observe a progressive increase in blood sTNFR1 level with increasing NYHA class in our patients. Blood sTNFR1 was inversely and exponentially correlated with blood glutathione, illustrating the ROC curve analysis for discriminating NYHA class I–IV patients from healthy controls was done for each sTNFR1 and blood glutathione marker. Assays for glutathione and sTNFR1 {P,0.05 vs NYHA class I; {P,0.05 vs NYHA class II; $P,0.05 vs NYHA class III. doi:10.1371/journal.pone.0004871.g001 Figure 2. Relation between atrial tissue glutathione content and NYHA class in cardiac patients. Atrial tissue glutathione Figure 2. Relation between atrial tissue glutathione content and NYHA class in cardiac patients. Atrial tissue glutathione content was significantly decreased in symptomatic patients of NYHA class IV compared to asymptomatic patients of NYHA class I. *P,0.05 vs NYHA class I. doi:10 1371/journal pone 0004871 g002 doi:10.1371/journal.pone.0004871.g002 100%. Using these thresholds, the composite index {glutathio- ne,1.835 mM or sTNFR1$0.33 nmol/ml} vs {glutathio- ne$1.835 mM and sTNFR1,0.33 nmol/ml} discriminated be- tween patients and controls with an improved sensitivity of 88.7% and a conserved 100% specificity. This result confirms the complementarity between both markers for discriminating patients from healthy controls. In fact, blood glutathione decrease allowed identification of NYHA class I patients from controls, whereas a large increase in blood sTNFR1 characterized patients of NYHA class IV. Assays for glutathione and sTNFR1 doi:10 1371/journal pone 0004871 t001 CAD: patients with coronary artery diseases; AS: patients with aortic valve stenosis; LVEF: left ventricular ejection fraction; LVEDD: left ventricular end diastolic diameter; iLVEDD: indexed LVEDD; ST: end-diastolic septal wall thickness; PWT: end-diastolic posterior wall thickness; LAD: left atrial diameter; PAP: pulmonary artery pressure; CRP: C-reactive protein. In CAD and AS, blood glutathione was not correlated with the age (p = 0.46). Data are given as mean or percentage 6sem. P-values refer to the comparisons between CAD and AS patients. doi:10.1371/journal.pone.0004871.t001 NYHA class II to IV displayed larger depletion in blood glutathione (P= 0.005) with a mean 40% decrease below the control value (P,0.0001). When considering separately the two CAD and AS groups of patients, blood glutathione level was found significantly lower than that of healthy controls, independently of the LVEF value (Fig. 4B).The decrease in blood glutathione was exponentially correlated with the increase in blood sTNFR1 level in the whole cohort of patients (r= 0.88; Fig. 5). Interestingly, significant depletion in blood glutathione occurred before detectable elevation in blood sTNFR1. To examine, whether or not ageing might influence our findings, patients were divided into two groups, younger patients (#65 years) and older patients (.65 years). The compared to the subgroup having preserved LVEF (.45%) (1.7260.2 vs 2.960.4 nmol glutathione/mg tissue, respectively; Fig. 3A). Indeed, atrial glutathione in CAD patients was positively correlated with LVEF (r= 0.48, P = 0.007) (Fig. 3B). In contrast, atrial glutathione content in AS patients was rather low, indepen- dently of the LV function (2.260.5 and 2.460.3 nmol glutathione/ mg tissue for .45% and #45% LVEF, respectively; Fig. 3A). Blood glutathione deficiency in patients with cardiac disease Compared to healthy controls, patients of NYHA class I displayed a significant 21% decrease in blood glutathione (P,0.0001) (Fig. 4A). Compared to patients of NYHA class I, patients of PLoS ONE | www.plosone.org March 2009 | Volume 4 | Issue 3 | e4871 3 Glutathione in Heart Diseases level of blood glutathione was not related with the age of the patients Figure 1. Relations between LVEF or blood sTNFR1 level and NYHA class in cardiac patients and healthy controls. The cohort displayed standard relations between functional NYHA class and LVEF (A) or blood sTNFR1 level (B). LVEF: left ventricular ejection fraction. sTNFR1: cleaved extracellular domain of TNFR1. Linear trends P,0.0001. *P,0.05 vs healthy controls (C). Discussion Blood glutathione level in cardiac patients and in the subgroups of patients with coronary artery diseases (CAD) or aortic stenosis (AS). (A) In patients undergoing cardiac surgery, the decrease in blood glutathione level was related to NYHA class (linear trend P,0.0001). (B) Compared to healthy controls, blood glutathione level in the CAD and AS subgroups of patients was depleted, independently of the LVEF value. *P,0.05 vs healthy controls (C); {P,0.05 vs NYHA class I. doi:10.1371/journal.pone.0004871.g004 Figure 4. Blood glutathione level in cardiac patients and in the subgroups of patients with coronary artery diseases (CAD) or aortic stenosis (AS). (A) In patients undergoing cardiac surgery, the decrease in blood glutathione level was related to NYHA class (linear trend P,0.0001). (B) Compared to healthy controls, blood glutathione level in the CAD and AS subgroups of patients was depleted, independently of the LVEF value. *P,0.05 vs healthy controls (C); {P,0.05 vs NYHA class I. doi:10.1371/journal.pone.0004871.g004 Figure 3. Atrial tissue glutathione content in patients with coronary artery diseases (CAD) or aortic stenosis (AS), according to preserved LVEF (.45%) or depressed LVEF (#45%). Patients with permanent atrial fibrillation were excluded. (A) Deficiency in atrial tissue glutathione was related to LV dysfunction in CAD patients. In contrast, atrial glutathione was rather low in AS patients, independently of the LVEF value. *P,0.05 vs LVEF.45%. (B) In CAD patients, atrial tissue glutathione content correlated significantly with the LVEF value (r = 0.45, P = 0.006). doi:10.1371/journal.pone.0004871.g003 We have previously reported that LV of patients with end-stage cardiomyopathies, undergoing orthotopic heart transplant or ventricular assist device, is depleted by 54% in gluthatione compared to control LV [19]. In the present study, we have used atrial appendage as a surrogate for LV tissue. We recognize that atrium does not undergo same physiological requests and stresses as the LV. However, atrial appendage is accessible in patients with a large range of symptoms and with different cardiac diseases. Interestingly atrial glutathione content is depleted by 58% in NYHA class IV patients compared with NYHA class I patients, which is a drop similar to that found in end-stage failing LV. A previous study has also reported that hemodynamic impairment in both right and left ventricles of patients with heart failure subsequent to myocardial infarction correlates with a decrease in glutathione antioxidant efficiency [31]. Discussion The optimal cut-off value of sTNFR1 level was 0.33 ng/ml with an AUROC-95%CI: 0.95 [0.9–1], a sensitivity of 85.5% and a specificity of 100%. The optimal cut-off value of blood glutathione level was 1.835 mM with an AUROC- 95%CI: 0.94 [0.89–0.99], a sensitivity of 81.5% and a specificity of PLoS ONE | www.plosone.org March 2009 | Volume 4 | Issue 3 | e4871 March 2009 | Volume 4 | Issue 3 | e4871 PLoS ONE | www.plosone.org 4 early decrease in systemic glutathione in the course of the cardiac disease. One may also note that deficiency in systemic glutathione in cardiac patients occurs well before the drop in cardiac tissue glutathione. Several studies provide evidence for a close relationship between blood glutathione decrease and the patho- genesis of different inflammatory chronic diseases [22,32]. In fact, We have previously reported that LV of patients with end-stage cardiomyopathies, undergoing orthotopic heart transplant or ventricular assist device, is depleted by 54% in gluthatione compared to control LV [19]. In the present study, we have used atrial appendage as a surrogate for LV tissue. We recognize that atrium does not undergo same physiological requests and stresses as the LV. However, atrial appendage is accessible in patients with a large range of symptoms and with different cardiac diseases. Figure 3. Atrial tissue glutathione content in patients with coronary artery diseases (CAD) or aortic stenosis (AS), according to preserved LVEF (.45%) or depressed LVEF (#45%). Patients with permanent atrial fibrillation were excluded. (A) Deficiency in atrial tissue glutathione was related to LV dysfunction in CAD patients. In contrast, atrial glutathione was rather low in AS patients, independently of the LVEF value. *P,0.05 vs LVEF.45%. (B) In CAD patients, atrial tissue glutathione content correlated significantly with the LVEF value (r = 0.45, P = 0.006). doi:10.1371/journal.pone.0004871.g003 Figure 4. Blood glutathione level in cardiac patients and in the subgroups of patients with coronary artery diseases (CAD) or aortic stenosis (AS). (A) In patients undergoing cardiac surgery, the decrease in blood glutathione level was related to NYHA class (linear trend P,0.0001). (B) Compared to healthy controls, blood glutathione level in the CAD and AS subgroups of patients was depleted, independently of the LVEF value. *P,0.05 vs healthy controls (C); {P,0.05 vs NYHA class I. doi:10.1371/journal.pone.0004871.g004 Glutathione in Heart Diseases Glutathione in Heart Diseases Figure 4. Discussion Accordingly in the present study, LVEF decline is associated with atrial glutathione deficiency early decrease in systemic glutathione in the course of the cardiac disease. One may also note that deficiency in systemic glutathione in cardiac patients occurs well before the drop in cardiac tissue glutathione. Several studies provide evidence for a close relationship between blood glutathione decrease and the patho- genesis of different inflammatory chronic diseases [22,32]. In fact, systemic glutathione provides many tissues in the body, and its deficiency is likely to affect vital functions including resistance to oxidative stress, mitochondrial function and integrity, immune response and cell survival [33]. In the cardiac myocyte, glutathione deficiency fuels a vicious TNF/ sTNFR1/ oxidative stress/ neutral sphingomyelinase/ apoptosis cycle [18,19,34–36]. Accordingly, early deficiency in systemic glutathione is likely to contribute to the progression of the cardiac disease. early decrease in systemic glutathione in the course of the cardiac disease. One may also note that deficiency in systemic glutathione in cardiac patients occurs well before the drop in cardiac tissue glutathione. Several studies provide evidence for a close relationship between blood glutathione decrease and the patho- genesis of different inflammatory chronic diseases [22,32]. In fact, systemic glutathione provides many tissues in the body, and its deficiency is likely to affect vital functions including resistance to oxidative stress, mitochondrial function and integrity, immune response and cell survival [33]. In the cardiac myocyte, glutathione deficiency fuels a vicious TNF/ sTNFR1/ oxidative stress/ neutral sphingomyelinase/ apoptosis cycle [18,19,34–36]. Accordingly, early deficiency in systemic glutathione is likely to contribute to the progression of the cardiac disease. PLoS ONE | www.plosone.org PLoS ONE | www.plosone. March 2009 | Volume 4 | Issue 3 | e4871 March 2009 | Volume 4 | Issue 3 | e4871 PLoS ONE | www.plosone.org 5 Glutathione in Heart Diseases Figure 5. Correlation between blood glutathione and blood sTNFR1 levels in cardiac patients and healthy controls. Blood glutathione level decrease was exponentially correlated with elevation in blood sTNFR1 level in the whole cohort of patients (r = 0.88). Open squares: controls; open circles: younger patients #65 years (mean age = 5561 years; r = 0.88); closed circles: older patients .65 years (mean age = 7461 years; r = 0.87). doi:10.1371/journal.pone.0004871.g005 glutathione precursor, N-acetylcysteine (NAC) [37]. Taken together, these results suggest that glutathione deficiency impinges on the whole damaged heart. References 11. Isaac DL (2008) Biomarkers in heart failure management. Curr Opin Cardiol 23: 127–133. 1. McCullough PA, Philbin EF, Spertus JA, Kaatz S, Sandberg KR, et al. (2002) Confirmation of a heart failure epidemic: findings from the Resource Utilization Among Congestive Heart Failure (REACH) study. J Am Coll Cardiol 39: 60–69. 12. Maisel A, Hollander JE, Guss D, McCullough P, Nowak R, et al. (2004) Primary results of the Rapid Emergency Department Heart Failure Outpatient Trial (REDHOT). A multicenter study of B-type natriuretic peptide levels, emergency department decision making, and outcomes in patients presenting with shortness of breath. J Am Coll Cardiol 44: 1328–1333. 2. Cleland JG, Swedberg K, Follath F, Komajda M, Cohen-Solal A, et al. (2003) The EuroHeart Failure survey programme– a survey on the quality of care among patients with heart failure in Europe. Part 1: patient characteristics and diagnosis. Eur Heart J 24: 442–463. 13. Morrow DA, de Lemos JA, Blazing MA, Sabatine MS, Murphy SA, et al. (2005) Prognostic value of serial B-type natriuretic peptide testing during follow-up of patients with unstable coronary artery disease. Jama 294: 2866–2871. 3. Jourdain P, Jondeau G, Funck F, Gueffet P, Le Helloco A, et al. (2007) Plasma brain natriuretic peptide-guided therapy to improve outcome in heart failure: the STARS-BNP Multicenter Study. J Am Coll Cardiol 49: 1733–1739. 4. Ferrari R, Bachetti T, Confortini R, Opasich C, Febo O, et al. (1995) Tumor necrosis factor soluble receptors in patients with various degrees of congestive heart failure. Circulation 92: 1479–1486. 14. Nishii M, Inomata T, Takehana H, Naruke T, Yanagisawa T, et al. (2008) Prognostic utility of B-type natriuretic peptide assessment in stable low-risk outpatients with nonischemic cardiomyopathy after decompensated heart failure. J Am Coll Cardiol 51: 2329–2335. 5. Nozaki N, Yamaguchi S, Shirakabe M, Nakamura H, Tomoike H (1997) Soluble tumor necrosis factor receptors are elevated in relation to severity of congestive heart failure. Jpn Circ J 61: 657–664. 15. Pfister R, Scholz M, Wielckens K, Erdmann E, Schneider CA (2004) Use of NT-proBNP in routine testing and comparison to BNP. Eur J Heart Fail 6: 289–293. 6. Keith M, Geranmayegan A, Sole MJ, Kurian R, Robinson A, et al. (1998) Increased oxidative stress in patients with congestive heart failure. J Am Coll Cardiol 31: 1352–1356. 16. Tang WH, Francis GS, Morrow DA, Newby LK, Cannon CP, et al. Acknowledgments in CAD patients (r = 0.45; P = 0.006). In contrast, AS patients display equal atrial glutathione content, independently of the LVEF value. Indeed, the latter experience early LV remodelling related to increased intra-cardiac pressures and without marked LVEF decline. Finally, in agreement with the previous observation made by Carnes et al. [28], we found a 50% decrease in atrial glutathione content in patients with permanent atrial fibrillation compared to other patients with sinus rhythm, which is also consistent with the decreased incidence of postoperative atrial fibrillation observed after intravenous supplementation with the in CAD patients (r = 0.45; P = 0.006). In contrast, AS patients display equal atrial glutathione content, independently of the LVEF value. Indeed, the latter experience early LV remodelling related to increased intra-cardiac pressures and without marked LVEF decline. Finally, in agreement with the previous observation made by Carnes et al. [28], we found a 50% decrease in atrial glutathione content in patients with permanent atrial fibrillation compared to other patients with sinus rhythm, which is also consistent with the decreased incidence of postoperative atrial fibrillation observed after intravenous supplementation with the The authors would like to thank S. Lotersztajn for helpful discussions and G. Guellae¨n for his permanent support. Author Contributions Conceived and designed the experiments: TD MK LK PLC JLDR LH CP FP. Performed the experiments: TD MK LK PC PLC FP. Analyzed the data: TD MK LK PC PLC FRT LH CP FP. Contributed reagents/ materials/analysis tools: TD MK PLC JLDR FP. Wrote the paper: TD MK LK FRT LH CP FP. Discussion They also suggest that glutathione supplementation may improve cardiac cell preservation in cardiac diseases, and be a complement to contemporary treatments. It should be mentioned that the present study is limited by the relatively small cohort size. In addition, although our results provide some evidence that the concomitant use of blood glutathione and blood sTNFR1 tests may improve the sensitivity of cardiac patient diagnostic, further comparison between blood glutathione and biomarkers of heart failure other than sTNFR1, in particular serum BNP peptides, is needed. Indeed, this was not possible in the present study in which only frozen blood samples were available. The present study did not either examine the prognostic impact of blood glutathione. In conclusion, although further studies are needed to verify the diagnostic and/ or predictive value of blood glutathione in heart failure as a part of a multi marker panel test, this study provides evidence that cardiac and systemic glutathione deficiency is related with the functional status and the structural cardiac abnormalities of patients with cardiac diseases. These data also encourage the development of blood glutathione test as a possible new diagnostic tool for detecting asymptomatic patients with structural cardiac abnormalities. Figure 5. Correlation between blood glutathione and blood sTNFR1 levels in cardiac patients and healthy controls. Blood glutathione level decrease was exponentially correlated with elevation in blood sTNFR1 level in the whole cohort of patients (r = 0.88). Open squares: controls; open circles: younger patients #65 years (mean age = 5561 years; r = 0.88); closed circles: older patients .65 years (mean age = 7461 years; r = 0.87). doi:10.1371/journal.pone.0004871.g005 ( g y ; ) doi:10.1371/journal.pone.0004871.g005 Glutathione in Heart Diseases 20. Yucel D, Aydogdu S, Cehreli S, Saydam G, Canatan H, et al. (1998) Increased oxidative stress in dilated cardiomyopathic heart failure. Clin Chem 44: 148–154. 27. Rahman I, Kode A, Biswas SK (2006) Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc 1: 3159–3165. 28. Carnes CA, Janssen PM, Ruehr ML, Nakayama H, Nakayama T, et al. (2007) Atrial glutathione content, calcium current, and contractility. J Biol Chem 282: 28063–28073. 21. Haddad JJ, Harb HL (2005) L-gamma-Glutamyl-L-cysteinyl-glycine (glutathi- one; GSH) and GSH-related enzymes in the regulation of pro- and anti- inflammatory cytokines: a signaling transcriptional scenario for redox(y) immunologic sensor(s)? Mol Immunol 42: 987–1014. 29. Mallat Z, Philip I, Lebret M, Chatel D, Maclouf J, et al. (1998) Elevated levels of 8-iso-prostaglandin F2alpha in pericardial fluid of patients with heart failure: a potential role for in vivo oxidant stress in ventricular dilatation and progression to heart failure. Circulation 97: 1536–1539. g ( ) 22. Franco R, Schoneveld OJ, Pappa A, Panayiotidis MI (2007) The central role of glutathione in the pathophysiology of human diseases. Arch Physiol Biochem 113: 234–258. 30. Giordano FJ (2005) Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Invest 115: 500–508. 23. Schiller NB, Shah PM, Crawford M, DeMaria A, Devereux R, et al. (1989) Recommendations for quantitation of the left ventricle by two-dimensional echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of Two-Dimensional Echocardio- grams. J Am Soc Echocardiogr 2: 358–367. 31. Hill MF, Singal PK (1997) Right and left myocardial antioxidant responses during heart failure subsequent to myocardial infarction. Circulation 96: 2414–2420. 32. Lang CA, Mills BJ, Mastropaolo W, Liu MC (2000) Blood glutathione decreases in chronic diseases. J Lab Clin Med 135: 402–405. 24. Dickstein K, Cohen-Solal A, Filippatos G, McMurray JJ, Ponikowski P, et al. (2008) ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: the Task Force for the diagnosis and treatment of acute and chronic heart failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM). Eur J Heart Fail 10: 933–989. 33. Wu G, Fang YZ, Yang S, Lupton JR, Turner ND (2004) Glutathione metabolism and its implications for health. J Nutr 134: 489–492. 34. References (2007) National Academy of Clinical Biochemistry Laboratory Medicine practice guidelines: Clinical utilization of cardiac biomarker testing in heart failure. Circulation 116: e99–109. 7. Rauchhaus M, Doehner W, Francis DP, Davos C, Kemp M, et al. (2000) Plasma cytokine parameters and mortality in patients with chronic heart failure. Circulation 102: 3060–3067. 17. Garvin P, Nilsson L, Carstensen J, Jonasson L, Kristenson M (2008) Circulating matrix metalloproteinase-9 is associated with cardiovascular risk factors in a middle-aged normal population. PLoS ONE 3: e1774. 8. Deswal A, Petersen NJ, Feldman AM, Young JB, White BG, et al. (2001) Cytokines and cytokine receptors in advanced heart failure: an analysis of the cytokine database from the Vesnarinone trial (VEST). Circulation 103: 2055–2059. 18. Bourraindeloup M, Adamy C, Candiani G, Cailleret M, Bourin MC, et al. (2004) N-acetylcysteine treatment normalizes serum tumor necrosis factor-alpha level and hinders the progression of cardiac injury in hypertensive rats. Circulation 110: 2003–2009. 9. Mann DL (2002) Inflammatory mediators and the failing heart. Past, present, and the foreseeable future. Circ Res 91: 988–998. 10. Valgimigli M, Ceconi C, Malagutti P, Merli E, Soukhomovskaia O, et al. (2005) Tumor necrosis factor-alpha receptor 1 is a major predictor of mortality and new-onset heart failure in patients with acute myocardial infarction: the Cytokine-Activation and Long-Term Prognosis in Myocardial Infarction (C- ALPHA) study. Circulation 111: 863–870. 19. Adamy C, Mulder P, Khouzami L, Andrieu-Abadie N, Defer N, et al. (2007) Neutral sphingomyelinase inhibition participates to the benefits of N- acetylcysteine treatment in post-myocardial infarction failing heart rats. J Mol Cell Cardiol 43: 344–353. PLoS ONE | www.plosone.org March 2009 | Volume 4 | Issue 3 | e4871 March 2009 | Volume 4 | Issue 3 | e4871 6 Glutathione in Heart Diseases PLoS ONE | www.plosone.org Glutathione in Heart Diseases Amadou A, Nawrocki A, Best-Belpomme M, Pavoine C, Pecker F (2002) Arachidonic acid mediates dual effect of TNF-alpha on Ca(2+) transients and contraction of adult rat cardiomyocytes. Am J Physiol Cell Physiol 282: C1339–1347. 25. Fuster V, Ryden LE, Cannom DS, Crijns HJ, Curtis AB, et al. (2006) ACC/ AHA/ESC 2006 guidelines for the management of patients with atrial fibrillation-executive summary: a report of the American College of Cardiolo- gy/American Heart Association Task Force on Practice Guidelines and the European Society of Cardiology Committee for Practice Guidelines (Writing Committee to Revise the 2001 Guidelines for the Management of Patients with Atrial Fibrillation). Eur Heart J 27: 1979–2030. 35. Cailleret M, Amadou A, Andrieu-Abadie N, Nawrocki A, Adamy C, et al. (2004) N-acetylcysteine prevents the deleterious effect of tumor necrosis factor-(alpha) on calcium transients and contraction in adult rat cardiomyocytes. Circulation 109: 406–411. 36. Defer N, Azroyan A, Pecker F, Pavoine C (2007) TNFR1 and TNFR2 signaling interplay in cardiac myocytes. J Biol Chem 282: 35564–35573. ) J 26. Tietze F (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 27: 502–522. 37. Ozaydin M, Peker O, Erdogan D, Kapan S, Turker Y, et al. (2008) N- acetylcysteine for the prevention of postoperative atrial fibrillation: a prospective, randomized, placebo-controlled pilot study. Eur Heart J 29: 625–631. PLoS ONE | www.plosone.org March 2009 | Volume 4 | Issue 3 | e4871 7 7
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WITHDRAWN: Does Green Finance Boost Carbon-Neutral Performance? -- Evidence from China
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Keywords: Posted Date: January 5th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-3132712/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Environmental Science and Pollution Research on September 25th, 2023. See the published version at https://doi.org/10.1007/s11356-023- 29921-8. EDITORIAL NOTE: The full text of this preprint has been withdrawn by the authors while they make corrections to the work. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author. Page 1/2 Page 1/2 Abstract The full text of this preprint has been withdrawn by the authors due to author disagreement with the posting of the preprint. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author. Full Text The authors have withdrawn this preprint from Research Square. Page 2/2
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Impact of wheat fractions differing by their dietary fibre content and incorporated in several cereal foods on their glycaemic and insulinaemic responses
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Impact of wheat fractions differing by their dietary fibre content and incorporated in several cereal foods on their glycaemic and insulinaemic responses glycaemic and insulinaemic responses Alexandra Meynier, Martine Champ, P Chauveau, Cecile Barron, Luc Saulnier, Sophie Vinoy lexandra Meynier, Martine Champ, P Chauveau, Cecile Barron, Luc Saulnier, Sophie Vinoy To cite this version: Alexandra Meynier, Martine Champ, P Chauveau, Cecile Barron, Luc Saulnier, et al.. Impact of wheat fractions differing by their dietary fibre content and incorporated in several cereal foods on their glycaemic and insulinaemic responses. 6. International Dietary Fibre Conference (DF15), Jun 2015, Paris, France. 2015. ￿hal-01602056￿ Methods: Figure 1 : Kinetics of glycaemia following the ingestion of the 9 tested cereal products Glycaemic responses following the 9 cereal products are all lower than the one obtained after the intake of the glucose solution. Kinetics of glycaemia confirm the ranking observed with GI values. However, peak values obtained with breads were lower than those following the ingestion of pastas and were delayed by about 15 minutes. Globally, kinetics of glycaemia were more spread over time with breads compared to the other cereal products. This lead to incremental Area Under the Curve values similar with breads and pastas and thus to similar values of GI. Conclusions : Significant differences were observed on GI, II and glycaemic and insulinaemic responses between the different cereal foods matrixes studied. However, high-fiber wheat fractions did not induced significant effects within a same food matrix. The level of enrichment in fibers (from 3 to 5g/portion) above the control products was potentially too ow to obtain a significant difference in GI. Further investigations are necessary to understand the reasons for delayed and spread kinetics observed following bread ngestion. Moreover, it appears interesting to investigate the impact of such glycaemic response profile on health. Figure 1 : Glycemic Index obtained for the 9 tested cereal products. Figure 1 : Glycemic Index obtained for the 9 tested cereal products. The 18 subjects recruited in the present study were aged between 20 – 34 years with BMI ranging from 19.1 to 25.0 kg/m². This population was recruited with a low HOMA-IR value ranging from 0.6 to 1.6. p The glycemic Index (Figure 1) of the products ranged between 44 and 74 with the biscuits having the lowest indexes (around 45). Unfortunately, no significant difference was observed between the fractions for a same type of cereal food product. On the other hand, significant differences were obtained between bread and biscuits for fractions R and F+ and between biscuits and pasta for fractions R and F+. Interestingly, pastas display quite high GI values despite their high content of Slowly Digestible Starch. The Insulin Index ranged between 57 and 91. No difference was observed between the fractions for a same type of food product. Significant differences were observed between bread and biscuits and between bread and pasta incorporating fraction F-. Methods: Insulin has been quantified using a commercial insulin EIA kit (Insulin EIA kit, Alpco). GI was measured according to the standard methodology (Brouns et al, 2005, ISO 26642-2010) using glucose solution as reference food tested on 3 separate occasions. Eighteen healthy normal-weight volunteers have been recruited for the study and 17 completed the 12 sessions. Glucose and Insulin concentrations have been measured on capillary blood samples at -5, 0, 15, 30, 45, 60, 90 and 120 minutes after eating commenced. Blood glucose concentrations have been measured using a Roche/Hitachi 912® automatic spectrophotometer employing the GOD-PAP method. Insulin has been quantified using a commercial insulin EIA kit (Insulin EIA kit, Alpco). GI was measured according to the standard methodology (Brouns et al, 2005, ISO 26642-2010) using glucose solution as reference food tested on 3 separate occasions. Eighteen healthy normal-weight volunteers have been recruited for the study and 17 completed the 12 sessions. Glucose and Insulin concentrations have been measured on capillary blood samples at -5, 0, 15, 30, 45, 60, 90 and 120 minutes after eating commenced. Blood glucose concentrations have been measured using a Roche/Hitachi 912® automatic spectrophotometer employing the GOD-PAP method. Insulin has been quantified using a commercial insulin EIA kit (Insulin EIA kit, Alpco). Results: The 18 subjects recruited in the present study were aged between 20 – 34 years with BMI ranging from 19.1 to 25.0 kg/m². This population was recruited with a low HOMA-IR value ranging from 0.6 to 1.6. The glycemic Index (Figure 1) of the products ranged between 44 and 74 with the biscuits having the lowest indexes (around 45). Unfortunately, no significant difference was observed between the fractions for a same type of cereal food product. On the other hand, significant differences were obtained between bread and biscuits for fractions R and F+ and between biscuits and pasta for fractions R and F+. Interestingly, pastas display quite high GI values despite their high content of Slowly Digestible Starch. The Insulin Index ranged between 57 and 91. No difference was observed between the fractions for a same type of food product. Significant differences were observed between bread and biscuits and between bread and pasta incorporating fraction F-. Figure 1 : Glycemic Index obtained for the 9 tested cereal products. Background and Objectives: Background and Objectives: The concept of glycemic Index developed in 1981 by Jenkins et al. allows to describe the variations in physiological responses produced by different carbohydrate- containing foods. Low Glycemic Index (GI) diets have been shown beneficial effects on the prevention of metabolic diseases such as cardio-vascular disease, type 2 diabetes and some types of cancers (Blaak et al, 2012). For cereal products, several studies have linked glycaemic response to the nutritional composition (lipid, protein and dietary fiber contents) and the digestibility rate of starch fraction (Meynier et al, 2015). The objective of the present study was to measure GI and Insulin Index (II) of different cereal food products enriched or not with two different wheat fractions differing by the Methods: Figure 1 : Kinetics of glycaemia following the ingestion of the 9 tested cereal products Glycaemic responses following the 9 cereal products are all lower than the one obtained after the intake of the glucose solution. Kinetics of glycaemia confirm the ranking observed with GI values. However, peak values obtained with breads were lower than those following the ingestion of pastas and were delayed by about 15 minutes. Globally, kinetics of glycaemia were more spread over time with breads compared to the other cereal products. This lead to incremental Area Under the Curve values similar with breads and pastas and thus to similar values of GI. Conclusions : Significant differences were observed on GI, II and glycaemic and insulinaemic responses between the different cereal foods matrixes studied. However, high-fiber wheat fractions did not induced significant effects within a same food matrix. The level of enrichment in fibers (from 3 to 5g/portion) above the control products was potentially too low to obtain a significant difference in GI. Further investigations are necessary to understand the reasons for delayed and spread kinetics observed following bread ingestion. Moreover, it appears interesting to investigate the impact of such glycaemic response profile on health. Figure 1 : Kinetics of glycaemia following the ingestion of the 9 tested cereal products Figure 1 : Kinetics of glycaemia following the ingestion of the 9 tested cereal products Glycaemic responses following the 9 cereal products are all lower than the one obtained after the intake of the glucose solution. Kinetics of glycaemia confirm the ranking observed with GI values. However, peak values obtained with breads were lower than those following the ingestion of pastas and were delayed by about 15 minutes. Globally, kinetics of glycaemia were more spread over time with breads compared to the other cereal products. This lead to incremental Area Under the Curve values similar with breads and pastas and thus to similar values of GI. Conclusions : Significant differences were observed on GI, II and glycaemic and insulinaemic responses between the different cereal foods matrixes studied. However, high-fiber wheat fractions did not induced significant effects within a same food matrix. The level of enrichment in fibers (from 3 to 5g/portion) above the control products was potentially too low to obtain a significant difference in GI. Further investigations are necessary to understand the reasons for delayed and spread kinetics observed following bread ingestion. MPACT OF WHEAT FRACTIONS DIFFERING BY THEIR DIETARY FIBER CONTENT AND SEVERAL CEREAL FOODS ON THEIR GLYCAEMIC AND INSULINAEMIC RES A. Meynier1, M. Champ2,3, P. Chauveau4, C. Barron5, L. Saulnier6 and S. Vinoy1 Mondelez France R&D, Saclay, France; 2 CRNH Ouest, Nantes, France; 3 UMR 1280 PhAN INRA, Nantes University, Nante France; 5 UMR 1208 IATE INRA, UMII, Cirad, Montpellier Supagro 2, Montpellier, France; 6 UR 1268 BIA INRA GI was measured according to the standard methodology (Brouns et al, 2005, ISO 26642-2010) using glucose solution as reference food tested on 3 separate occasions. Eighteen healthy normal-weight volunteers have been recruited for the study and 17 completed the 12 sessions. Glucose and Insulin concentrations have been measured on capillary blood samples at -5, 0, 15, 30, 45, 60, 90 and 120 minutes after eating commenced. Blood glucose concentrations have been measured using a Roche/Hitachi 912® automatic spectrophotometer employing the GOD-PAP method. Insulin has been quantified using a commercial insulin EIA kit (Insulin EIA kit, Alpco). Methods: 3 types of cereal products (bread, biscuits and pasta) have been prepared incorporating only refined wheat (reference products R) or two wheat fractions differing by the fermentescibility of the fiber they contain: one fraction containing mainly pericarp (products F-) and one fraction rich in aleurone layer (products F+). The composition and portion tested for each product is presented in Table 1. 3 types of cereal products (bread, biscuits and pasta) have been prepared incorporating only refined wheat (reference products R) or two wheat fractions differing by the fermentescibility of the fiber they contain: one fraction containing mainly pericarp (products F-) and one fraction rich in aleurone layer (products F+). The composition and portion tested for each product is presented in Table 1. 3 types of cereal products (bread, biscuits and pasta) have been prepared incorporating only refined wheat (reference products R) or two wheat fractions differing by the fermentescibility of the fiber they contain: one fraction containing mainly pericarp (products F-) and one fraction rich in aleurone layer (products F+). The composition and portion tested for each product is presented in Table 1. p p Table 1 : Composition and portion size of the 9 tested cereal products to provide 50g of available CHO. Products Moisture (g/portion) Proteins (g/portion) Lipids (g/portion) Sugars (g/portion) Dietary fibers (g/portion) Available starch (g/portion) Slowly Digestible Starch (g/portion) Tested portion (g) Bread R 36 7 1 3 3 43 1 95 Bread F- 49 10 2 3 8 42 1 119 Bread F+ 53 11 2 4 6 42 1 123 Pasta R 126 11 2 1 3 44 20 195 Pasta F- 126 10 2 1 6 45 9 192 Pasta F+ 121 11 2 1 5 45 16 190 Biscuits R 1 4 12 15 2 31 13 69 Biscuits F- 1 5 12 18 5 28 13 73 Biscuits F+ 1 7 14 20 5 27 10 78 GI was measured according to the standard methodology (Brouns et al, 2005, ISO 26642-2010) using glucose solution as reference food tested on 3 separate occasions. Eighteen healthy normal-weight volunteers have been recruited for the study and 17 completed the 12 sessions. Glucose and Insulin concentrations have been measured on capillary blood samples at -5, 0, 15, 30, 45, 60, 90 and 120 minutes after eating commenced. Blood glucose concentrations have been measured using a Roche/Hitachi 912® automatic spectrophotometer employing the GOD-PAP method. HAL Id: hal-01602056 https://hal.science/hal-01602056v1 Submitted on 3 Jun 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution - ShareAlike 4.0 International License IMPACT OF WHEAT FRACTIONS DIFFERING BY THEIR DIETARY FIBER CONTENT AND INCORPORATED IN IMPACT OF WHEAT FRACTIONS DIFFERING BY THEIR DIETARY FIBER CONTENT AND INCORPORATED IN SEVERAL CEREAL FOODS ON THEIR GLYCAEMIC AND INSULINAEMIC RESPONSES A. Meynier1, M. Champ2,3, P. Chauveau4, C. Barron5, L. Saulnier6 and S. Vinoy1 1 Mondelez France R&D, Saclay, France; 2 CRNH Ouest, Nantes, France; 3 UMR 1280 PhAN INRA, Nantes University, Nantes, France; 4 Biofortis, Nantes, France; 5 UMR 1208 IATE INRA, UMII, Cirad, Montpellier Supagro 2, Montpellier, France; 6 UR 1268 BIA INRA, Nantes, France Methods: Moreover, it appears interesting to investigate the impact of such glycaemic response profile on health. Significant differences were observed on GI, II and glycaemic and insulinaemic responses between the different cereal foods matrixes studied. However, high-fiber wheat fractions did not induced significant effects within a same food matrix. The level of enrichment in fibers (from 3 to 5g/portion) above the control products was potentially too low to obtain a significant difference in GI. Further investigations are necessary to understand the reasons for delayed and spread kinetics observed following bread ingestion. Moreover, it appears interesting to investigate the impact of such glycaemic response profile on health. Poster Reference Number : 403
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https://europepmc.org/articles/pmc3543457?pdf=render
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Immediate and Heterogeneous Response of the LiaFSR Two-Component System of Bacillus subtilis to the Peptide Antibiotic Bacitracin
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Abstract Background: Two-component signal transduction systems are one means of bacteria to respond to external stimuli. The LiaFSR two-component system of Bacillus subtilis consists of a regular two-component system LiaRS comprising the core Histidine Kinase (HK) LiaS and the Response Regulator (RR) LiaR and additionally the accessory protein LiaF, which acts as a negative regulator of LiaRS-dependent signal transduction. The complete LiaFSR system was shown to respond to various peptide antibiotics interfering with cell wall biosynthesis, including bacitracin. Methodology and Principal Findings: Here we study the response of the LiaFSR system to various concentrations of the peptide antibiotic bacitracin. Using quantitative fluorescence microscopy, we performed a whole population study analyzed on the single cell level. We investigated switching from the non-induced ‘OFF’ state into the bacitracin-induced ‘ON’ state by monitoring gene expression of a fluorescent reporter from the RR-regulated liaI promoter. We found that switching into the ‘ON’ state occurred within less than 20 min in a well-defined switching window, independent of the bacitracin concentration. The switching rate and the basal expression rate decreased at low bacitracin concentrations, establishing clear heterogeneity 60 min after bacitracin induction. Finally, we performed time-lapse microscopy of single cells confirming the quantitative response as obtained in the whole population analysis for high bacitracin concentrations. Conclusion: The LiaFSR system exhibits an immediate, heterogeneous and graded response to the inducer bacitracin in the exponential growth phase. ation: Kesel S, Mader A, Ho¨fler C, Mascher T, Leisner M (2013) Immediate and Heterogeneous Response of the LiaFSR Two-Compon btilis to the Peptide Antibiotic Bacitracin. PLoS ONE 8(1): e53457. doi:10.1371/journal.pone.0053457 Editor: Tarek Msadek, Institut Pasteur, France Received August 31, 2012; Accepted November 30, 2012; Published January 11, 2013 Copyright:  2013 Kesel 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. Funding: Funding to ML, the authors thank excellence cluster Nano Initiative Munich and the Center for Nanoscience for funding. Funding to TM, funding is gratefully acknowledged by the Deutsche Forschungsgemeinschaft (MA2873/3-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: madeleine.leisner@physik.uni-muenchen.de Immediate and Heterogeneous Response of the LiaFSR Two-Component System of Bacillus subtilis to the Peptide Antibiotic Bacitracin Sara Kesel1, Andreas Mader1, Carolin Ho¨ fler2, Thorsten Mascher2, Madeleine Leisner1* 1 Center for NanoScience, Ludwig-Maximilians-University, Fakulta¨t fu¨r Physik, Munich, Germany, 2 Department Biology I, Microbiology, Ludwig-Maximilians-University Munich, Planegg-Martinsried, Germany reas Mader1, Carolin Ho¨ fler2, Thorsten Mascher2, Madeleine Leisner1* Gene expression increases at high bacitracin concentrations In this study, we aimed at a deeper understanding of the response of the LiaFSR system to various concentrations of the peptide antibiotic bacitracin. We used the B. subtilis strain TMB 1172 [35], which carries a translational fusion of PliaI with the green fluorescent protein GFPmut1. This GFP reporter has been integrated chromosomally in addition to the naturally occurring genes under the control of PliaI and regulated by the RR LiaR (Figure 1). Therefore, we were able to study the response of the LiaFSR system by analyzing the expression of the GFP reporter, as it represents the expression of the LiaR regulated target genes. In particular, we studied the fluorescence development of the GFP reporter in dependence of bacitracin, a model component used to study cell envelope stress response modules of Bacillus subtilis [19,36]. We chose the stable GFP variant, GFPmut1, shown to have a half-life of more than 24 h [37,38], as we were only interested in the onset of gene expression. Thereby, we excluded possible variations in gene expression due to GFP decay. Figure 1. Core of the LiaFSR system. Arrows denote upregulation and T-shaped lines indicate inhibition. The LiaFSR system of Bacillus subtilis consists of the two-component signal transducing system LiaRS and the accessory membrane protein LiaF, a LiaRS-specific inhibitor. Stress represented e.g. by cell wall antibiotics such as bacitracin is sensed by LiaS/F and leads to expression of the liaIH - liaGFSR (‘‘lia locus’’ in the Figure) locus mediated by LiaR. To study the response of the Lia system to external stressors, we report activity of PliaI using the fluorescent marker GFP expressed under the control of the liaI promoter, chromosomally inserted ectopically in addition to the native Lia system. CM indicates the cytoplasmic membrane. doi:10.1371/journal.pone.0053457.g001 p g p y Our cells were grown until mid-exponential phase before being induced with bacitracin to ensure that the recorded PliaI response was only due to external induction via bacitracin rather than intrinsic induction via the transition state regulator AbrB or the master regulator of sporulation Spo0A as present in the stationary phase [39]. Prior to bacitracin induction, we quantified the fluorescence intensity (FI) of non-induced cells representing the autofluorescence level (FIauto) and found it to be narrowly distributed with FIauto 861 FU (Figure 2A). After bacitracin induction, we monitored the fluorescence development for two hours with five to seven minute intervals. Response of B. subtilis LiaFSR System to Bacitracin Independent of the source of the noise, the arising heterogeneity can be manifested in broad gene expression distributions or by bifurcation into distinct subpopula- tions [32], as has been observed in B. subtilis in case of the transition state and stationary phase differentiation [32,33]. Response of B. subtilis LiaFSR System to Bacitracin Activation of LiaR results in induction of the liaI promoter (PliaI) resulting in a strong upregulation of the liaIH operon, but also the complete lia locus (Figure 1) [21,22]. The exact physiological role of LiaI and LiaH is not well understood, but the proteins seem to be involved in sensing and counteracting membrane damage [22]. In contrast to other cell wall antibiotic sensors of B. subtilis, such as the BceRS and PsdRS systems that directly sense peptide antibiotics and specifically mediate resistance against them [26], the Lia system seems to respond only indirectly to some quality of the damage caused by the diverse set of inducing conditions [27]. For the LiaFSR system, averaged data obtained by whole population studies revealed that the response of the PliaI is dependent on the external antibiotic concentration [23]. However, a quantitative single cell analysis of the Lia response addressing heterogeneity in gene expression has not yet been performed. Using quantitative fluorescence microscopy [33,34], we focused on a whole population study analyzed at the single cell level. We monitored gene expression from PliaI over time and found heterogeneity at low bacitracin concentrations. While expression levels from PliaI increased with the externally provided bacitracin amount, we found the immediate response of the LiaFSR system independent of the antibiotic concentration. We defined a switching threshold from the non-induced ‘OFF’ state to the bacitracin-induced ‘ON’ state. The number of cells in the ‘ON’ state, as well as the basal expression rate of the PliaI increased with bacitracin concentration. In addition, a well defined time window for switching into the ‘ON’ state was observed at all bacitracin concentrations. Here we focus on the activation of the PliaI by LiaR in response to the external stimulus bacitracin, which is the strongest and most robust inducer of LiaRS activity [23,26]. As seen recently in other studies [28,29], signal transduction of TCS can result in heterogeneous expression of genes regulated by these TCS. Heterogeneous gene expression in genetically identical cells can result in phenotypic different outcomes, a phenomenon also known as phenotypic heterogeneity [30]. Gene expression in itself is a stochastic or ‘noisy’ process [31]. Two different kinds of noise can be distinguished: intrinsic noise, due to noise in transcription or translation of the particular gene studied; or extrinsic noise as caused by fluctuations in the amount of other cellular components affecting gene expression [31]. Response of B. subtilis LiaFSR System to Bacitracin Response of B. subtilis LiaFSR System to Bacitracin LiaF interferes with LiaRS-dependent signal transduction is not yet understood. The genes of the LiaFSR system, together with a forth protein of unknown function, LiaG, are encoded in the liaGFSR operon, which is expressed from the constitutive liaG promoter (PliaG) in the absence of inducing conditions [21]. Activation of LiaR results in induction of the liaI promoter (PliaI) resulting in a strong upregulation of the liaIH operon, but also the complete lia locus (Figure 1) [21,22]. The exact physiological role of LiaI and LiaH is not well understood, but the proteins seem to be involved in sensing and counteracting membrane damage [22]. In contrast to other cell wall antibiotic sensors of B. subtilis, such as the BceRS and PsdRS systems that directly sense peptide antibiotics and specifically mediate resistance against them [26], the Lia system seems to respond only indirectly to some quality of the damage caused by the diverse set of inducing conditions [27]. Here we focus on the activation of the PliaI by LiaR in response to the external stimulus bacitracin, which is the strongest and most robust inducer of LiaRS activity [23,26]. As seen recently in other studies [28,29], signal transduction of TCS can result in heterogeneous expression of genes regulated by these TCS. Heterogeneous gene expression in genetically identical cells can result in phenotypic different outcomes, a phenomenon also known as phenotypic heterogeneity [30]. Gene expression in itself is a stochastic or ‘noisy’ process [31]. Two different kinds of noise can be distinguished: intrinsic noise, due to noise in transcription or translation of the particular gene studied; or extrinsic noise as caused by fluctuations in the amount of other cellular components affecting gene expression [31]. Independent of the source of the noise, the arising heterogeneity can be manifested in broad gene expression distributions or by bifurcation into distinct subpopula- tions [32], as has been observed in B. subtilis in case of the transition state and stationary phase differentiation [32,33]. LiaF interferes with LiaRS-dependent signal transduction is not yet understood. The genes of the LiaFSR system, together with a forth protein of unknown function, LiaG, are encoded in the liaGFSR operon, which is expressed from the constitutive liaG promoter (PliaG) in the absence of inducing conditions [21]. Introduction response [7], others result in an all-or-nothing response [14]. The latter is only triggered after a particular stimulus concentration has been overcome. The response itself can thereby be homogeneous (the whole population behaves in the same way) or heterogeneous with parts of the population behaving differently than the others. Regardless of the observed output, regulation of both types of systems can involve a number of auxiliary protein components. Systems involving accessory proteins [15–17], often referred to as three-component systems, also include peptide antibiotic-sensing systems of Gram-positive bacteria [18,19,20]. Two-component systems (TCS) are a fundamental principle of bacterial signal transduction that enables cells to respond to environmental stimuli [1–3]. These phosphotransfer systems involve two conserved components, a histidine protein kinase (HK) and a response regulator protein (RR). Extracellular stimuli are sensed by the HK, leading to its autophosphorylation [4]. The phosphoryl group is then transferred from the HK to the RR. The RR, now in its ‘active’ form, elicits the specific response. Bacteria such as Escherichia coli or Bacillus subtilis posses about 30 HKs and RRs [5,6], including well-known systems such as the EnvZ/ OmpR TCS of the osmosensing pathway [7] or the HK CheA of the chemotaxis system phosphorylating two RRs, CheB and CheY [8]. In addition to functional characterization of TCS focusing on phosphorylation rates [9] accompanied by theoretical studies [10,11], specificity and crosstalk of TCS is of great interest [12] and several methods for two-component research have been developed to accommodate such studies [13]. While some TCS mediate differential expression of the output genes by a graded One such system is the LiaFSR cell envelope stress response module of Bacillus subtilis [21,22], which strongly responds to various peptide antibiotics such as bacitracin, nisin, vancomycin or daptomycin [23], but also to other less specific envelope perturbating conditions, such detergents or alkaline shock (summarized in [24] and [25]). The Lia system, is comprised of the LiaRS TCS, with the HK LiaS and the RR LiaR, and additionally the accessory protein LiaF (Figure 1). The latter is associated with all LiaRS-like TCS and acts as a negative regulator of LiaR-mediated gene regulation [21]. The mechanism by which January 2013 | Volume 8 | Issue 1 | e53457 1 January 2013 | Volume 8 | Issue 1 | e53457 PLOS ONE | www.plosone.org Gene expression increases at high bacitracin concentrations At high bacitracin concentrations all cells shifted from the autofluorescence level to intermediate and finally high GFP expression levels. The maximal fluorescence intensities were reached at 60 min after bacitracin induction as shown in Figure 2B–F. While at 30 mg/ml bacitracin maximal fluorescence intensities of 272 FU on average were reached, FImax decreased with lower bacitracin concentrations (Table 1). FImax thereby represents the average FI of all cells at time point 60 min (see Materials and Methods). As seen in earlier publications [23,36], we verified that even the highest bacitracin concentrations used had no negative effects on cell growth, thereby ruling out the risk of affecting gene expression (Figure S1). In addition, we performed control experiments using a promoter-less GFP mutant to ensure that the observed increase in fluorescence is Figure 1. Core of the LiaFSR system. Arrows denote upregulation and T-shaped lines indicate inhibition. The LiaFSR system of Bacillus subtilis consists of the two-component signal transducing system LiaRS and the accessory membrane protein LiaF, a LiaRS-specific inhibitor. Stress represented e.g. by cell wall antibiotics such as bacitracin is sensed by LiaS/F and leads to expression of the liaIH - liaGFSR (‘‘lia locus’’ in the Figure) locus mediated by LiaR. To study the response of the Lia system to external stressors, we report activity of PliaI using the fluorescent marker GFP expressed under the control of the liaI promoter, chromosomally inserted ectopically in addition to the native Lia system. CM indicates the cytoplasmic membrane. doi:10.1371/journal.pone.0053457.g001 Figure 1. Core of the LiaFSR system. Arrows denote upregulation and T-shaped lines indicate inhibition. The LiaFSR system of Bacillus subtilis consists of the two-component signal transducing system LiaRS and the accessory membrane protein LiaF, a LiaRS-specific inhibitor. Stress represented e.g. by cell wall antibiotics such as bacitracin is sensed by LiaS/F and leads to expression of the liaIH - liaGFSR (‘‘lia locus’’ in the Figure) locus mediated by LiaR. To study the response of the Lia system to external stressors, we report activity of PliaI using the fluorescent marker GFP expressed under the control of the liaI promoter, chromosomally inserted ectopically in addition to the native Lia system. CM indicates the cytoplasmic membrane. doi:10.1371/journal.pone.0053457.g001 January 2013 | Volume 8 | Issue 1 | e53457 January 2013 | Volume 8 | Issue 1 | e53457 2 PLOS ONE | www.plosone.org Response of B. subtilis LiaFSR System to Bacitracin Figure 2. Gene expression increases at high bacitracin concentrations Expression profiles of the PliaI response in dependence of the bacitracin concentration. Addition of bacitracin induced GFP expression. At T60 all cells reached their maximum fluorescence intensities. While at high bacitracin concentrations all cells shifted to high fluorescence values, at low bacitracin concentrations (1 and 0.3 mg/ml) a fraction of cells did not express GFP. The observed decrease of fluorescence intensities after T60 is attributed to ongoing cell division. A) Autofluorescence (,8 FU) of Bacillus subtilis cells recorded shortly before bacitracin addition at T0. B) Representative images of B. subtilis cells 60 min after bacitracin induction. Bacitracin concentration is given in the right upper corner of each image in mg/ml. C)–F) Histograms of GFP expression from the liaI promoter for different time points, at C) 30 mg/ml bacitracin (T7 = 7 min after bacitracin induction), D) 3 mg/ml bacitracin, E) 1 mg/ml bacitracin, and F) 0.3 mg/ml bacitracin. doi:10.1371/journal.pone.0053457.g002 p y Figure 2. Expression profiles of the PliaI response in dependence of the bacitracin concentration. Addition of bacitracin induced GFP expression. At T60 all cells reached their maximum fluorescence intensities. While at high bacitracin concentrations all cells shifted to high fluorescence values, at low bacitracin concentrations (1 and 0.3 mg/ml) a fraction of cells did not express GFP. The observed decrease of fluorescence intensities after T60 is attributed to ongoing cell division. A) Autofluorescence (,8 FU) of Bacillus subtilis cells recorded shortly before bacitracin addition at T0. B) Representative images of B. subtilis cells 60 min after bacitracin induction. Bacitracin concentration is given in the right upper corner of each image in mg/ml. C)–F) Histograms of GFP expression from the liaI promoter for different time points, at C) 30 mg/ml bacitracin (T7 = 7 min after bacitracin induction), D) 3 mg/ml bacitracin, E) 1 mg/ml bacitracin, and F) 0.3 mg/ml bacitracin. doi:10.1371/journal.pone.0053457.g002 due to bacitracin induction. As expected no GFP expression could be detected in the promoter-less mutant (data not shown). The general response of PliaI was similar for all bacitracin concentrations (Figure 2C–F). First, the whole cell population responded within less than 10 min as at T10 a clear shift to higher fluorescence values was observable. Only at very low bacitracin concentrations (0.1 mg/ml) hardly any fluorescence could be detected within the 120 min observation period, as cells stayed at FIauto = 861 FU (Figure S2). Second, FImax was reached within due to bacitracin induction. January 2013 | Volume 8 | Issue 1 | e53457 Switching into the ‘ON’ state occurs within 20 min Interestingly, no bimodality was observed at any time point for low bacitracin concentrations, as FI levels of cells expressing GFP ranged continuously from FIauto to high FI values, making it difficult to separate the non-induced cells from cells with induced GFP expression corresponding to higher GFP levels. Therefore, we defined the switching threshold from the non-induced ‘OFF’ state to the induced ‘ON’ state in the following way: At high bacitracin induction all cells switched into the induced ‘‘ON’’ state. Although FImax was not reached until T60, all cells had clearly shifted away from the autofluoresce level FIauto at T7 (30 mg/ml bacitracin) and T10 (3 mg/ml bacitracin). We used these intermediate states as seen in experiments with high inducer concentrations (30 and 3 mg/ml bacitracin) to determine the switching threshold by applying a Gaussian fit to the histograms shown in Figure 3 (see Material and Methods, Table S1). This resulted in a switching threshold of 30 FU: cells showing expression levels above 30 FU ( = three-fold above background) were considered as being in the ‘ON’ state. This threshold definition best reflected the observed fluorescence expression distributions (Figure 2C–F). Subsequently, we determined the fraction of cells in the ‘ON’ state as a function of time (fON(T)) (see Materials and Methods), which was well described by a sigmoid function (Figure 4 left, Table 1, Table S2). Around 20 min after bacitracin induction, the fraction of cells in the ‘ON’ state saturated at fONmax, ranging from 100% for high bacitracin concentrations to 2.3% for very low (0.1 mg/ml) antibiotic concentrations (Figure 4 left, Table 1, Figure S2). After these 20 min no further increase of the fraction of cells in the ‘ON’ state could be detected. The observed decrease of fluorescence intensities, and with it the fraction of cells in the ‘ON’ state, seen for low bacitracin concentrations (1 and 0.3 mg/ml), can be attributed to ongoing cell division. Our data show that the number Independent on the bacitracin concentration added, switching into the ‘ON’ state started approximately five minutes after bacitracin induction, ending 20 min later. This indicates the presence of a well-defined switching window of about 20 min in which cells can enter the ‘ON’ state. As soon as bacitracin, or any damage caused by it, is sensed by the LiaFSR system, cells start to switch into the ‘ON’ state. Switching into the ‘ON’ state occurs within 20 min We next investigated the time needed by the whole population to switch into the ‘ON’ state by analyzing the switching rate (Materials and Methods). We determined the switching rate (PfON) as the first derivative of the fraction of cells in the ‘ON’ state with respect to time (Figure 4, right), which was well described by a Gaussian function (Material and Methods, Table S3). Maximal switching into the ‘ON’ state was observed at about 11 min for 3 and 30 mg/ml bacitracin and about 14 min for 1 and 0.3 mg/ml bacitracin. One possible explanation for this observation is heterogeneous timing [36]. Here, the time point of switching for individual cells is distributed over a longer time period. As the fraction of cells in the ‘ON’ state saturated 20 min after bacitracin induction, even for low bacitracin concentrations, and no further increase of the fraction of cells in the ‘‘ON’’ state could be observed thereafter, we find this explanation unlikely. Instead, we assume that cells still responding at low antibiotic concentrations need more time to do so (Figure 4 left, Table S4). The maximal switching rate (PfONmax) was about 10 to 20%/min for high bacitracin concentrations (Table 1, Table S5), and was signifi- cantly reduced at 0.3 mg/ml bacitracin with about 4%/min. Therefore, the small number of cells entering the ‘ON’ state at this bacitracin concentration can be ascribed to the reduced switching rate. As we had observed that FImax decreased with lower bacitracin concentrations, the question arose whether this was due to general lower fluorescence intensities in all cells at T60 or due to a heterogeneous GFP expression in the population at low inducer concentrations, with only a fraction of cells expressing GFP at high levels. While for high bacitracin concentrations (30 and 3 mg/ml) all cells switched from FIauto to FImax by 60 min post-induction, this could not be observed at low bacitracin concentrations (1 and 0.3 mg/ml). Here, parts of the population were not induced by bacitracin, as indicated by fluorescence levels in the range of the autofluorescence. Therefore, a clear heterogeneity in gene expression levels was present at 60 min after bacitracin induction at low antibiotic concentrations (Figure 2B). Gene expression increases at high bacitracin concentrations doi:10.1371/journal.pone.0053457.t001 system where for low inducer concentrations cells responded only 20 min after induction [40]. of cells switching into the ‘ON’ state is dependent on the external antibiotic concentration and reaches a saturating level at 3 mg/ml bacitracin. Above this concentration all cells enter the ‘ON’ state. Gene expression increases at high bacitracin concentrations As expected no GFP expression could be detected in the promoter-less mutant (data not shown). 60 min. Third, after 60 min fluorescence levels decreased again probably due to ongoing cell division. Taken together our data demonstrate that the LiaFSR system exhibits a graded and fast response to the external stimulus bacitracin: The FImax as obtained after 60 min of induction increased with the stimulus concentra- tion. In addition, cells started expression of the fluorescent protein even at low inducer concentrations within less than 10 min, in contrast to other systems such as e.g. the arabinose utilization The general response of PliaI was similar for all bacitracin concentrations (Figure 2C–F). First, the whole cell population responded within less than 10 min as at T10 a clear shift to higher fluorescence values was observable. Only at very low bacitracin concentrations (0.1 mg/ml) hardly any fluorescence could be detected within the 120 min observation period, as cells stayed at FIauto = 861 FU (Figure S2). Second, FImax was reached within January 2013 | Volume 8 | Issue 1 | e53457 PLOS ONE | www.plosone.org 3 Response of B. subtilis LiaFSR System to Bacitracin Table 1. Quantitative Analysis of the LiaFSR response. Bacitracin [mg/ml] FImax [FU] fONmax [%] PfONmax [%/min] t(PfONmax) [min] FIbasalmax [FU] Pamax [FU/min] t(Pamax) [min] 30 27262 9960.2 10.661.1 11.361.3 NA NA NA 3 13964 10060 19.5613.8 10.661.9 NA NA NA 1 4464 7862.4 9.762.9 14.261.1 21.660.7 2.360.4 8.060.35 0.3 2662 2662 3.962.6 14.361.5 11.960.4 0.360. 3 6.261.3 0.1 861 2.360.1 NA NA NA NA NA FImax = average maximal fluorescence intensity at T60, fONmax = maximal fraction of cells in the ‘ON’ state, pfONmax = maximal switching rate, t(pfONmax) = time point of maximal switching, FIbasalmax = average maximal basal fluorescence intensity, Pamax = maximal expression rate, t(Pamax) = time point of maximal expression rate. doi:10.1371/journal.pone.0053457.t001 scence intensity at T60, fONmax = maximal fraction of cells in the ‘ON’ state, pfONmax = maximal switching rate, t(pfONmax) = time point of average maximal basal fluorescence intensity, Pamax = maximal expression rate, t(Pamax) = time point of maximal expression rate. 457 t001 FImax = average maximal fluorescence intensity at T60, fONmax = maximal fraction of cells in the ‘ON’ state, pfONmax = maximal switching rate, t(pfONmax) = time point of maximal switching, FIbasalmax = average maximal basal fluorescence intensity, Pamax = maximal expression rate, t(Pamax) = time point of maximal expression rate. January 2013 | Volume 8 | Issue 1 | e53457 Basal expression rate of PliaI is dependent on bacitracin concentration times as compared with fON(T), indicating that the basal expression rate Pa had a maximum and that the maximum expression rate was shifted to earlier times as compared with the maximum switching rate PfON. The maximal fluorescence values of not-induced cells as obtained at 20 min after bacitracin induction showed significantly higher values as compared to the autofluorescence (Figure 5 A,C), with about 22 and 12 FU for 1 and 0.3 mg/ml bacitracin, respectively (Table 1). Basal expression rate of PliaI is dependent on bacitracin concentration We observed that the maximal switching rate PfONmax was reduced at 0.3 mg/ml bacitracin as compared to higher bacitracin concentrations and was reached at later time points. This raised the question whether the smaller switching rate at low bacitracin concentrations was due to a reduced PliaI promoter activity. We addressed this question by analyzing the basal expression rate (Pa). As GFPmut1 and LiaI represent two different proteins, it is possible that GFPmut1 and LiaI have different proteolysis rates. Therefore, the concentration of GFPmut1 controlled by PliaI is not necessarily a direct measure for the concentration of LiaI. However, the expression rates, i.e. the production rate of LiaI and GFPmut1, are expected to be similar, as the complete native PliaI including all native signals for LiaI expression is present. g p y ( ) We determined the basal expression rate Pa as the first derivative with respect to time of the mean grey value of those cells that had not entered the ‘ON’ state (Figure 5 B and D), which was well described by a Gaussian function (Table S7). The maximum basal expression rate, Pamax (Material and Methods), at 1 mg/ml was 2.360.4 FU/min exceeding the value of 0.360.3 FU/min at 0.3 mg/ml bacitracin by a factor of eight (Table S8). This indicated that the graded response of the LiaFSR system was merely due to a decreased basal expression rate at low bacitracin concentrations. As the maximal basal expression rate was reached at about 7 min at 1 and 0.3 mg/ml bacitracin as compared to the maximal switching rate at about 14 min (Table 1, Table S9), switching into the ‘ON’ state can be attributed to the increase of the basal expression rate at these bacitracin concen- trations. Switching into the ‘ON’ state occurs within 20 min The shut-down of the LiaFSR response can be understand in the context of the complete bacitracin stress response network that the Lia system is embedded in: several TCS are present in B. subtilis [19] that sense the antibiotic bacitracin leading to the activation of bacitracin detoxification systems that remove the antibiotic from its site of action [19,32]. This in turn lowers the inducing stress that is sensed by the LiaFSR system, resulting in the observed ‘switch-off’ at about 20 min. Although, the fraction of cells in the ‘ON’ state does not increase any further 20 min after bacitracin induction, an increase in fluorescence intensities can be observed until T60. We attribute this to the stability of the GFP-mRNA: as long as GFP-mRNA is present, translation can occur, resulting in the obtained increase in fluorescence intensity. January 2013 | Volume 8 | Issue 1 | e53457 PLOS ONE | www.plosone.org 4 Response of B. subtilis LiaFSR System to Bacitracin Figure 3. Definition of the switching threshold. Histograms of GFP fluorescence intensity at various time points. A) 30 mg/ml bacitracin, B) 3 mg/ml bacitracin. T0: time point of bacitracin induction representing the autofluorescence with ,8 FU. T7 and T10: Time points 7 and 10 min after bacitracin induction representing the phase at which cells are switching into the ‘ON’ state. At T14 and T15 (14 and 15 min after bacitracin induction) all cells have switched and the fluorescence distribution is clearly shifted towards higher fluorescence values. T7 and T10 therefore represent intermediate switching states and have therefore been used to determine the switching threshold as described in the Materials and Methods section. Red line: Gaussian fit. For details on the fit parameters see Table S1. doi:10.1371/journal.pone.0053457.g003 Figure 3. Definition of the switching threshold. Histograms of GFP fluorescence intensity at various time points. A) 30 mg/ml bacitracin, B) 3 mg/ml bacitracin. T0: time point of bacitracin induction representing the autofluorescence with ,8 FU. T7 and T10: Time points 7 and 10 min after bacitracin induction representing the phase at which cells are switching into the ‘ON’ state. At T14 and T15 (14 and 15 min after bacitracin induction) all cells have switched and the fluorescence distribution is clearly shifted towards higher fluorescence values. T7 and T10 therefore represent intermediate switching states and have therefore been used to determine the switching threshold as described in the Materials and Methods section. Red line: Gaussian fit. Switching into the ‘ON’ state occurs within 20 min For details on the fit parameters see Table S1. doi:10.1371/journal.pone.0053457.g003 January 2013 | Volume 8 | Issue 1 | e53457 Basal expression rate of PliaI is dependent on bacitracin concentration As the basal expression rate is reduced again to zero As a first step, we selected the cells that had not switched into the ‘ON’ state, as present in experiments with 1 and 0.3 mg/ml bacitracin. The average basal fluorescence value of cells that had not switched (FIbasal) shifted to higher values with time, saturating at the maximal basal fluorescence value FIbasalmax. This increase of fluorescence values of not-induced cells could be well described by a sigmoid fit function FI(T) (Table S6), similar to the fraction of cells in the ‘ON’ state. However, FI(T) was shifted towards earlier January 2013 | Volume 8 | Issue 1 | e53457 PLOS ONE | www.plosone.org January 2013 | Volume 8 | Issue 1 | e53457 5 Response of B. subtilis LiaFSR System to Bacitracin Figure 4. Fraction of cells in the ‘ON’ state as a function of time (fON(T)) and switching rate (PfON). For definition of the switching threshold see description in the Materials and Method section. The fraction of cells in the ‘ON’ state (fON) increased with time, finally saturating at its maximal level. The maximal fraction of cells in the ‘ON’ state (fONmax) decreased with the bacitracin concentration. Similarly, the maximal switching rate (PfONmax) decreased at low bacitracin concentrations (e.g. 0.3 mg/ml). A, C, E, G) Fraction of cells in the ‘ON’ state as a function of time (fON). Solid line: best fit to a sigmoid function as previously described in [33] (Table S2). B, D, F, H) Switching rate (PfON). The switching rate was determined as the first derivative with respect to time of the fraction of cells in the ‘ON’ state. Solid line: best fit to a Gaussian function (Table S3). A and B: 30 mg/ml bacitracin; C and D: 3 mg/ml bacitracin; E and F: 1 mg/ml bacitracin, G and H: 0.3 mg/ml bacitracin. doi:10.1371/journal.pone.0053457.g004 Figure 4. Fraction of cells in the ‘ON’ state as a function of time (fON(T)) and switching rate (PfON). For definition of the switching threshold see description in the Materials and Method section. The fraction of cells in the ‘ON’ state (fON) increased with time, finally saturating at its maximal level. The maximal fraction of cells in the ‘ON’ state (fONmax) decreased with the bacitracin concentration. Similarly, the maximal switching rate (PfONmax) decreased at low bacitracin concentrations (e.g. 0.3 mg/ml). Basal expression rate of PliaI is dependent on bacitracin concentration A, C, E, G) Fraction of cells in the ‘ON’ state as a function of time (fON). Solid line: best fit to a sigmoid function as previously described in [33] (Table S2). B, D, F, H) Switching rate (PfON). The switching rate was determined as the first derivative with respect to time of the fraction of cells in the ‘ON’ state. Solid line: best fit to a Gaussian function (Table S3). A and B: 30 mg/ml bacitracin; C and D: 3 mg/ml bacitracin; E and F: 1 mg/ml bacitracin, G and H: 0.3 mg/ml bacitracin. doi:10.1371/journal.pone.0053457.g004 Switching initiation is similar for individual cells S f h i i l l d h P approximately 15–20 min after bacitracin induction, the duration of the switching window is well defined. The time delay between Pamax and PfONmax of about 6 to 8 min (Figure 5 E, F) is in the range of the maturation time of the used fluorescent protein GFPmut1 with 8 min (Figure S3, Table S10), demonstrating the immediate response of the LiaFSR system to the antibiotic bacitracin. So far, we have quantitatively analyzed the PliaI response of the whole bacterial population grown in stirred liquid cultures as given by the averaged values of the single cells. In order to study the switching behavior of individual cells we developed a new protocol for fluorescent time-lapse microscopy of exponentially growing B. subtilis cells. Bacteria were fixed via attachment to microfluidic chambers coated with a specific silane (Materials and Methods) and flushed with fresh medium including the antibiotic bacitracin. PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org January 2013 | Volume 8 | Issue 1 | e53457 6 Response of B. subtilis LiaFSR System to Bacitracin Figure 5. Basal expression rate (Pa) of PliaI at 1 and 0.3 mg/ml bacitracin. The average fluorescence intensities (FIbasal) of cells in the ‘OFF’ state increased with time, saturating shortly thereafter. This enabled us to determine the basal expression rate (Pa) as described in the Material and Methods section. The maximal basal fluorescence intensity decreased with lower bacitracin concentrations. Similarly, the basal expression rate was significantly reduced in experiments with 0.3 mg/ml bacitracin as compared to 1 mg/ml bacitracin. A) and C) Fluorescence development of cells being in the ‘OFF’ state (FIbasal). Solid line: best fit to a sigmoid function (Table S6). B) and D) Expression rate of PliaI as the first derivative of fluorescence development given in A) and C). Solid line: best fit to a Gaussian function (Table S7). A) and B): 1 mg/ml bacitracin, C) and D) 0.3 mg/ml bacitracin. E) and F) comparison of switching rate PfON (grey) and basal expression rate Pa (black). E) 1 mg/ml bacitracin. F) 0.3 mg/ml bacitracin. doi:10.1371/journal.pone.0053457.g005 Figure 5. Basal expression rate (Pa) of PliaI at 1 and 0.3 mg/ml bacitracin. The average fluorescence intensities (FIbasal) of cells in the ‘OFF’ state increased with time, saturating shortly thereafter. This enabled us to determine the basal expression rate (Pa) as described in the Material and Methods section. The maximal basal fluorescence intensity decreased with lower bacitracin concentrations. Switching initiation is similar for individual cells S f h i i l l d h P Similarly, the basal expression rate was significantly reduced in experiments with 0.3 mg/ml bacitracin as compared to 1 mg/ml bacitracin. A) and C) Fluorescence development of cells being in the ‘OFF’ state (FIbasal). Solid line: best fit to a sigmoid function (Table S6). B) and D) Expression rate of PliaI as the first derivative of fluorescence development given in A) and C). Solid line: best fit to a Gaussian function (Table S7). A) and B): 1 mg/ml bacitracin, C) and D) 0.3 mg/ml bacitracin. E) and F) comparison of switching rate PfON (grey) and basal expression rate Pa (black). E) 1 mg/ml bacitracin. F) 0.3 mg/ml bacitracin. doi:10.1371/journal.pone.0053457.g005 As bleaching of the GFPmut1 molecules in single cells was significant, we corrected the obtained fluorescent values as described in the Materials and Methods section. Since the GFP expression levels for low bacitracin concentrations were in the range of the bleaching, we were only able to monitor the switching behavior of individual cells over time at 30 mg/ml bacitracin. Analyzing bleach-corrected fluorescence values (Material and Methods), we observed that cells started switching at about five minutes after bacitracin induction and all cells had switched into the ‘ON’ state within 15 min, as seen in experiments performed in liquid cultures. As expected, individual cells reached fluorescence values at 60 min post-induction between 200 and 600 FU (Figure 6). But in contrast to the experiments of whole populations described above, FI values increased until 80 min (200–800 FU) indicating that cell division was reduced for cells grown directly on the microscopic slide rather than in flask cultures. Nevertheless, the same overall switching behavior could be observed for individual cells growing in the microfluidic chamber as compared to cells grown in liquid culture, demonstrating the suitability of this approach. In a next step we compared the individual switching curves by applying a sigmoid function to the fluorescence development of single cells over time. This study revealed that cells initiated switching into the ‘ON’ state within the same time frame, but the individual switching curves showed a high variation with individual switching rates ranging from 6–15 FU/min (Figure 6). In accordance with our findings of whole population studies, our single cell data obtained by time-lapse microscopy demonstrate the fast response of the LiaFSR system to bacitracin. Switching initiation is similar for individual cells S f h i i l l d h P As bleaching of the GFPmut1 molecules in single cells was significant, we corrected the obtained fluorescent values as described in the Materials and Methods section. Since the GFP expression levels for low bacitracin concentrations were in the range of the bleaching, we were only able to monitor the switching behavior of individual cells over time at 30 mg/ml bacitracin. Analyzing bleach-corrected fluorescence values (Material and Methods), we observed that cells started switching at about five minutes after bacitracin induction and all cells had switched into the ‘ON’ state within 15 min, as seen in experiments performed in liquid cultures. As expected, individual cells reached fluorescence values at 60 min post-induction between 200 and 600 FU (Figure 6). But in contrast to the experiments of whole populations described above, FI values increased until 80 min (200–800 FU) indicating that cell division was reduced for cells grown directly on the microscopic slide rather than in flask cultures. Nevertheless, the same overall switching behavior could be observed for individual cells growing in the microfluidic chamber as compared Response of B. subtilis LiaFSR System to Bacitracin coli revealed a wide span in auto-phosphorylation rates of the HK ranging from about 2 min to 10 min. Phospo-transfer to RRs by phosphorylated cognate HKs took place within less than K min [9]. As maximal switching into the ‘ON’ state of the LiaFSR system can be observed within 15 min after bacitracin addition, even at the lowest bacitracin concentration, this demonstrates that no further regulatory elements are involved in the bacitracin- dependent LiaFSR response. This is in line with our finding that the basal expression rate of the liaI promoter is dependent on the bacitracin concentration, indicating that the LiaR concentration is directly affecting gene expression from PliaI. Recently, it was found that even at very high bacitracin concentrations (50 mg/ml) only about 20 molecules of LiaR are present within a single cell [43], while in the absence of bacitracin LiaR was not detectable. The amount of available LiaR controlling expression from PliaI is therefore dependent on the bacitracin concentration. The low number of LiaR molecules can explain the observed variations in gene expression, in particular the heterogeneity present at low bacitracin concentrations, as cell-to-cell differences (noise [31]) in the exact number of LiaR directly affect gene expression from PliaI. Performing a population study analyzed at the single cell level Figure 6. Switching characteristics of single cells at 30 mg/ml bacitracin. Fluorescence development of single cells over time at 30 mg/ml bacitracin was comparable to the data obtained by single cell analysis of the above described population study: All cells switched into the induced ‘ON’ state, exceeding the threshold fluorescence intensity within 15 min. In contrast to the whole population study the maximal fluorescence intensity was reached only after 80 min. A) Fluorescence development of one individual cell is shown. Top: bright field images at different time points. Bottom: fluorescence images at different time points. B) Fluorescence development of 13 individual cells is shown. C) Sigmoidal fits have been applied to eight fluorescence intensity traces in Figure 6B. The fluorescence intensity was normalized to the maximum fluorescence intensity and the time axis was shifted to T45, where cells had half-maximum fluorescence intensity. Blue and red line: two individual fluorescence traces representing cells with the slowest and highest individual switching rates in this cell batch. Response of B. subtilis LiaFSR System to Bacitracin doi:10.1371/journal.pone.0053457.g006 particular case it was shown that the sensitivity of this approach is high enough to detect an increase of promoter activity by a factor of two. This result was confirmed independently, using fluores- cence in situ hybridization (FISH), demonstrating the usability of quantitative fluorescence microscopy [41]. Another quantitative method to analyze single cells is flow cytometry. We performed flow cytometry experiments in order to study the LiaFSR response to various bacitracin concentrations (Figure S4), confirming our results obtained by QFM. Fluorescence values of single cells obtained by flow cytometry for low bacitracin concentrations were difficult to separate from the buffer background even after applying gating procedures. Therefore, we chose to focus on quantitative fluorescence microcopy to analyze our data in order Response of B. subtilis LiaFSR System to Bacitracin Response of B. subtilis LiaFSR System to Bacitracin to obtain the complete information of the LiaFSR response for high and low bacitracin concentrations. Figure 6. Switching characteristics of single cells at 30 mg/ml bacitracin. Fluorescence development of single cells over time at 30 mg/ml bacitracin was comparable to the data obtained by single cell analysis of the above described population study: All cells switched into the induced ‘ON’ state, exceeding the threshold fluorescence intensity within 15 min. In contrast to the whole population study the maximal fluorescence intensity was reached only after 80 min. A) Fluorescence development of one individual cell is shown. Top: bright field images at different time points. Bottom: fluorescence images at different time points. B) Fluorescence development of 13 individual cells is shown. C) Sigmoidal fits have been applied to eight fluorescence intensity traces in Figure 6B. The fluorescence intensity was normalized to the maximum fluorescence intensity and the time axis was shifted to T45, where cells had half-maximum fluorescence intensity. Blue and red line: two individual fluorescence traces representing cells with the slowest and highest individual switching rates in this cell batch. doi:10.1371/journal.pone.0053457.g006 We observed an immediate response of the system with cells switching in the bacitracin-induced ‘ON’ state within 20 min, irrespective of the externally provided bacitracin concentration. The switching rate shows its maximum approximately 7 min after the maximum of the basal expression rate. Importantly, this response time is in the range of the maturation time of the green fluorescent reporter with 8 min [42], indicating an almost instant burst of LiaR-dependent transcription initiation at PliaI. This is in contrast to other studies, in which maximum RR-regulated mRNA concentrations [1] or the concentration of promoter- bound RR [10] could be detected only within 20–30 min after exposure to the externally provided signal. Functional character- ization of all two-component signal transduction systems in E. coli revealed a wide span in auto-phosphorylation rates of the HK ranging from about 2 min to 10 min. Phospo-transfer to RRs by phosphorylated cognate HKs took place within less than K min [9]. As maximal switching into the ‘ON’ state of the LiaFSR system can be observed within 15 min after bacitracin addition, even at the lowest bacitracin concentration, this demonstrates that no further regulatory elements are involved in the bacitracin- dependent LiaFSR response. Response of B. subtilis LiaFSR System to Bacitracin This is in line with our finding that the basal expression rate of the liaI promoter is dependent on the bacitracin concentration, indicating that the LiaR concentration is directly affecting gene expression from PliaI. Recently, it was found that even at very high bacitracin concentrations (50 mg/ml) only about 20 molecules of LiaR are present within a single cell [43], while in the absence of bacitracin LiaR was not detectable. The amount of available LiaR controlling expression from PliaI is therefore dependent on the bacitracin concentration. The low number of LiaR molecules can explain the observed variations in gene expression, in particular the heterogeneity present at low bacitracin concentrations, as cell-to-cell differences (noise [31]) in the exact number of LiaR directly affect gene expression from PliaI. Performing a population study analyzed at the single cell level, in combination with time-lapse microscopy, we quantitatively analyzed the response of the LiaFSR two-component system to bacitracin. As described above, the LiaFSR system responds within less than 15 min to the external stimulus. Cell-to-cell differences are present at all bacitracin concentrations and decrease at low bacitracin levels. The maximum switching rate as well as basal expression rate depends on the bacitracin concentration, reflecting the graded response of the LiaFSR system. For a stress sensor system, this kind of response is reasonable. Changing environmental conditions, including the presence of stressors, require fast stress sensing systems such as the LiaFSR system, that are shut-off as soon as the stressor is no longer present. Taken together, our data demonstrate that the LiaFSR system exhibits an immediate, heterogeneous and graded response to the peptide antibiotic bacitracin in the exponential growth phase. We observed an immediate response of the system with cells switching in the bacitracin-induced ‘ON’ state within 20 min, irrespective of the externally provided bacitracin concentration. The switching rate shows its maximum approximately 7 min after the maximum of the basal expression rate. Importantly, this response time is in the range of the maturation time of the green fluorescent reporter with 8 min [42], indicating an almost instant burst of LiaR-dependent transcription initiation at PliaI. This is in contrast to other studies, in which maximum RR-regulated mRNA concentrations [1] or the concentration of promoter- bound RR [10] could be detected only within 20–30 min after exposure to the externally provided signal. Functional character- ization of all two-component signal transduction systems in E. Discussion In this report, we quantitatively investigated the response of the LiaFSR system to an external signal, the peptide-antibiotic bacitracin, by performing a population study analyzed on the single cell level. Quantitative fluorescence microscopy (QFM) as described in this study, has been used previously to analyze switching of Bacillus subtilis into the competent state [33]. In this January 2013 | Volume 8 | Issue 1 | e53457 PLOS ONE | www.plosone.org January 2013 | Volume 8 | Issue 1 | e53457 7 January 2013 | Volume 8 | Issue 1 | e53457 Fluorescence Microscopy Cells were sampled throughout growth as indicated in the main text. For image acquisition of the whole cell population, cells were permitted to attach to microscopic slides (eight-well IBIDI chamber, uncoated) and covered with 1% Agarose-patches. For time-series of single cells, cells were allowed to attach to microfluidic chambers coated with 100% 1-[3-(Trimethoxysilyl)- propyl]urea (Sigma). Cells were induced already attached to the microfluidic channels and washed with fresh medium in the presence of bacitracin at a flow-rate of 0.3 ml/h. PfON: average switching rate of cells switching into the ‘ON’ state. The switching rate was determined as the first derivative of the fraction of cells in the ‘ON’ state with respect to time. To reduce the error, the maximal switching rate (PfONmax) was determined using two different calculation methods: a) Pfonmax = maximum of the 1st derivative of the exact data points of fON. b) by obtaining A of the Gaussian fit applied to the data Figure 4 right according to PfON = y0+Aexp (2((x2x0)/width)2) (Table S5). The high error for data determined at 3 mg/ml bacitracin is attributed to the steep increase of the fraction of cells in the ‘ON’ state leading to a high fitting error. Additional data points in order to reduce the error could not be attained, as cells stored on ice for later image acquisition tended to lyse at bacitracin concentrations .1 mg/ml. Therefore image acquisition was performed immedi- ately after sampling of the cells. The exact results of both calculation methods as well as the average values are given in Table S5. The error of the switching rate was calculated according to: Error PfON at time point t2 = square root of ((error at (t2))2+(error at (t1))2), with t1 and t2 the time points of the derivated time interval. The individual errors here are the errors of fON as described above. Please note that error propagation has to be Image acquisition was done using a Zeiss Axiovert 200 M microscope equipped with an Andor Digital Camera and a Zeiss EC Plan-Neofluar 1006/1.3 Oil immersion objective. Andor software was used for image acquisition. The stability of the absolute fluorescence values was verified using a microscope image intensity calibration kit (Invitrogen, FokalCheckTM fluorescence microscope test slide #3). Microspheres showed a deviation of mean grey value of less than 1% under the experimental conditions used for detection of GFP fluorescence. Fluorescence Microscopy Homogeneity of illumination was tested using fluorescent slides and the maximum deviation was less than 5%. Growth conditions Bacillus subtilis strain TMB 1172 [35] carries a translational fusion of PliaI with the green fluorescent reporter protein GFPmut1. TMB 1172 was grown in LB medium at 37uC, shaken at 300 rpm. Overnight cultures were diluted to OD600 of 0.1. Cells were grown to mid-logarithmic phase, then were again diluted to OD600 of 0.1 into fresh medium and grown for additional 30 min to ensure optimal growth conditions before induction with the peptide-antibiotic bacitracin (Sigma) at T0 = 30 min and applying January 2013 | Volume 8 | Issue 1 | e53457 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org January 2013 | Volume 8 | Issue 1 | e53457 8 Response of B. subtilis LiaFSR System to Bacitracin the cells to the microscopic slides. This way any cross-over from intrinsic stationary phase induction [35] could be avoided. Experiments for each bacitracin concentration were performed in triplicates on three different days. For each time point a minimum of 100 cells was analyzed. The bacitracin concentrations used in this study are far below the minimal inhibitory concentration (MIC) [23,36] and have been shown to have no effect on growth (Figure S1). and transferred to the fluorescence image using the ROI manager of ImageJ. Only cells that were fully lying within the bright field image and were not in the process of cell division were considered. Furthermore, dead cells as observable by different contrast in the bright field image as compared to healthy cells were excluded from the single cell analysis. The remaining single cells were than analyzed with respect to their mean grey value. Data preparation was performed using the Software IGOR PRO 4.06 and Adobe Illustrator CS4. Definitions and calculation methods The promoter less vector pGFPamy [44] was transformed into B. subtilis as a negative control. The vector carries a chloram- phenicol resistance cassette for selection in B. subtilis, and integrates into the amyE locus by double crossing-over, resulting in a stable integration of the promoter-less-gfp fusion. The plasmid was linearized with PstI and used to transform B. subtilis 168 with chloramphenicol selection (5 mg/ml). Successful integration into the amyE locus was confirmed by starch test. FIauto: average autofluorescence/fluorescence intensity of cells not induced by bacitracin, given as fluorescence units [FU] as obtained by the mean grey value. The average autofluorescence level of cells prior bacitracin induction was FIauto = 861 FU (Figure 2A). FImax: average maximal GFP expression/fluorescence intensity as observed at T60. Upon induction with bacitracin cells expressed the GFP reporter. The resulting fluorescence intensities were obtained as the mean grey value of each single cell. The error of FImax is given as the standard error. Flow cytometry For flow cytometry experiments, the cultures were grown as described above. Samples were taken every 10 min for 120 min and diluted 1:100 in PBS (phosphate buffered saline). The experiments were performed using a Partec CyFlow Space instrument and the software FlowMax. GFP was excited with a laser at 488 nm and its emission measured at 518 nm. The analysis of the cells was done at a flow-rate of 2 ml/s. In between measurements, the instrument was rinsed with PBS to eliminate cross-contamination. In addition to the different concentrations of bacitracin, not induced samples and PBS alone were analyzed for control purposes. To discriminate dead from healthy cells, appropriate gating procedures have been applied. 50000 cells lying in the appropriate gate have been analyzed for each time point. Switching threshold: The switching threshold separates cells being in the ‘OFF’ state (no/basal expression) from cells being in the ‘ON’ state (induced GFP-expression). We used the intermediate states seen in experiments with high inducer concentrations (30 and 3 mg/ml bacitracin) to determine the switching threshold. A Gaussian fit was applied to the histograms shown in Figure 3 (Table S1). The values of the center of these distributions, in addition to the average fluorescence values of all cells at this time point, were averaged. The resulting value of 30 FU was then defined to be the switching threshold: any cell with fluorescent value above 30 FU (mean grey value) was considered as being in the ‘ON’ state. fON: fraction of cells in the ‘ON’ state. We determined the fraction of cells in the ‘ON’ state as a function of time using the switching threshold. The fraction of cells in the ‘ON’ state was well defined by a sigmoid function with fON(T) = fbase+fmax/1+exp(k(Thalf2T)). The fit parameter of this function can be found in Table S2. The maximal fraction of cells in the ‘ON’ state (fONmax) was determined using this fit function (Table S2). The error of the fraction of cells in the ‘ON’ state has been calculated according to: square root of (p(12p)/(n21)). Image Analysis Images were processed using ImageJ software. Image back- ground was corrected using a rolling ball algorithm with a radius of 50. An intensity threshold tool was used to delimit the boundaries of the cells in the bright field image. The boundaries of the cells were obtained with the wand tool of the ImageJ software January 2013 | Volume 8 | Issue 1 | e53457 January 2013 | Volume 8 | Issue 1 | e53457 PLOS ONE | www.plosone.org 9 Response of B. subtilis LiaFSR System to Bacitracin after 80 min must therefore be due to folding of already synthesized GFP (Figure S3). Assuming a first-order kinetic we fitted the data with a single exponential function and obtained a characteristic maturation time of 7.960.69 min. taken into account when deriving data points, leading to the high errors in Figure 4, F and H. t(PfONmax): Time point of maximal switching rate. To reduce the error the time point of the maximal switching rate has been determined in three different ways: a) Thalf of the sigmoidal fit applied to Figure 4 left according to fON(T) = fbase+fmax/1+ex- p(k(Thalf2T)). b) Time point of PfONmax = maximum of the 1st derivative of the exact data points of fON. c) by obtaining x0 of the Gaussian fit applied to the data in Figure 4 right according to PfON(T) = y0+A exp (2((x2x0)/width)2) (Table S4). Supporting Information t(Pamax): The time point of the maximal basal expression rate has been determined in three different ways: a) Thalf of the sigmoidal fit applied to Figure 5 A, C according to FI(T) = fbase+fmax/ 1+exp(k(Thalf2T)) (Table S9). b) Pamax = maximum of the 1st derivative of the exact data points of FIbasal. c) by obtaining x0 of the Gaussian fit applied to Figure 5 right according to Pa(T) = y0+Aexp (2((x2x0)/width)2) (Table S9). Table S9 Time point of maximal basal expression rate t(Pamax). (DOC) Bleach correction of single cell time-series In time-series of individual cells, bleaching of GFP in these cells occurred. Hence, we applied a bleach correction to our time-series data. After each time-series a new spot was chosen at an appropriate distance to ensure that no bleaching had occurred yet on this spot. Twenty successive images were taken. One image was immediately taken after the previous one. For each cell of this spot the obtained ‘bleach curve’ was fitted exponentially. The resulting rates were averaged. The data obtained in the actual time-series were then divided by e2nk, with n being the number of pictures already taken of this spot and k the average of the rates determined by the exponential fit of the ‘bleach curves’. Figure S2 Expression profiles of the Lia response at 0.1 mg/ml bacitracin. At these very low inducing concentra- tion of bacitracin nearly all cells stay in the non-induced ‘OFF’ state. A) Representative image of B. subtilis cells 60 min after bacitracin induction. B) Histograms of GFP expression from the liaI promoter for different time points (T10 = 10 min after bacitracin induction). Red arrows indicate the few cells in the ‘ON’ state at this bacitracin concentration. (EPS) Figure S3 Maturation of GFPmut1. Arrow indicates the addition of 400 mg/ml erythromycin at 80 min leading to immediate translation inhibition. Therefore any fluorescence development arising after erythromycin addition can be attributed to the maturation of the GFP fluorophore. Grey: cells grown in the absence of erythromycin. Black: Cells grown in the presence of erythromycin. Solid lines: best fit to an exponential function (Table S10). Table S10 Maturation of GFPmut1. (DOC) Figure S1 Influence of bacitracin on cell growth. Cells were grown as described in the Material and Methods section in the presence of bacitracin at different final concentrations (Black: 0 mg/ml, grey: 0.1 mg/ml, blue: 0.3 mg/ml, yellow: 1 mg/ml, green: 3 mg/ml, red: 30 mg/ml). At these concentrations bacitracin has no influence on cell growth. (EPS) A summery of all data described here, as well as the average data obtained from the different calculation methods for t(PfONmax), t(Pamax), PfONmax and Pamax can be found in Table 1. The obtained data for each calculation method for t(PfONmax), t(Pamax), PfONmax and Pamax are given in Figure S5. A summery of all data described here, as well as the average data obtained from the different calculation methods for t(PfONmax), t(Pamax), PfONmax and Pamax can be found in Table 1. The obtained data for each calculation method for t(PfONmax), t(Pamax), PfONmax and Pamax are given in Figure S5. Supporting Information Table S1 Fit parameter for the fluorescence distribu- tions given in Figure 3. (DOC) ( ) y p ( (( ) ) ) ( ) FIbasal: average basal fluorescence intensity of cells in the ‘OFF’ state. The error is given as the standard error. Table S2 Fit parameters for the fraction of cells in the ‘ON’ state fON. (DOC) FIbasalmax: maximal average basal fluorescence intensity as obtained by applying a sigmoidal fit function FI (T) = fbase+fmax/ 1+exp(k(Thalf2T)), with fbase baseline, fmax maximum basal fluorescence intensity, Thalf half time and k rate (Table S6). Table S3 Fit parameter for the switching rate PfON. (DOC) Pa: average basal expression rate of cells in the non-induced ‘OFF’ state. We determined the Pa as the first derivative with respect to time of the mean grey value of those cells that had not entered the ‘ON’ state. To reduce the error the maximal basal expression rate (Pamax) has been determined in two different ways: a) Pa = maximum of the 1st derivative of the exact data points of FIbasal. b) by obtaining x0 of the Gaussian fit applied to Figure 5 B, D according to Pa(T) = y0+Aexp (2((x2x0)/width)2) (Table S8). The error of the basal expression rate was calculated according to: Error Pa at time point t2 = square root of ((error at (t2))2+(error at (t1))2), with t1 and t2 the time points of the derivated time interval. The individual errors here are the errors of FIbasal as described above. Please note that error propagation has to be taken into account when deriving data points, leading to the high errors in Figure 5 B and D. Table S4 Time point of maximal switching rate t(PfON- max). (DOC) Table S5 Maximal switching rate PfONmax. (DOC) Table S6 Fit parameters for the basal fluorescence level FIbasal. (DOC) Table S7 Fit parameter for the basal expression rate Pa. (DOC) Table S8 Maximal basal expression rate Pamax. (DOC) t(Pamax): The time point of the maximal basal expression rate has been determined in three different ways: a) Thalf of the sigmoidal fit applied to Figure 5 A, C according to FI(T) = fbase+fmax/ 1+exp(k(Thalf2T)) (Table S9). b) Pamax = maximum of the 1st derivative of the exact data points of FIbasal. c) by obtaining x0 of the Gaussian fit applied to Figure 5 right according to Pa(T) = y0+Aexp (2((x2x0)/width)2) (Table S9). References 17. De Orue Lucana DO, Groves MR (2009) The three-component signaling system HbpS-SenS-SenR as an example of a redox-sensing pathway in bacteria. Amino Acids 37:479–486. 1. Kato A, Mitrophanov Y, Groisman EY (2007) A connector of two-component regulatory systems promotes signal amplification and persistence of expression. Proc Natl Acad Sci U S A 104: 12063–12068. 2. 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Washington, DC: ASM press pp. 105–127. 23. Mascher T, Zimmer SL, Smith T-A, Helmann JD (2004) Antibiotic-inducible promoter regulated by the cell envelope stress-sensing two-component system LiaRS of Bacillus subtilis. AAC 48:2888–2896. 8. Acknowledgments We thank Prof. J. O. Ra¨dler for the opportunity to use laboratory equipment, in particular the inverse microscope. We thank Sonja Westermayer, Andrea Ebert and Elke Hebisch for fruitful discussions. Figure S5 Maximal switching rate PfONmax and maximal basal expression rate Pamax for various bacitracin concentrations. As switching into the ‘ON’ state took place in a very short time period of less than 10–15 min, only few data points between the ‘OFF’ and the ‘ON’ state could be obtained. As cells treated with high bacitracin concentrations, although not showing any fitness defects, tended to lyse when stored on ice, a shorter experimental time resolution was not possible. Therefore, Response of B. subtilis LiaFSR System to Bacitracin Response of B. subtilis LiaFSR System to Bacitracin to reduce the error by simply fitting to the data, the maximal switching rate as well as the maximal basal expression rate was determined using several calculation methods as described in the Material and Methods section. This Figure gives an overview of the data obtained by the various methods used. A) Time point of maximum switching rate t(PfONmax); Black, grey and light grey bars represent data obtained as described in Table S4 a–c. Blue: averaged data of the time point of maximal switching. B) Maximal switching rate PfONmax: Black, and light grey bars represent data obtained as described in Table S5 a and b. Blue: averaged data of maximal switching rate PfONmax. C) Time point of maximum basal expression rate t(Pamax); Black, grey and light grey bars represent data obtained as described in Table S9 a–c. Blue: average data of t(Pamax). D) Maximum basal expression rate Pamax; Black and light grey bars represent data as described in Table S8 a and b. Blue: average date of maximal Pamax. (EPS) Figure S4 Flow cytometry analysis of the PliaI response of LiaFSR to bacitracin. Flow cytometry analysis verified the results obtained by quantitative fluorescence microcopy as shown in main Figure 2. Addition of bacitracin induced GFP expression. At T60 all cells reached their maximum fluorescence intensities. While at high bacitracin concentrations all cells shifted to high fluorescence values, at low bacitracin concentrations (1 and 0.3 mg/ml) a fraction of cells did not express GFP and stayed at the autofluorescence value. As low fluorescence intensities of induced cells were hard to distinguish from the background fluorescence of not induced cells using flow cytometry, we chose quantitative fluorescence microscopy for detailed analysis of the LiaFSR response. Data shown here represent the mean grey value of each single cell: mean FI [FU]. A) Background signal of the buffer PBS in the gated area. B) Autofluorescence of not induced Bacillus subtilis cells C)–F) Histograms of GFP expression from the liaI promoter for different time points, at C) 30 mg/ml bacitracin (T30 = 30 min after bacitracin induction), D) 3 mg/ml bacitracin, E) 1 mg/ml bacitracin, and F) 0.3 mg/ml bacitracin. (EPS) References Li J, Swanson RV, Simon MI, Weis RM (1995) The response regulators CheB and CheY exhibit competitive binding to the kinase CheA. Biochemistry 34: 14626–14636. 24. Jordan S, Hutchings MI, Mascher T (2008) Cell envelope stress response in Gram-positive bacteria. FEMS Microbiol Rev 32:107–146. 9. Yamamoto K, Hirao K, Oshima T, Aiba H, Utsumi R, et al. (2005) Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli. J Biol Chem 280:1448–1456. 25. Schrecke K, Staron A, Mascher T (2012) Two-Component Signaling in the Gram-positive Envelope Stress Response: Intramembrane-sensing Histidine Kinases and Accessory Membrane Proteins. Two-component Systems in Bacteria. Book: EAN: 9781908230089. 10. Ray JCJ, Igoshin OA (2010) Adaptable functionality of transcriptional feedback in bacterial two-component systems. Plos Comp Biol 6(2): e1000676. Accessed 06 August 2012. 26. Staron A, Finkeisen DE, Mascher T (2011) Peptide antibiotic sensing and detoxification modules of Bacillus subtilis. Antimicrob Agents Chemother 55:515– 525. 11. Mitrophanov AY, Churchward G, Borodovsky M (2007) Control of Streptococcus pyogenes virulence: Modeling of the CovR/S signal transduction system. J Theo Biol 246:113–128. 27. Wolf D, Dominguez-Cuevas P, Daniel RA, Mascher T (2012). Cell envelope stress response in cell wall-deficient L-forms of Bacillus subtilis. Antimicrob Agents Chemother. DOI:10.1128. 12. Groban ES, Clarke EJ, Salis HM, Miller SM, Voigt CA (2009) Kinetic buffering of cross talk between bacterial two-component sensors. J Mol Biol 390:380–393. 28. Ghosh S, Sureka K, Ghosh B, Bose I, Basu J, et al. (2011) Phenotypic heterogeneity in mycobacterial stringent response. BMC Systems Biology 5:18. Available: http://www.biomedcentral.com/1752-0509/5/18. Accessed 2012 July 9. 13. Scharf BE (2010) Summary of useful methods for two-component system research. Curr Opin Microbiol 13:246–252. 14. Hoch JA (1995) Two-component signal transduction. Washington, DC: Am Soc Microbiol. pp. 129–144. 29. Botella E, Hu¨bner S, Hokamp K, Hansen A, Bisicchia P, et al. (2011) Cell envelope gene expression in phosphate-limited Bacillus subtilis cells. Microbiology 157:2470–2484. 15. Szurmant H, Bu L, Brooks CLIII Hoch JA (2008) An essential sensor histidine kinase controlled by transmembrane helix interactions with its auxiliary proteins. Proc Natl Acad Sci U S A 105: 5891–5896. 30. Smits WK, Kuipers OP, Veening J-W (2006) Phenotypic variation in bacteria: the role of feedback regulation. Nature Reviews Microbiol 4:259–271. 16. Elsen S, Duche O, Colbeau A (2003) Interaction between the sensor HupUV and the histidine kinase HupT controls HupSL hydrogenase synthesis in Rhodobacter capsulatus. J Bacteriol 185: 7111–7119. 31. Author Contributions Conceived and designed the experiments: TM ML. Performed the experiments: CH AM SK. Analyzed the data: SK ML. Wrote the paper: TM ML. GFPmut1 maturation To determine the time delay between expression of the GFP reporter and the onset of fluorescence, strain TMB 1172 was grown in LB medium as described above. For induction of GFPmut1 expression bacitracin was added after 60 min at a final concentration of 30 mg/ml and erythromycin was added at 80 min, inhibiting protein biosynthesis. Increase of fluorescence January 2013 | Volume 8 | Issue 1 | e53457 PLOS ONE | www.plosone.org 10 Response of B. subtilis LiaFSR System to Bacitracin References Swain PS, Elowitz MB, Siggia ED (2002) Intrinsic and extrinsic contributions to stochasticity in gene expression. Proc Natl Acad Sci U S A 99:12795–12800. PLOS ONE | www.plosone.org 11 January 2013 | Volume 8 | Issue 1 | e53457 32. Dubnau D, Losick R (2006) Bistability in bacteria. Mol Microbiol 61:564–572. Response of B. subtilis LiaFSR System to Bacitracin Response of B. subtilis LiaFSR System to Bacitracin 32. Dubnau D, Losick R (2006) Bistability in bacteria. Mol Microbiol 61:564–572. 39. Jordan S, Rietko¨tter E, Strauch MA, Kalamorz F, Butcher BG, et al. (2007) LiaRS-dependent gene expression is embedded in the transition state regulation in Bacillus subtilis. Microbiology 153:2530–2540. 33. 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English
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Ultra-early response assessment in lymphoma treatment: [18F]FDG PET/MR captures changes in glucose metabolism and cell density within the first 72 hours of treatment
European journal of nuclear medicine and molecular imaging
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cc-by
7,845
Abstract Purpose To determine whether, in patients with Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL), [18F]FDG PET/ MR can capture treatment effects within the first week after treatment initiation, and whether changes in glucose metabolism and cell density occur simultaneously. Methods Patients with histologically proven HL or NHL were included in this prospective IRB-approved study. Patients underwent [18F]FDG PET/MR before, and then 48–72 h after (follow-up 1, FU-1) and 1 week after (FU-2) initiation of the first cycle of their respective standard chemotherapy (for HL) or immunochemotherapy (for NHL). Standardized [18F]FDG uptake values (SUVmax, SUVmean) and apparent diffusion coefficients (ADCmin, ADCmean) based on diffusion-weighted MRI, and metabolic and morpho- logical tumour volumes (MTV, VOL) were assessed at each time-point. Multilevel analyses with an unstructured covariance matrix, and pair-wise post-hoc tests were used to test for significant changes in SUVs, ADCs, MTVs and VOLs between the three time-points. Results A total of 58 patients (11 with HL and 47 with NHL) with 166 lesions were analysed. Lesion-based mean rates of change in SUVmax, SUVmean, ADCmin, ADCmean, MTVand VOL between baseline and FU-1 were −46.8%, −33.3%, +20.3%, +14%, −46% and −12.8%, respectively, and between baseline and FU-2 were −65.1%, −49%, +50.7%, +32.4%, −61.1% and −24.2%, respectively. These changes were statistically significant (P < 0.01) except for the change in VOL between baseline and FU-1 (P = 0.079). Conclusion In lymphoma patients, [18F]FDG PET/MR can capture treatment-induced changes in glucose metabolism and cell density as early as 48–72 h after treatment initiation. Keywords Lymphoma . PET/MRI . FDG . Chemotherapy . Immunotherapy Keywords Lymphoma . PET/MRI . FDG . Chemotherapy . Immunotherapy Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00259-018-3937-z) contains supplementary material, which is available to authorized users. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00259-018-3937-z) contains supplementary material, which is available to authorized users. * Marius E. Mayerhoefer marius.mayerhoefer@meduniwien.ac.at European Journal of Nuclear Medicine and Molecular Imaging (2018) 45:931–940 https://doi.org/10.1007/s00259-018-3937-z European Journal of Nuclear Medicine and Molecular Imaging (2018) 45:931–940 https://doi.org/10.1007/s00259-018-3937-z European Journal of Nuclear Medicine and Molecular Imaging (2018) 45:931–940 https://doi.org/10.1007/s00259-018-3937-z ORIGINAL ARTICLE Ultra-early response assessment in lymphoma treatment: [18F]FDG PET/MR captures changes in glucose metabolism and cell density within the first 72 hours of treatment Marius E. Mayerhoefer1 & Markus Raderer2 & Ulrich Jaeger2 & Philipp Staber2 & Barbara Kiesewetter2 & Daniela Senn3 & Ferdia A. Gallagher4,5 & Kevin Brindle4 & Edit Porpaczy2 & Michael Weber1 & Dominik Berzaczy1 & Ingrid Simonitsch-Klupp6 & Christian Sillaber2 & Cathrin Skrabs2 & Alexander Haug3 Marius E. Mayerhoefer1 & Markus Raderer2 & Ulrich Jaeger2 & Philipp Staber2 & Barbara Kiesewetter2 & Daniela Senn3 & Ferdia A. Gallagher4,5 & Kevin Brindle4 & Edit Porpaczy2 & Michael Weber1 & Dominik Berzaczy1 & Ingrid Simonitsch-Klupp6 & Christian Sillaber2 & Cathrin Skrabs2 & Alexander Haug3 Received: 13 October 2017 /Accepted: 4 January 2018 /Published online: 26 February 2018 # The Author(s) 2018. This article is an open access publication * Marius E. Mayerhoefer marius.mayerhoefer@meduniwien.ac.at 4 Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK 5 Department of Radiology, Addenbrooke’s Hospital, Cambridge, UK Introduction Positron emission tomography (PET) using the radio- tracer [18F]FDG (2-[18F]fluoro-2-deoxy-D-glucose) is the current imaging technique of choice to assess treat- ment response in the majority of lymphoma subtypes [1–5]. It allows direct assessment of the glycolytic ac- tivity at the cellular level, which correlates with cell proliferation [6]. Diffusion-weighted imaging (DWI), an advanced magnetic resonance imaging (MRI) tech- nique, can be used to assess tissue diffusivity which is an indirect measure of cell density. Since extracellular water movement in tumours is affected by cell density [7], DWI has recently been suggested as a potent 1 Department of Biomedical Imaging and Image-guided Therapy, Division of General and Pediatric Radiology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria 2 Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria 2 Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria 3 Department of Biomedical Imaging and Image-guided Therapy, Division of Nuclear Medicine, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria 4 Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK 5 Department of Radiology, Addenbrooke’s Hospital, Cambridge, UK 6 Institute of Pathology, Medical University Vienna, Vienna, Austria Eur J Nucl Med Mol Imaging (2018) 45:931–940 932 lymphoid malignancies, in combination with the following specified treatment schemes: alternative to [18F]FDG PET/CT to assess treatment re- sponse in lymphoma patients [8–12]. p y p p [ ] Previous studies have demonstrated that a reduction in gly- colytic activity (i.e. [18F]FDG uptake on PET), and an in- crease in diffusivity (i.e. reduction of diffusion restriction on DWI, chiefly reflecting a reduction in cell density) can be observed after only a small number of therapy cycles [10, 12–15]. Notably, interim PET, which is typically performed after two to four therapy cycles and is included in the Lugano classification of the International Conference on Malignant Lymphoma (ICML) [1, 13], is currently under evaluation as a prognostic marker for different lymphoma subtypes, treat- ments and time-points, but so far with mixed results [15–22]. However, little is known about in vivo changes at the onset of therapy – in particular, to what extent do changes in glycolytic activity and cell density correlate with each other, and which method detects the earliest changes. In a murine lymphoma model, it has been shown that [18F]FDG PET can capture changes after treatment with etoposide as early as 16 h after treatment initiation [23]. Imaging protocols All pretherapy and follow-up [18F]FDG PET/MR examina- tions were performed using a commercially available fully CE-certified integrated simultaneous PET/MR system (Biograph mMR; Siemens, Erlangen, Germany). This PET/ MR system comprises a PET detector system inserted into a 3-T MRI system with high-performance gradient systems (45 mT/m) and a slew rate of 200 T/m/s. It is equipped with Total Imaging Matrix coil technology (Siemens) covering the body (from the vertex to the upper thighs) with multiple integrated radiofrequency surface coils that do not require repositioning. The PET system offers an axial field of view (FOV) of 258 mm, and a sensitivity of 13.2 cps/kBq. Introduction So far, no comparable in vivo data in lymphoma patients are available. DWI has been shown to capture treatment response in lymphoma in vivo as early as 1 week after therapy initiation, but no earlier time-points were evaluated [24]. Finally, in a study in patients with Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBCL), no significant changes in apparent diffusion coef- ficients (ADC) were observed approximately 2 days after ther- apy initiation, whereas [18F]FDG PET revealed treatment re- sponses after 19 days [25]. – Patients with HL scheduled for treatment with escalated BEACOPP – Patients with DLBCL scheduled for treatment with either R-CHOP in those with an International Prognostic Index (IPI) of 1–3, or dose-adjusted (DA) EPOCH-R in those with an IPI >3 or double-hit lymphomas – Patients with follicular lymphoma (FL) scheduled for treatment with R-BENDA – Patients with mantle cell lymphoma (MCL) scheduled for treatment with R-BENDA – Patients with mantle cell lymphoma (MCL) scheduled for treatment with R-BENDA Patients with any of the following were excluded: women with a positive pregnancy test or clinically confirmed preg- nancy; inability to understand the study goals or outline, or to consent to participation; age below the specified minimum of 18 years; known contraindication to MRI (e.g. implantable medical device according to the MRI Safety Guidelines, or conditions such as claustrophobia); or a blood glucose level >150 mg/dL. Patients who fulfilled the above requirements for participation underwent [18F]FDG PET/MR at three time- points: 1. Baseline: before treatment (with a specified range of 1– 7 days before the first therapy cycle) 2. Follow-up 1 (FU-1): 48–72 h after initiation of the first therapy cycle 3. Follow-up 2 (FU-2): 1 week after initiation of the first therapy cycle Here, we investigated whether standard chemotherapy or immunochemotherapy led to significant changes in glucose metabolism, cell density, and lesion volume in lymphoma patients, at two time-points during the first week after treat- ment initiation. We particularly aimed to determine whether such ultra-early effects of treatment on glycolytic activity and cell density of lymphomas occur simultaneously, and whether they differ among histological subtypes, therapeutic regimens, and individuals with the same histology and treatment. Treatment protocols Escalated BEACOPP was administered to patients with ad- vanced stage HL and the presence of risk factors, according to the guidelines of the German and Austrian societies of haematology and oncology (DGHO and OeGHO; https:// www.onkopedia.com). A 21-day cycle consisted of bleomycin 10 mg/m2 intravenously (i.v.) on day 8, etoposide 200 mg/m2 i.v. on days 1–3, doxorubicin 35 mg/m2 i.v. on day 1, cyclophosphamide 1,250 mg i.v. on day 1, vincristine 1. 4 mg/m2 i.v. on day 8, procarbazine 100 mg/m2 orally (p.o.) on days 1–7, prednisone 100 mg/m2 p.o. on days 1–14. In these patients, FU-1 PET/MR was performed on day 3 follow- ing administration of the third BEACOPP dose. Maximum and mean standardized [18F]FDG uptake values (SUVmax, SUVmean) and metabolic tumour volumes (MTV, cm3) of these lesions were measured on the pretherapy (baseline)and the two follow-up (FU-1, FU-2) [18F]FDG PET/MR examinations, based on isocontour volumes of inter- est (VOIs) constructed using a 41% SUVmax threshold (as recommended by the European Association of Nuclear Medicine, and applied in previous studies [26–28]), using the syngo Multimodality Workplace environment (Siemens). Similarly, minimum and mean ADCs (ADCmin, ADCmean, ×10−3 mm2/s) of the target lesions were measured on pretherapy and follow-up ADC maps, based on manually de- fined three-dimensional VOIs, using the syngo environment (Siemens). Finally, manually defined VOIs were constructed on the T1-weighted images (guided, if necessary, by DWI), again using the syngo environment, to measure the morpho- logical volumes (VOL, cm3) of the target lesions at baseline, FU-1 and FU-2. For focal bone and bone marrow lesions, which frequently remain visible as structural defects even in the absence of viable disease, MTVs were calculated, but not VOLs; whereas for diffuse bone marrow involvement, neither MTVs nor VOLs could be obtained due to the lack of clearly defined margins. For diffuse bone marrow involvement, SUVs and ADCs were extracted from spherical VOIs with a 1-cm diameter that were placed in pelvic bone marrow, avoiding the site of the biopsy, where reactive changes are known to occur. In DLBCL patients, the choice between R-CHOP and DA- EPOCH-R treatments was based on each patient’s risk profile. High-risk patients with an IPI of >3 and patients with double- hit lymphomas were assigned to treatment with DA-EPOCH- R, whereas patients with an IPI of 1–3 were treated with R- CHOP. An R-CHOP cycle consisted of rituximab 375 mg/m2 i.v. Image analysis All [18F]FDG PET/MR examinations were evaluated by a board-certified radiologist and a board-certified nuclear med- icine physician side-by-side to ensure that the same lesions were chosen for the quantitative analysis on [18F]FDG PET and DWI. The largest nodal or extranodal lymphoma mani- festations of each involved anatomic region (with a specified maximum of five regions) that were visible on both [18F]FDG PET images (with a clear, focal, nonphysiological tracer accu- mulation) and DWI images (with a clear diffusion restriction, according to previously published criteria for malignancy [11]) were defined as target lesions on pretherapy [18F]FDG PET/MR, provided that they showed a long-axis lesion diam- eter of >1.0 cm for extranodal manifestations, or >1.5 cm for nodal manifestations on the T1-weighted or DWI images, as recommended in the Lugano/ICML guidelines [1]. Only for diffuse bone marrow involvement, for which histological ver- ification prior to baseline imaging was required, could no size criteria be applied due to the lack of clear margins. Materials and methods separation into air, lungs, fat, and soft tissue) with a repetition time (TR) of 3.6 ms and echo times (TE) of 1.23 and 2.46 ms, one average, a 10° flip angle, a 79 × 192 matrix with a 328 × 500 mm FOV, and a 3-mm slice thickness with a 0.6-mm gap. An axial two-point Dixon T1-weighted VIBE 3D sequence was used for anatom- ic evaluation with a TR of 4.02 ms and TEs of 1.23 and 2.46 ms, a 10° flip angle, a 296 × 430 mm FOVand an image matrix of 154 × 320 with a 3-mm slice thickness with a 20% gap. In addition, an axial echoplanar DWI sequence with spectral adiabatic inversion recovery (SPAIR) was used dur- ing free-breathing for the entire anatomy, using b-values of 50 and 800, a TR of 6,800 ms and a TE of 63 ms, six averages and one echo, a 180° flip angle, a 168 × 104 matrix with a 440 × 340 mm FOV; and a 6-mm slice thickness with a 1.2- mm gap. ADC maps were generated automatically by the operational software supplied with the PET/MR scanner. In patients with FL and MCL, an R-BENDA cycle consisted of rituximab 375 mg/m2 on day 1 and bendamustine 90 mg/m2 on days 2 and 3. In these patients, FU-1 PET/MR was performed on day 3 following administration of the sec- ond bendamustine dose. Materials and methods Patients with histologically proven, previously untreated ma- lignant lymphoma who were referred to our institution for routine pretherapy [18F]FDG PET/MR were eligible for par- ticipation in our prospective exploratory study. Ethics com- mittee approval and written informed consent from all patients were obtained prior to imaging. Inclusion criteria were the presence of one of the following histological lymphoma sub- types, as verified by a reference pathologist who analysed tissue samples (obtained by biopsy or during surgery) accord- ing to the current WHO classification of haematological and [18F]FDG PET was performed 60 min after intravenous administration of a target dose of 3 MBq/kg (minimum injected dose 200 MBq) [18F]FDG with 5 min per bed posi- tion, three iterations and 21 subsets, a 4.2-mm slice thickness, and a 172 × 172 matrix, using the point spread function-based reconstruction algorithm HD-PET (Siemens). A coronal two- Eur J Nucl Med Mol Imaging (2018) 45:931–940 933 point Dixon T1-weighted VIBE 3D sequence was used for PET attenuation correction (i.e. separation into air, lungs, fat, and soft tissue) with a repetition time (TR) of 3.6 ms and echo times (TE) of 1.23 and 2.46 ms, one average, a 10° flip angle, a 79 × 192 matrix with a 328 × 500 mm FOV, and a 3-mm slice thickness with a 0.6-mm gap. An axial two-point Dixon T1-weighted VIBE 3D sequence was used for anatom- ic evaluation with a TR of 4.02 ms and TEs of 1.23 and 2.46 ms, a 10° flip angle, a 296 × 430 mm FOVand an image matrix of 154 × 320 with a 3-mm slice thickness with a 20% gap. In addition, an axial echoplanar DWI sequence with spectral adiabatic inversion recovery (SPAIR) was used dur- ing free-breathing for the entire anatomy, using b-values of 50 and 800, a TR of 6,800 ms and a TE of 63 ms, six averages and one echo, a 180° flip angle, a 168 × 104 matrix with a 440 × 340 mm FOV; and a 6-mm slice thickness with a 1.2- mm gap. ADC maps were generated automatically by the operational software supplied with the PET/MR scanner. point Dixon T1-weighted VIBE 3D sequence was used for PET attenuation correction (i.e. Treatment protocols on day 1, and cyclophosphamide 750 mg/m2 i.v., doxoru- bicin 50 mg/m2 i.v. and a vincristine 2 mg i.v. bolus on day 1 along with prednisone 50 mg/m2 p.o. on days 1–5. These patients had already received the full immunochemotherapy cycle (with the exception of prednisone, which was continued until day 5). In these patients, FU-1 PET/MR was performed on day 3. A DA-EPOCH-R cycle consisted of rituximab 375 mg/m2 i.v. on day 1, continuous infusion of etoposide 50 mg/m2, doxorubicin 10 mg/m2 and vincristine 0.4 mg/m2 on days 1–4, and cyclophosphamide 750 mg/m2 i.v. on day 5, along with 60 mg/m2 prednisone p.o. twice daily on days 1–5. In these patients, FU-1 PET/MR was performed on day 3 following administration of the third chemotherapy dose. In DLBCL patients, the choice between R-CHOP and DA- EPOCH-R treatments was based on each patient’s risk profile. 934 Eur J Nucl Med Mol Imaging (2018) 45:931–940 (R-CHOP arm) underwent [18F]FDG PET/MR at baseline and FU-2, but not at FU-1. A total of 166 target lesions (134 nodal, 32 extranodal; Table 1) were analysed. If a lesion was no longer visible on the follow-up [18F]FDG PET images or ADC map, but was still visible as a residual mass/lesion on the respective T1-weighted MRI images, the latter were used for VOI definition and then copied. If a lesion was no longer visible on follow-up [18F]FDG PET images, the ADC map, or T1-weighted MRI images, SUVs, MTVs and VOLs were set to zero, whereas for ADCs for which no ab- solute maximum value existed, a spherical VOI with a 1-cm diameter was placed in the region where the lesion was ob- served at baseline taking care not to include areas of physio- logically low ADCs (e.g. bowel contents). (R-CHOP arm) underwent [18F]FDG PET/MR at baseline and FU-2, but not at FU-1. A total of 166 target lesions (134 nodal, 32 extranodal; Table 1) were analysed. On a per-lesion basis, pooled SUVmax, SUVmean, ADCmin and ADCmean values from all lymphoma subtypes differed significantly between baseline and FU-1 (−46.8%, −33.3%, +20.3% and +14%, respectively; P < 0.001), be- tween baseline and FU-2 (−65.1%, −49%, +50.7% and +32.4%, respectively; P < 0.001), and between FU-1 and FU-2 (−34.5%, −23.5%, +24.2% and +15.2%, respectively; P < 0.001). Statistical analysis Mean rates of change in SUV, ADC, MTVand VOL between baseline and follow-up examinations (ΔSUVmax, ΔSUVmean, ΔADCmin, ΔADCmean, ΔMTV and ΔVOL) were determined for the target lesions, and also on a per- patient basis, using estimated marginal means of the patients’ target lesions. Lesion-based Pearson correlation coefficients (r) between ΔSUV and ΔADC were calculated. Multilevel analyses with an unstructured covariance matrix which showed the best model fit according to the Akaike information criterion were performed on a per-lesion basis; descriptive statistics were based on model estimates. Bonferroni- corrected post-hoc tests were used for pair-wise lesion-based comparisons of SUV, ADC, MTV and VOL among the three time-points (baseline, FU-1, and FU-2) based on pooled data from all lymphoma subgroups. Repeated-measures analysis of variance with post-hoc tests was used to compare baseline and FU-1 and FU-2 patient-based SUVs, ADCs, MTVs and VOLs using pooled data from all lymphoma subgroups. In the DLBCL subgroup, patient-based rates of change of the imag- ing parameters between baseline and FU-1 and between base- line and FU-2 were compared between the two treatment arms (R-CHOP vs. DA-EPOCH-R). The specified level of signifi- cance was P ≤0.05 for all tests. All statistical tests were per- formed using IBM SPSS Statistics 21.0 (IBM Corp., Armonk, NY). On a per-patient basis, pooled SUVmax, SUVmean, ADCmin and ADCmean values from all lymphoma subtypes differed significantly between baseline and FU-1 (−54.9%, −41.7%, +27.8% and +17.8%, respectively; P < 0.001), and between baseline and FU-2 (−70.7%, −55.9%, +58% and +34.4%, respectively; P < 0.001). Treatment protocols MTV also differed significantly between baseline and FU-1 (−46%; P = 0.002), and between baseline and FU-2 (−61.1%; P < 0.001), but not between FU-1 and FU-2 (−28%; P = 0.79) whereas VOL differed significantly only between baseline and FU-2 (−24.2%; P < 0.001), and not between baseline and FU-1 (−12.8%; P = 0.079), or between FU-1 and FU-2 (−13.1%; P = 0.154; Table 2, Figs. 1 and 2). Statistical analysis SUVmean, ADCmin and ADCmean, but not SUVmax, differed significantly between FU-1 and FU-2 (−24.6%, +23.6%, +14.1% and −35%, Table 1 Absolute numbers of analysed target lesions by anatomic region Region HL DLBCL FL MCL Total Nodal Cervical 1 2 2 4 9 Infraclavicular 4 2 0 0 6 Axillary 0 6 1 1 8 Mediastinal 14 4 2 2 22 Hilar 8 2 0 1 11 Mesenteric 2 8 2 3 15 Periaortic 1 8 4 4 17 Pelvic 0 8 13 8 29 Inguinal 0 5 8 4 17 Extranodal Waldeyer’s ring 0 1 0 0 1 Lungs 0 1 0 4 5 Spleen 1 0 0 0 1 Stomach 0 2 0 1 3 Small intestine 0 1 0 0 1 Large intestine 0 1 0 0 1 Bones 1 6 5 0 12 Soft tissues 0 2 0 0 2 Other 0 5 1 0 6 HL Hodgkin lymphoma, DLBCL diffuse large B-cell lymphoma. FL follicular lymphoma, MCL mantle cell lymphoma Table 1 Absolute numbers of analysed target lesions by anatomic region R i HL DLBCL FL MCL T t l Table 1 Absolute numbers of analysed target lesions by anatomic region HL Hodgkin lymphoma, DLBCL diffuse large B-cell lymphoma. FL follicular lymphoma MCL mantle cell lymphoma HL Hodgkin lymphoma, DLBCL diffuse large B-cell lymphoma. FL follicular lymphoma, MCL mantle cell lymphoma Results Of 65 patients who fulfilled the inclusion criteria, seven were excluded at baseline due to elevated blood glucose levels (>150 mg/dL). Thus, a total of 58 patients (28 women and 30 men; mean age 58.9 ± 17.6 years, range, 25–92 years) ful- filled the criteria for participation in the study. Of these 58 patients, 11 were diagnosed with HL, 25 with DLBCL (15 in the R-CHOP arm, and 10 in the DA-EPOCH-R arm), 12 with FL, and 10 with MCL. One patient with FL and one patient with MCL underwent [18F]FDG PET/MR at baseline and FU-1, but not at FU-2; whereas one patient with DLBCL HL Hodgkin lymphoma, DLBCL diffuse large B-cell lymphoma. FL follicular lymphoma, MCL mantle cell lymphoma Eur J Nucl Med Mol Imaging (2018) 45:931–940 935 Table 2 Lesion-based pooled arithmetic means and 95% confidence intervals of standardized uptake values (SUV), apparent diffusion coefficients (ADC), metabolic tumour volumes (MTV), and morphological volumes (VOL) before (baseline), 48–72 h after, and 1 week after treatment initiation Table 2 Lesion-based pooled arithmetic means and 95% confidence intervals of standardized uptake values (SUV), apparent diffusion coefficients (ADC), metabolic tumour volumes (MTV), and morphological volumes (VOL) before (baseline), 48–72 h after, and 1 week after treatment initiation ( ), ( ), p g ( ) ( ), , Baseline 48–72 h 1 week SUVmax 10.9 (10.1–11.8) 5.8 (5.0–6.7) 3.8 (3.0–4.7) SUVmean 5.1 (0–13.8) 3.4 (0–12.1) 2.6 (0–11.0) ADCmin (×10−3 mm2/s) 518.6 (409.5–627.7) 629.2 (520.1–738.4) 781.7 (672.4–891.0) ADCmean (×10−3 mm2/s) 925.0 (898.9–971.1) 1,054.6 (1,018.5–1,090.8) 1,215.2 (1,178.6–1,251.7) MTV (cm3) 75.4 (0–438.4) 40.7 (0–395.2) 29.3 (0–343.8) VOL (cm3) 103.7 (0–422.2) 90.4 (0–408.9) 78.6 (0–397.1) A closer look at the rates of change of the imaging param- eters clearly suggests that, regardless of histological subtype, glucose metabolism-based measures (SUVand MTV) show a more pronounced response to treatment during the first 48– 72 h after treatment initiation, whereas the changes between 72 h and 1 week appeared to be less pronounced. For diffusivity/cell density, as reflected by ADCs, the opposite effect was generally observed, with a less pronounced re- sponse to treatment during the first 48–72 h after treatment initiation, and a more pronounced response between 72 h and 1 week; only DLBCL, in which changes in ADC over time more closely resembled those in SUV, differed in this regard. Results Significant changes in morphological lesion volumes were also observed at these early time-points, although they were only small-to-moderate, particularly during the first 48–72 h after treatment initiation, when, contrary to changes after 1 week, they did not reach statistical significance. This very early reduction in lesion size under treatment confirms the results of Horger et al. [24], who found a significant reduction in lesion size in a small sample of responding lymphoma patients 1 week after treatment initiation. The fact that, in the latter study, lesion shrinkage was less pronounced than in the present study may possibly be explained by the fact that transaxial lesion diameters, instead of volumes, were used, and also the study population of Horger et al. included only three patients with DLBCL, which was the subtype that showed by far the largest volume decreases in our study. respectively; P = 0.015, P < 0.001, P < 0.001 and P = 0.094). MTV also differed significantly between baseline and FU-1 (−42.9%; P = 0.01), and between baseline and FU-2 (−53.9%; P = 0.001), but not between FU-1 and FU-2 (−19.2%; P = 1.0), whereas VOL differed significantly only between base- line and FU-2 (−18.7%; P = 0.002), and not between baseline and FU-1 (−12%; P = 0.069), or between FU-1 and FU-2 (−7.6%; P = 0.62). Line graphs revealed major differences in treatment response between individual patients, and even be- tween patients with the same lymphoma subtype and treat- ment regimen (Online Resource 1). In the DLBCL group, all patient-based imaging parameter changes from baseline to FU-1 except for ADCmin differed significantly between the two treatment arms (R-CHOP vs. DA-EPOCH-R), whereas only changes in SUVmean and VOL between baseline and FU-2 differed significantly be- tween the two treatment arms (Table 3, Online Resource 2). Weak, statistically significant negative correlations were observed between lesion-based ΔSUVmax and ΔADCmin (baseline to FU-1 r = −0.32, P < 0.001; baseline to FU-2 r = −0.32, P < 0.001), whereas moderate significant negative cor- relations were observed between ΔSUVmean and ΔADCmean (baseline to FU-1 r = −0.54, P < 0.001; baseline to FU-2 r = −0.41, P < 0.001). Discussion Clear differences in treatment responses and associated changes in imaging parameters were seen among the four histological lymphoma subtypes in our study. DLBCL showed the largest changes, followed by HL, followed by MCL, with FL showing the smallest changes. On the other hand, only patients with FL and MCL received the same treat- ment regimen, whereas treatment regimens differed among those with DLBCL and HL, and hence the latter two subtypes cannot be directly compared with each other, or with FL and MCL. Thus, while it is striking that the different degrees of treatment response among the four histological subtypes ap- pear to reflect the order of aggressiveness of these lympho- mas, the main finding on this point is that both glucose The observed decrease in lymphoma glucose metabolism (i.e. FDG uptake, measured in terms of SUV and MTV) and in- crease in tissue diffusivity (i.e. ADC, chiefly reflecting cell density) in our study clearly suggest the presence of treatment effects, which can be measured in vivo by [18F]FDG PET/MR as early as 48–72 h after treatment initiation. Further imaging parameter changes reflecting treatment effects clearly oc- curred during the first week. Notably, significant changes dur- ing the first week after treatment initiation were found not only in SUVs (and the associated MTVs) and ADCs, but also in morphological volumes, albeit to a lesser degree. 936 Eur J Nucl Med Mol Imaging (2018) 45:931–940 Eur J Nucl Med Mol Imaging (2018) 45:931–940 936 Eur J Nucl Med Mol Imaging (2018) 45:931–940 937 Table 3 Patient-based mean rates of change (%) of the six imaging parameters relative to baseline for the two DLBCL treatment arms (R- CHOP, DA-EPOCH-R) ƒFig. Discussion 1 Lesion-based rates of change of standardized [18F]FDG uptake values (SUVmax, SUVmean), apparent diffusion coefficients (ADCmin, ADCmean), metabolic tumour volumes (MTV) and morphological tumour volumes (VOL) between baseline and 1 week separately for the four histological lymphoma subtypes R-CHOP DA-EPOCH-R P value ΔSUVmax 48–72 h −69.9 ± 27.9 −41.1 ± 17.2 0.005 1 week −78.7 ± 23.2 −67.7 ± 18.4 0.20 ΔSUVmean 48–72 h −65.4 ± 23.6 −34.0 ± 13.2 <0.001 1 week −74.8 ± 24.4 −57.0 ± 21.3 0.007 ΔADCmin 48–72 h 65.9 ± 71.9 50.7 ± 49.9 0.55 1 week 103.8 ± 83.3 93.5 ± 79.0 0.76 ΔADCmean 48–72 h 41.8 ± 28.7 17.0 ± 18.0 0.016 1 week 57.9 ± 32.7 35.4 ± 23.9 0.059 ΔMTV 48–72 h −73.2 ± 30.5 −41.2 ± 32.1 0.023 1 week −84.6 ± 19.8 −75.5 ± 18.3 0.25 ΔVOL 48–72 h −38.1 ± 28.6 −7.9 ± 17.3 0.005 1 week −60.2 ± 24.0 −30.2 ± 24.2 0.009 metabolism and cell density of the lesions show a marked response to their respective standard therapy regimens in all examined lymphoma subtypes as early as 48–72 h after treat- ment initiation. Notably, in DLBCL, for which two different treatment reg- imens (R-CHOP and DA-EPOCH-R) were used, the observed differences between the two treatment groups point towards a stronger and/or more rapid effect of R-CHOP on glucose me- tabolism and diffusivity/cell density of the lymphomas in this early phase, which seems plausible because, with R-CHOP, both rituximab and the chemotherapeutic agents were admin- istered on day 1, whereas with DA-EPOCH-R, rituximab was administered on day 1, and the chemotherapeutic agents were administered over 5 days. Thus, at the first PET/MR follow- up at 48–72 h after treatment initiation, patients in the DA- EPOCH-R arm had only received a fraction of the total che- motherapy dose, and lower peak levels. However, assignment to the two treatment groups was based on IPI score or the presence of double-hit lymphomas, i.e. only high-risk patients were chosen for treatment with DA-EPOCH-R, in accordance with previous studies [29, 30]. As a consequence, it is quite possible that the clinical entities in themselves are not compa- rable, and this led to, or at least contributed to, the observed differences in ultra-early responses on [18F]FDG PET/MR. Discussion Secondly, a possible treatment-induced inflammatory reaction would not explain the obvious differences in ADC increase among the four histological lymphoma subtypes, as this would require a histology-dependent or treatment- dependent degree of inflammation for which there is presently no evidence; even more so as three of the lymphoma sub- groups received rituximab-based treatment. Here, it is also important to note that treatment regimens for HL and DLBCL included the corticosteroid prednisone, which has been shown to lead to suppression of [18F]FDG uptake caused by the inflammatory influx early (i.e. on day 9) after cytotoxic treatment [32]. Thus, the decrease in [18F]FDG uptake early after treatment that was observed at the same time as the in- crease in diffusivity (i.e. ADCs), also supports our theory that DWI does indeed capture treatment-induced cell necrosis, rather than inflammatory changes. This is also in agreement with the results of Papaevangelou et al. [31], who observed significant ADC increases in the presence of necrosis in hu- man colon carcinoma xenografts on days 3 and 5 after treat- ment with irinotecan. in SUVand MTV, compared with the changes in ADC, espe- cially since doxorubicin was also part of the DLBCL and HL treatment regimens in the present study. However, stunning would not have affected ADC and lesion volume changes, which showed the same general trends including differences in response among lymphoma subtypes. Thus, the observed reduction in FDG uptake after treatment initiation can at least not entirely be attributed to stunning and anti-inflammatory treatment effects. Only in three patients was an atypical be- haviour observed (initial strong decreases in SUV and MTV from baseline to 48–72 h, followed by increases in SUV and MTV from 48–72 h to 1 week). Unfortunately, in these three patients, we were not able to distinguish between an initial stunning effect that was captured by [18F]FDG PET at 48– 72 h and an inflammatory Bflare^ reaction that occurred be- tween 48–72 h and 1 week, as this would have required his- tological evaluation. Several studies have demonstrated the prognostic value of interim [18F]-FDG PET, typically performed after two to four therapy cycles, in different lymphoma subtypes, most con- vincingly in HL [14, 34]. For interim DWI, on the other hand, relatively few data are presently available with regard to its possible value for clinical outcome prediction. Discussion Based on a small series of 14 lymphoma patients (12 with DLBCL), De Paepe et al. found that changes in ADC between baseline and follow-up after 2 weeks (as well as 4 weeks) of treatment were significantly correlated with progression-free survival [35]. In a mixed population of 20 patients, 7 with HL and 13 with NHL including aggressive and indolent subtypes, Horger et al. found that changes in ADC after 1 week of treatment predicted outcome as assessed 6 months after the end of treat- ment [24]. Finally, in a series of 15 patients with FDG-avid MALT lymphoma, Mayerhoefer et al. found that changes in ADC between baseline and interim imaging differed signifi- cantly between patients with complete remission and those with residual disease at the end of treatment [10]. To our knowledge, no long-term data regarding the predictive value of therapy-induced ADC changes in lymphoma are presently available. Conversely, the decrease in FDG uptake observed at the ultra-early follow-up in our study was probably also caused, at least in part, by cell death, as indicated by the simultaneous increase in diffusivity and lesion shrinkage. Nevertheless, par- ticularly in the context of HL with its pronounced hyperplastic lymphoid reaction, the anti-inflammatory and lympholytic ef- fects of prednisone on [18F]FDG uptake and on lesion volume must be taken into account. Furthermore, a previous in vitro study in breast cancer cells established the concept of treatment-induced Bstunning^ (i.e. a discrepancy between FDG uptake and viable cell number) 24 h after doxorubicin treatment, and thus before the earliest follow-up at 48–72 h after treatment initiation in our study [33]. In the latter study, the stunning effect was explained by declines in GLUT-1 and HKII levels [33]. Similarly, in a murine lymphoma model, a reduction in FDG uptake 16 h after initiation of etoposide treatment was explained by a decrease in the plasma mem- brane presentation of GLUT-1 and GLUT-3 [23]. It is there- fore difficult to judge whether, and to what degree, a possible stunning effect (in combination with the anti-inflammatory effect of prednisone in HL and DLBCL) may have contributed to the observed reduction in FDG uptake in our study, and whether it could partly explain the more pronounced changes Our study had several limitations. Discussion As mentioned above, DWI enables (indirect) assessment of cell density through assessment of tissue diffusivity, based on the underlying mechanism that in hypercellular tumours such as lymphoma, the extracellular space is compressed, limiting the local Brownian motion of water molecules. As a Fig. 2 A 68-year-old woman with DLBCL of the left adrenal gland (blue arrows). The lymphoma shows intense glucose metabolism (high [18F]FDG uptake; high SUVs) at baseline on the axial fused [18F]FDG PET/MR image, and a clear diffusion restriction on the DWI image (high signal) and the ADC map (low signal). The PET/MR image at 48–72 h after treatment initiation (R-CHOP) shows an almost complete reduction in [18F]FDG uptake (reduced glucose metabolism) and a clear increase in tissue diffusivity (increased ADC, indicating reduced cell density) at this time-point. The PET/MR image at 1 week after treatment initiation shows an additional moderate decrease in lesion size relative to the baseline image Fig. 2 A 68-year-old woman with DLBCL of the left adrenal gland (blue arrows). The lymphoma shows intense glucose metabolism (high [18F]FDG uptake; high SUVs) at baseline on the axial fused [18F]FDG PET/MR image, and a clear diffusion restriction on the DWI image (high signal) and the ADC map (low signal). The PET/MR image at 48–72 h after treatment initiation (R-CHOP) shows an almost complete reduction in [18F]FDG uptake (reduced glucose metabolism) and a clear increase in tissue diffusivity (increased ADC, indicating reduced cell density) at this time-point. The PET/MR image at 1 week after treatment initiation shows an additional moderate decrease in lesion size relative to the baseline image Eur J Nucl Med Mol Imaging (2018) 45:931–940 938 consequence, DWI may be used to capture treatment effects in terms of cell necrosis [31]. It is highly unlikely that, in our study, an isolated treatment-induced inflammatory fluid influx (which might have led to a T2-shine-through effect), rather than cell necrosis, was responsible for the observed ADC in- crease after treatment for several reasons. Firstly, a fluid influx in the absence of cell necrosis would lead to an increase in lesion size, which we did not observe. On the contrary, there was a small-to-moderate statistically significant decrease in morphological lesion volumes, which is indicative of cell ne- crosis. Discussion No posttreatment histo- logical data were available for comparison with the quantita- tive imaging parameters of the target lesions, because biopsies are rarely performed in routine clinical practice, and purely study-related biopsies were not performed because of ethical considerations. For calculation of MTVs, we used a 41% SUVmax cut-off, as previously recommended [26–28]. However, it is well-known that major differences in glucose metabolism (and the associated [18F]FDG uptake) exist be- tween different lymphoma subtypes [36, 37]. We therefore cannot rule out the possibility that the use of a 41% SUVmax threshold led to a small-to-moderate MTVoveresti- mation in patients with indolent lymphomas (i.e. FL, MCL) which frequently show less-pronounced FDG uptake. 939 Eur J Nucl Med Mol Imaging (2018) 45:931–940 In conclusion, our preliminary data clearly suggest that patients with HL and common NHL subtypes may show a s u b s t a n t i a l r e s p o n s e t o s t a n d a r d c h e m o - o r immunochemotherapy as early as during the first 48–72 h after treatment initiation, which can be captured in vivo by [18F]FDG PET/MR. Treatment-induced changes in glucose metabolism appear to precede changes in diffusivity/cell den- sity of lymphoma tissue, which in turn precede changes in lesion volume. Flare reactions that may be detrimental to [18F]FDG PETappear to be rare events during the investigated time period early after treatment initiation. 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Trends in fatalities from road traffic injuries and associated factors of patients admitted to the emergency department of St. Luke Hospital, South West Shewa Zone, Ethiopia
Frontiers in disaster and emergency medicine
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TYPE Original Research PUBLISHED 12 January 2024 DOI 10.3389/femer.2023.1328817 TYPE Original Research PUBLISHED 12 January 2024 DOI 10.3389/femer.2023.1328817 TYPE Original Research PUBLISHED 12 January 2024 DOI 10.3389/femer.2023.1328817 road trafc injury, road trafc injury fatality, factors, South West Shewa, Ethiopia Trends in fatalities from road trafc injuries and associated factors of patients admitted to the emergency department of St. Luke Hospital, South West Shewa Zone, Ethiopia OPEN ACCESS EDITED BY Hao Wang, JPS Health Network, United States REVIEWED BY Nobuhiro Sato, Niigata City General Hospital, Japan Ismet Çelebi, Gazi University, Türkiye *CORRESPONDENCE Getahun Fetensa fetensagetahun@gmail.com RECEIVED 27 October 2023 ACCEPTED 27 December 2023 PUBLISHED 12 January 2024 CITATION Beyera TM, Serbessa SD, Fetensa G and Woldemichael K (2024) Trends in fatalities from road trafc injuries and associated factors of patients admitted to the emergency department of St. Luke Hospital, South West Shewa Zone, Ethiopia. Front. Disaster Emerg. Med. 1:1328817. d i 10 3389/f 2023 1328817 Teshale Merga Beyera1, Sheleme Duga Serbessa2, Getahun Fetensa3,4* and Kifle Woldemichael5 1Department of South West Shewa Zonal Health, Oromia Health Bureau, Addis Ababa, Ethiopia, 2Waliso Woreda Health Ofce, Oromia Health Bureau, Addis Ababa, Ethiopia, 3Institute of Health Science, Wollega University, Nekemte, Ethiopia, 4Department of Health Behaviour and Society, Faculty of Public Health, Jimma Institute of Health, Jimma Medical Center, Jimma, Ethiopia, 5Department of Epidemiology, Faculty of Public Health, Jimma Institute of Health, Jimma University, Jimma, Ethiopia Beyera TM, Serbessa SD, Fetensa G and Woldemichael K (2024) Trends in fatalities from road trafc injuries and associated factors of patients admitted to the emergency department of St. Luke Hospital, South West Shewa Zone, Ethiopia. Front. Disaster Emerg. Med. 1:1328817. doi: 10.3389/femer.2023.1328817 Introduction: Road trafc injuries are a major global public health problem. They are fatal or non-fatal injuries resulting from a road trafc accident involving at least one moving vehicle on a path or road open to public trafc. Therefore, this study aimed to determine the trends in fatalities from road trafc injuries and the associated factors of patients admitted to the emergency department of St. Luke Hospital. Eligibility criteria Inclusion criteria: ­ All road traffic–injured patients who attended the emergency department at St. Luke Hospital between January 1, 2018, and December 31, 2018. The 2015 WHO report on road safety highlights that the situation is the worst in low-income countries and that there is a disproportionate number of deaths relative to the lower level of motorization. The African region continues to have the highest rates of road traffic deaths, while the lowest rates are in the European region (2). RTIs pose a high burden over the entire life course of people in sub-Saharan Africa, impacting not just young adults but also children and older adults. Nigeria, Ethiopia, South Africa, and Sudan together account for half of the RTI deaths in sub-Saharan Africa (15). Exclusion criteria: ­ Those road traffic–injured patients who were transferred immediately to other hospitals and without outcome variables were excluded. ­ The information indicating that patients died instantly at the scene was excluded. ­ The information indicating that patients died instantly at the scene was excluded. The Ethiopia Demographic Health Survey 2016, indicated that RTAs in Ethiopia accounted for the highest percentages of accidental injuries and deaths, accounting for 23% of all injuries (16). Therefore, this study aimed to identify the trends in fatalities from RTIs and the associated factors of patients admitted to the emergency department of St. Luke Catholic Hospital, South West Shewa Zone, Central Ethiopia, during 2019. COPYRIGHT © 2024 Beyera, Serbessa, Fetensa and Woldemichael. 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. Methods: An institution-based cross-sectional study was conducted from July 10 to 30, 2019. The study on the trends in road trafc injuries was conducted monthly bases from January 1 to December 31, 2018. A total of 364 road trafc– injured victim records were included. A simple random sampling technique was used for the record selection. A structured data abstraction format was used to gather the required data. Binary logistic regression and multivariate logistic regression were used to determine the association between the outcome and explanatory variables. Adjusted odds ratios (AORs) at 95% confidence intervals were used to report independent variables. Results and conclusions: The average annual fatalities from road trafc injuries was 8.34% deaths/month. The magnitude of road trafc injury fatalities was 8.79%. Of all victims who visited the hospital, 75.5% were male. The median age of the victims was 26 (51.6%), with a range from 6 to 70. The identified risk factors for road trafc injury fatalities were a lack of ambulance for transportation to the hospital after the accident (AOR = 7.093, 95% CI [1.950, 25.793], p = 0.003) and head injury (AOR = 2.515, 95% CI [1.075, 5.885], p = 0.034). Road trafc injury fatalities are an important public health problem that needs special attention. A shortage of ambulance service for transportation and injury sites related to head injury were associated factors. So it is important to establish and strengthen advanced prehospital care and an efective ambulance system for transporting victims with road trafc injuries. road trafc injury, road trafc injury fatality, factors, South West Shewa, Ethiopia Frontiers in Disaster and Emergency Medicine 01 frontiersin.org Beyera et al. Beyera et al. Beyera et al. 10.3389/femer.2023.1328817 Source population All RTI patients who attended the emergency department of St. Luke Hospital from January 1 to December 31, 2018, were considered. For every 10 lives lost to traffic injuries, nine occur in low- and middle-income countries (11). RTIs are a growing public health issue that disproportionately affects vulnerable groups of road users, including the poor (2). Ninety percent of the world’s fatalities on roads occur in low- and middle-income countries, even though these countries have approximately 54% of the world’s vehicles (12). Introduction Road transportation provides great benefits to both nations and individuals by facilitating the movement of goods and people. However, road transportation has also placed a considerable burden on people’s health in the form of road traffic injuries and the consequences of these injuries (1). Although the World Health Organization (WHO) reported the overall trend in the number of fatalities caused by road traffic collisions in low in middle-income countries, it has shown a rising trend since 2010 (2). This study was conducted in St. Luke Catholic Hospital and College of Nursing and Midwifery located in Woliso town of Oromia Regional State from July 10 to 30, 2019. The hospital has 200 beds and serves more than 400 outpatients per day. It is the only referral hospital in the South West Shewa Zone, serving a population of ∼1.3 million people. The outpatient department is fully operational 5 days a week for routine outpatient consultations, and emergency room services are available 24 h every day, with 1 health officer on duty and 3 nurses, 1 surgeon, and 1 internist on call (17). A road traffic injury (RTI) is defined as a fatal or non-fatal injury that occurs as a result of a road traffic crash that occurs on a way or street open to public traffic and involves at least one moving vehicle (3, 4). It is a major global public health problem that occurs due to collisions between vehicles, vehicles and pedestrians, vehicles and animals, or vehicles and geographical or architectural obstacles and has been a leading cause of mortality for many years that requires concerted efforts for effective and sustainable prevention (3, 5–8). Study population All RTI patients who attended the emergency department of St. Luke Hospital who were admitted from January 1 to December 31, 2018, and fulfilled the inclusion criterion. pp y Without immediate action being taken, the global number of deaths and injuries from road traffic accidents (RTAs) was predicted to be the third-leading cause of death by 2020 (13). Deaths from RTAs are also projected to increase from 1.2 million in 2004 to 2.4 million in 2030, primarily due to the increased motor vehicle ownership and use associated with economic growth in low- and middle-income countries (14). Study design An institution-based cross-sectional study was conducted. The factors for the increase in global road traffic injury fatalities are the growing number of motor vehicles, speeding, people not using seat belts and motorcyclists not using helmets, alcohol and other substance use, road type, weather conditions, and other human-related factors (9). However, there are no facilities for pedestrians and cyclists, and the increasing number of motor vehicles generally leads to reductions in walking and cycling (10). Then, the calculated sample size is n = 364. Then, the calculated sample size is n = 364. To determine the required sample size for the second specific objective of this study, different factors significantly associated with RTIs were considered with the following assumptions: 95% confidence level and a power of 80% using the Epi Info software program, version 7.2.2.2. The calculated sample size for selected variables and the maximum sample size are used for the final required sample size (Supplementary Table 1). Data collection tools and procedures The data for the study were extracted from routinely registered client medical records and the emergency department registration book. The data were collected using a pretested format from the sample population according to the inclusion criterion. The data collection tool was adapted from the injury surveillance guideline document developed by the WHO in 2004 (19). The data were collected and supervised by two diploma holders and one degree- holding nurse, respectively. Finally, the required sample size for this study was determined by taking the maximum sample size from the first objective 364 with a magnitude of RTI fatalities. Sampling technique and procedure The study subjects were selected using a simple random sampling technique and were included in the study until the sample size was obtained. First, the card numbers of 866 victims from January 1 to December 31, 2018, were collected from the emergency room registration book to get the main files of the patients from Dependent variable RTI fatality. Dependent variable RTI fatality. FIGURE 1 Schematic presentation of the sampling procedure from January 1 to December 30, 2018, of monthly road trafc–injured patients at St. Luke Hospital, South West Shewa Zone, Oromia. FIGURE 1 Schematic presentation of the sampling procedure from January 1 to December 30, 2018, of monthly road trafc–injured patients at St. Luke Hospital, South West Shewa Zone, Oromia. Sample size determination The sample size was determined based on the single population proportion formula. Based on the study conducted in Dilchora Hospital in 2016, the magnitude of RTI fatalities was 9.4% (18). The Frontiers in Disaster and Emergency Medicine frontiersin.org frontiersin.org 02 Beyera et al. 10.3389/femer.2023.1328817 level of confidence (α) was taken as 95% (z α/2) = 1.96; to maximize the sample size, the margin of error was taken as 0.03, using the equation (Z ∝ 2 )2 ∗p(1−p) d2 , the records room. Next, using the medical record numbers of patients, cards were collected from the card room. Road traffic– injured victims registered from January 1 to December 31, 2018, at the emergency department were listed based on the sequence of their card numbers for each month. To select the charts of victims, a sampling frame was developed from the victims’ registration books, and each RTI patient card was accessed based on a simple random sampling technique. The injured patient charts were selected by a lottery method within the sampling frame (Figure 1). zα/2 is the 95% confidence interval two-tailed test is equal to 1.96, and d is the margin of error, which is taken as 3%, or 0.03. Data processing procedure The collected data were entered into Epi-data Manager 4.4.2 and then exported to SPSS, version 23.0, for statistical analysis. The data were checked for completeness and inconsistencies, cleaned, and coded. The data were then entered into the Epi-Data Manager software, version 4.4.2, and were cleaned and edited. The frequency distribution of the variables was examined for data entry errors. Independent variables Demographic and socioeconomic factors included age, sex, residence, and type of injured person (pedestrian, passenger, driver, motorbike driver). The environmental factor was the mode of transportation to the hospital after the injury occurred. The characteristics of RTI victims during admission included the site of injury, whether first aid was received before arriving at the hospital, the source of referral, and the time between the RTA and admission. Frontiers in Disaster and Emergency Medicine Frontiers in Disaster and Emergency Medicine 03 frontiersin.org Beyera et al. 10.3389/femer.2023.1328817 Data quality management A descriptive analysis was done by computing proportions and summary statistics. Simple frequencies, summary measures, tables, and figures were used to present the data. A binary logistic regression was used to describe the association between each independent variable with the dependent variable. Variables with a p < 0.25 were used for the multivariate logistic regression model to control for the effects of other confounders, and finally, the variables that have an association with road traffic injury fatality were identified based on adjusted odds ratios (AORs), with 95% confidence values and p < 0.05. The variables were entered in the multivariable model using the backward stepwise regression method. Model fitness was checked by using the To ensure the quality of data, the format was pretested using 5% of the sample size, which is 18 records, before the actual data collection. Necessary modifications were made based on the pretest findings. To maintain data quality, 1 day of training was given to data collectors and a supervisor. The completeness and consistency of the data were checked daily by both the supervisor and the principal investigator. Correctly completed formats were collected from data collectors and entered into the Epi-Data Manager software, version 4.4.2. FIGURE 2 Trends in road trafc injuries from January 1 to December 30, 2018, in St. Luke Hospital, South West Shewa Zone, Oromia Region. FIGURE 2 Trends in road trafc injuries from January 1 to December 30, 2018, in St. Luke Hospital, South West Shewa Zone, Oromia Region. Frontiers in Disaster and Emergency Medicine Results A total of 364 RTI victims were included in the study, making the response rate of 100%. Of all victims who visited the hospital, 275 (75.5%) were male. The median age of victims was 26, with a range from 6 to 70. The highest number of victims was 188 (51.6%) in the 15–29-year-old age group, followed by those in the 30– 44-year-old age group (92, 25.3%). RTA victims presenting to the hospital more commonly occurred as victims coming from outside Woliso (242, 66.5%) than from Woliso (122, 33.5%). Of the total victims of RTA participants in the study, 177 (48.6%) were pedestrians, followed by passengers and motorbike drivers, which accounted for 130 (35.7%) and 49 (13.5%), respectively. The highest number of RTIs occurred on weekdays (136, 37.36%). From a total of 364 cases of RTIs, only 16 (4.3%) were transported by ambulance to the hospital, whereas 11 (3%) and 337 (92.6%) were transported to the hospital by police car and another method, respectively (Table 1). Ethical consideration Ethical clearance was secured from the institutional review board (IRB) of the Institute of Health of Jimma University on February 27, 2019, reference number IHRPGD/181/2019. The IRB of the institute waived the informed consent as the study does not directly work with study participants. No informed consent was obtained from subjects and/or their legal guardian(s) as the study used secondary data. A support letter was written from the South West Shewa Zonal Health Office to St. Luke Hospital. Then a letter of permission was obtained from St. Luke Hospital. All methods were carried out according to relevant guidelines and regulations. To ensure confidentiality, names and other identifiers of patients, physicians, and other health care workers’ recommendations were not recorded in the data abstraction format. Characteristics of RTI victims during admission RTA, road traffic accident. Of the total RTA victims included in this study, 199 (54.7%) were admitted to the emergency department of the hospital within less than 1 h, and 165 (45.3%) were admitted 1–12 h after the accident. Of those admitted to the hospital, 27 (7.7%), 128 (35.2%), and 208 (57.1%) were referred from the hospital, a health center, or did not have a referral, respectively. Of the admitted RTA victims, 191 (52.5%) received first aid before they were sent to the hospital. Frontiers in Disaster and Emergency Medicine Frontiers in Disaster and Emergency Medicine 04 frontiersin.org Beyera et al. 10.3389/femer.2023.1328817 TABLE 1 General characteristics of the study population from January 1 to December 30, 2018, in St. Luke Hospital, South West Shewa Zone, Oromia Region. TABLE 1 General characteristics of the study population from January 1 to December 30, 2018, in St. Luke Hospital, South West Shewa Zone, Oromia Region. Hosmer–Lemeshow goodness-of-fit test. Finally, AORs and 95% confidence intervals were used to report the independent variables associated with RTI fatalities. Variables Frequency (n = 364) Percentage (%) Age 5–14 25 6.9 15–29 188 51.6 30–44 92 25.3 45–59 40 11.0 60 and above 19 5.2 Sex Male 275 75.5 Female 89 24.5 Place of residence From Woliso 122 33.5 Outside Woliso 242 66.5 Day of the week Weekend 136 37.36 Weekday 228 62.64 Head injury Yes 273 75.0 No 91 25.0 Upper extremity injury Yes 103 28.3 No 261 71.7 Chest injury Yes 45 12.4 No 319 87.6 Abdominal injury Yes 83 22.8 No 281 77.2 Pelvic injury Yes 27 7.4 No 337 92.6 Lower extremity injury Yes 161 44.2 No 203 55.8 Spinal cord injury Yes 62 17.0 No 302 83.0 Unknown Yes 14 3.8 No 350 96.2 RTA victim type Pedestrian 179 49 Passenger 131 36 Driver 7 2 Motorbike driver 47 13 RTA, road traffic accident. Trends in RTI fatalities The trends in RTI fatalities vary from month to month; the highest number of road traffic injuries was recorded in November, followed by August and October 2018. From January to December 2018, 866 RTIs occurred. The trends in RTI fatalities also varied seasonally (Figure 2). According to the injury site of the victim, head injuries accounted for the highest (196, 53.8%), followed by lower extremity injuries (162, 44.5%). Frontiers in Disaster and Emergency Medicine frontiersin.org frontiersin.org 05 10.3389/femer.2023.1328817 Beyera et al. TABLE 2 Cross-tabulation of RTI fatalities from January 1 to December 30, 2018, in St. Luke Hospital, South West Shewa Zone, Oromia Region. TABLE 2 (Continued) TABLE 2 Cross tabulation of RTI fatalities from January 1 to December 30, 2018, in St. Luke Hospital, South West Shewa Zone, Oromia Region. Factors associated with RTI fatalities A binary logistic regression analysis was undertaken to screen candidate variables for the final model using a p < 0.25. In the binary logistic regression analysis, RTI fatality was significantly associated with variables like victim type, the day the injury occurred, the duration between the occurrence of the injury and the patient reaching the hospital, and the location of injury (head, upper extremity, chest, abdomen, pelvis, lower extremity, and/or spinal cord injury) at a p < 0.05 (Table 3). The results of the multivariable analysis showed that the mode of transportation to the hospital after the injury occurred and a head injury were independently associated with RTI fatalities. Accordingly, fatalities were seven times more likely for those transported by a public transportation facility compared with those transported by ambulance (AOR 7.093, 95% CI [1.950, 25.793]). RTI victims who had a head injury were 2.5-fold more likely to die compared to those who did not have a head injury (AOR 2.515, 95% CI [1.075, 5.885]; Table 4). Trends in RTI fatalities Variables RTI fatality (n = 364) Yes (%) No (%) Residence of injured patients Woliso 7 (5.8) 113 (94.2) Outside Woliso 25 (10.2) 219 (89.8) Gender Male 26 (9.5) 249 (90.5) Female 6 (6.7) 83 (93.3) Age of victims 5–14 3 (12) 22 (88) 15–29 20 (10.5) 170 (89.5) 30–44 7 (7.6) 85 (92.4) 45 and above 2 (3.5) 55 (96.5) Victim type Pedestrian 10 (5.6) 167 (94.4) Passenger 10 (7.7) 120 (92.3) Driver 1 (12.5) 7 (87.5) Motorbike driver 11 (22.4) 38 (77.6) Day of the week Weekday 20 (8.8) 208 (92.2) Weekend 12 (8.8) 124 (92.2) Season injury occurred Winter 7 (8.4) 76 (91.6) Spring 7 (9) 71 (91) Summer 7 (8.2) 78 (91.8) Autumn 11 (9.3) 107 (90.7) Mode of transportation after accident to hospital Ambulance 5 (31.3) 11 (68.8) Police care 1 (9.1) 10 (90.9) Public transport 26 (7.7) 311 (92.3) Duration at which injured patient reached hospital <1 h 12 (6) 187 (94) 1–12 h 20 (12.1) 145 (87.9) Source of referral Hospital 2 (7.1) 26 (92.9) Health center 4 (3.1) 124 (96.9) Without referral 26 (12.5) 182 (87.5) First aid before admitted Yes 7 (18.9) 151 (81.1) No 25 (7.6) 181 (92.4) Head injury Yes 19 (7) 254 (93) No 13 (14.3) 78 (85.7) Upper extremity Yes 4 (3.9) 99 (96.1) No 28 (10.7) 233 (89.3) Chest injury Yes 4 (8.9) 41 (91.1) No 28 (8.8) 291 (91.2) (Continued) Variables RTI fatality (n = 364) Yes (%) No (%) Abdominal injury Yes 10 (12) 73 (88) No 22 (7.8) 259 (92.2) Pelvic injury Yes 4 (14.8) 23 (85.2) No 28 (8.3) 309 (91.7) Lower extremity injury Yes 10 (6.2) 151 (93.8) No 22 (10.8) 181 (89.2) Spinal cord injury Yes 10 (16.1) 52 (83.9) No 22 (7.3) 280 (92.7) Unknown Yes 2 (14.3) 12 (85.7) No 30 (8.6) 320 (91.4) RTI, road traffic injury. Road trafc fatality cross-tabulation Many cases of fatality recorded in the current study occurred outside of the study area (Woliso, 10.2%), males (9.5%) were most often RTI fatality victims, and those in the 15–29-year-old age range experienced the most deaths (10.5%). The most frequent RTI fatality victims were motor vehicle drivers (22.4%), there was no significant variation of fatality among the seasons in Ethiopia in the study area, and among all fatalities, most victims (12.1%) arrived at the hospital within 1–12 h of the accident occurring (Table 2). (Continued) Discussion The trend of RTI fatalities in this study shows that between January and December 2018, there were 32 deaths, Frontiers in Disaster and Emergency Medicine Frontiers in Disaster and Emergency Medicine frontiersin.org 06 10.3389/femer.2023.1328817 Beyera et al. ic regression of factors associated with RTI fatalities from January 1 to December 30, 2018, in St. Luke Hospital, South West Shewa Variables RTI fatality (n = 364) COR [95% CI] p-value Yes (%) No (%) Residence of injured patients Woliso 7 (5.8) 113 (94.2) 1 Outside Woliso 25 (10.2) 219 (89.8) 1.84 [0.77, 4.39] 0.168∗ Gender Male 26 (9.5) 249 (90.5) 1.44 [0.71, 5.09] 0.199∗ Female 6 (6.7) 83 (93.3) 1 Age of victims 5–14 3 (12) 22 (88) 0.30 [0.04, 1.71] 0.16∗ 15–29 20 (10.5) 170 (89.5) 0.31 [0.07, 1.37] 0.12∗ 30–44 7 (7.6) 85 (92.4) 0.44 [0.09, 2.20] 0.32 45 and above 2 (3.5) 55 (96.5) 1 Victim type Pedestrian 10 (5.6) 167 (94.4) 1 Passenger 10 (7.7) 120 (92.3) 0.72 [0.29, 1.78] 0.475 Driver 1 (12.5) 7 (87.5) 0.42 [0.05, 3.75] 0.437 Motorbike driver 11 (22.4) 38 (77.6) 0.21 [0.08, 0.52] 0.001∗ Mode of transportation after accident to hospital Ambulance 5 (31.3) 11 (68.8) 1 Police care 1 (9.1) 10 (90.9) 4.55 [0.45, 45.86] 0.199∗ Public transport 26 (7.7) 311 (92.3) 5.44 [1.76, 16.84] 0.003∗ Duration at which injured patient reached hospital <1 h 12 (6) 187 (94) 2.15 [1.02, 4.54] 0.045∗ 1 h−12 h 20 (12.1) 145 (87.9) 1 First aid before admitted Yes 7 (18.9) 151 (81.1) 1 No 25 (7.6) 181 (92.4) 0.34 [0.14, 0.79] 0.01∗ Head injury Yes 19 (7) 254 (93) 2.23 [1.05, 4.72] 0.04∗ No 13 (14.3) 78 (85.7) 1 Upper extremity Yes 4 (3.9) 99 (96.1) 2.97 [1.02, 8.70] 0.047∗ No 28 (10.7) 233 (89.3) 1 Abdominal injury Yes 10 (12) 73 (88) 0.62 [0.28, 1.37] 0.24 No 22 (7.8) 259 (92.2) 1 Lower extremity injury Yes 10 (6.2) 151 (93.8) 1.84 [0.84, 4.00] 0.126∗ No 22 (10.8) 181 (89.2) 1 Spinal cord injury Yes 10 (16.1) 52 (83.9) 0.41 [0.18, 0.91] 0.029∗ No 22 (7.3) 280 (92.7) 1 RTI, road traffic injury; COR, crude odds ratio; CI, confidence interval. p < 0.05 which is significantly associated with binary logistic regression. RTI, road traffic injury; COR, crude odds ratio; CI, confidence interval. p < 0.05 which is significantly associated with binary logistic regression. Frontiers in Disaster and Emergency Medicine Discussion Fatalities in this study were lower than those reported in the same study conducted in Dilchora referral hospital, Dire Dawa (9.4%) (18), and at the tertiary care center in Lucknow, India (13.1%) (23). Because the hospital where the study was conducted was used as a referral center and many victims were linked to another health facility, life-threatening cases may have led to higher death rates. In this study, both males and females were exposed to RTI fatalities. The study’s results showed that males were highly affected by RTI fatalities, accounting for 26 (81.25%) among RTI victims. It is in line with a similar study conducted in Iran in 2016 that showed males accounted for almost 78.7% of RTIs (27). Similarly, a study conducted in Tanzania in 2012 also showed that the male- to-female ratio of RTI fatalities was 2.1:1 (26). A cross-sectional study conducted at Tikur Anbesa Specialized Hospital composed of 71.7% male and 28.3% female RTA victims resulted in a male-to- female fatality ratio of 2.6:1 (21). That more males than females die from RTIs may be explained by the movement of males to support their families and their exposure to high-risk activities. This study revealed that RTA victims referred to the hospital who had not gotten an ambulance as a mode of transportation after the occurrence of injury were more than seven times more likely to die from an RTI compared to those who did get an ambulance (AOR = 7.093, 95% CI [1.950, 25.793]). According to a 2013 WHO road safety report on the African region, five countries have no ambulance services. In 22 countries (50%), experts estimate that <10% of seriously injured patients benefited from ambulance evacuation. In only nine of the 44 countries (22%) are a reasonable proportion (50% and above) of injured patients taken to hospital by ambulance (24). This may be because those who got an ambulance received first aid before they reached the hospital, minimizing the chance of death. According to this study, RTI fatalities were higher among those aged 15–29 (20, 10.5%), followed by those aged 30–44 (7, 7.6%). A similar study done in India in 2013 also showed that the highest number of victims of RTI fatalities (51.8%) were 16–30 years of age, followed by those aged 31–45 years (25.1%) (28). Discussion ∗Indicates a p < 0.25 and a candidate for multiple logistic regressions. enforcement of traffic safety rules, and poor road-use behavior of pedestrians. or ∼3 deaths per month on average. The highest RTI rate was observed during November, which accounted for 13% of total injuries compared to an average of 8.3% per month during the study period. A similar study conducted in the emergency department of Zewditu Memorial Hospital showed that between September 2014 and August 2015, ∼779 RTI cases were admitted to the emergency department, which was about 8.34% cases per month on average (20). The main reasons for the increase in RTIs may be the increase in the number of vehicle owners, the risky driving behaviors of drivers, poor This study revealed that there were 32 (8.79%) RTI fatalities out of the 364 study participants admitted to the emergency department of the hospital because of an RTI. A higher death rate was observed in this study compared to a study conducted in Tikur Anbesa Specialized Hospital on injury characteristics and outcomes that revealed that in all RTI victims reaching the hospital, 7.4% died (21). Other studies conducted at Wolayita Sodo Hospital and Zewditu Memorial Hospital indicated that 6% and 1%, respectively, Frontiers in Disaster and Emergency Medicine 07 frontiersin.org 10.3389/femer.2023.1328817 Beyera et al. logistic regression of factors associated with RTI fatality from January 1 to December 30, 2018, in St. Luke Hospital, South West Variables RTI fatality AOR [95% CI] p-value Yes (%) No (%) Mode of transportation after accident to hospital Ambulance 5 (31.3) 11 (68.8) 1 Police care 1 (9.1) 10 (90.9) 8.781 [0.726, 106.160] 0.088 Public transport 26 (7.7) 311 (92.3) 7.093 [1.950, 25.793] 0.003∗ First aid before admitted Yes 7 (18.9) 30 (81.1) 1 No 25 (7.6) 302 (92.4) 2.529 [0.901, 7.096] 0.078 Head injury Yes 19 (7) 254 (93) 2.515 [1.075, 5.885] 0.034∗ No 13 (14.3) 78 (85.7) 1 Upper extremity Yes 4 (3.9) 99 (96.1) 3.129 [0.985, 9.943] 0.053 No 28 (10.7) 233 (89.3) 1 Lower extremity injury Yes 10 (6.2) 151 (93.8) 2.348 [0.970, 5.686] 0.059 No 22 (10.8) 181 (89.2) 1 RTI, road traffic injury; AOR, adjusted odds ratio. ∗Statistically significant. RTI, road traffic injury; AOR, adjusted odds ratio. ∗Statistically significant. upper and lower extremity injuries with emergency department treatment fatalities of RTA victims. died (20, 22). Discussion The study conducted in Burrayu town in 2016 also showed that those aged 18–30 accounted for 59% of the total fatalities, followed by those aged 31–50, who accounted for 20% (29). The higher vulnerability of those aged 15–29 may be because productive age groups, in the effort to earn income for their family and themselves, expose themselves to traffic injuries. In contrast to this study, a study in India in 2016 showed that the 30–44-year-old age group is the most vulnerable to road accident fatalities (35%), followed by the 15–29-year-old age group, which accounted for 29.9% (30). Injuries to the head are the main cause of death from RTIs. This study showed that RTA victims who had head injuries were more than two times more likely to die compared to those who did not have a head injury (AOR = 2.515, 95% CI [1.075, 5.885]). This finding is lower than the study conducted in Taiwan in 2016 that showed that those who had head injuries were >14 times more likely to die compared to those who did not have a head injury (AOR = 14.6, 95% CI [7.7, 27.8]) (25). A study done in Tanzania also reported that head injuries are the most common type of injury sustained, which predisposed victims to prolonged hospitalization and mortality (26). This may be due to the seriousness of a head injury, which has the potential to cause death in injured victims. Frontiers in Disaster and Emergency Medicine 3. Ghaffar A, Hyder AA, Masud TI. The burden of road traffic injuries in developing countries : the 1st national injury survey of Pakistan. Public Health. (2004) 118:211– 7. doi: 10.1016/j.puhe.2003.05.003 Limitations Because secondary data were used, the current study was unable to identify crash characteristics, and drivers’ behaviors like using substances (alcohol, khat, and others) were not studied. And the The study results showed that there is no significant association between the victims’ residence area, gender, age, source of referral, or injury characteristics like chest injuries, abdominal injuries, or Frontiers in Disaster and Emergency Medicine frontiersin.org frontiersin.org 08 Beyera et al. 10.3389/femer.2023.1328817 Author contributions records also lack some important data, such as driver’s level, the quality of roads, and the utilization of seat belts, helmets, and mobile phones. Also, we did not consider any variables with clinical significance unless it was statistically significant. Therefore, generalizing these results should be done with caution. TB: Conceptualization, Formal analysis, Investigation, Methodology, Software, Supervision, Visualization, Writing— original draft, Writing—review & editing, Data curation, Funding acquisition, Project administration, Resources, Validation. SS: Data curation, Formal analysis, Supervision, Validation, Visualization, Writing—original draft, Writing—review & editing. GF: Formal analysis, Supervision, Visualization, Writing— original draft, Writing—review & editing, Conceptualization, Investigation, Methodology, Software. KW: Conceptualization, Investigation, Methodology, Supervision, Validation, Visualization, Writing—original draft, Writing—review & editing. 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 original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author. Funding Appropriate preventive measures targeting reducing the occurrence of RTIs are necessary to reduce deaths from RTIs and reduce the number of families exposed to social crises due to the death of their family member due to an accident or RTI. The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article. The Oromia Regional Health Bureau and the Ministry of Health should organize the availability and accessibility of prehospital care services for RTI victims. Supplementary material Ethical clearance was secured from the Institutional Review Board of the Institute of Health of Jimma University on February 27, 2019, reference number, IHRPGD/181/2019. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/femer.2023. 1328817/full#supplementary-material Acknowledgments Moreover, the Transport Authority and traffic police should work with other stakeholders and take appropriate measures to reduce the problems caused by RTIs in the region. They should work on the provision of important messages to all members of the community regarding knowledge and practices of road safety measures, such as the appropriate use of the road by pedestrians and avoiding risky driving behaviors. Besides this, the traffic police and Transport Authority should make the use of motorcycle helmets compulsory for motorbike users to decrease RTAs. The authors acknowledge St. Luke Catholic Hospital and College of Nursing and Midwifery stafffor their facilitation role during the data collection process. Conclusion and recommendations RTI fatalities are an important public health problem. The trends of RTIs varied from month to month, and the highest number of deaths occurred from August to November 2018. A lack of ambulance services for transporting RTI victims to the hospital after the accident and head injuries were the main causes of death from an RTI. 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. Further research should be conducted to explore the risk factors that hinder and/or aggravate the frequency of RTAs in the community. 1. WHO. Global Status Report On Road Safety. Geneva: WHO. (2013). 2. WHO. Global Status Report on Road Safety 2015. Geneva: WHO. (2015). 1. WHO. Global Status Report On Road Safety. Geneva: WHO. (2013). 2. WHO. Global Status Report on Road Safety 2015. Geneva: WHO. (2015). References 1. WHO. Global Status Report On Road Safety. Geneva: WHO. (2013). 2. WHO. Global Status Report on Road Safety 2015. Geneva: WHO. (2015). Frontiers in Disaster and Emergency Medicine frontiersin.org 09 Beyera et al. 10.3389/femer.2023.1328817 4. WHO. World Report on Child Injury Prevention. Geneva: World Health Organization. (2008). 20. Getachew S, Ali E, Silkondez W, Abebe D, Deressa W, Enquessilase F, et al. The burden of road traffic injuries in an emergency department in Addis Ababa, Ethiopia. (2016) 6:66–71. doi: 10.5588/pha.15.0082 5. WHO. Global Status Report on Road Safety. Geneva: World Health Organization. (2004). 21. Seid M, Azazh A, Enquselassie F, Yisma E. Injury characteristics and outcome of road traffic accident among victims at Adult Emergency Department of Tikur Anbessa specialized hospital, Addis Ababa, Ethiopia : a prospective hospital based study. BMC Emerg Med. (2015) 15:1–9. doi: 10.1186/s12873-015-0035-4 6. Bener A, Burgut HR, Sidahmed H, Albuz R, Sanya R. Khan WA. Road traffic injuries and risk factors. California J Health Prom. (2009) 7:92–101. doi: 10.32398/cjhp.v7i2.2017 22. Hailemichael F, Suleiman M, Wondimagegn P. Magnitude and outcomes of road traffic accidents at Hospitals in Wolaita Zone, SNNPR, Ethiopia. BMC Res Notes. (2015) 8:1–5. doi: 10.1186/s13104-015-1094-z 7. Chisholm D, Naci H. Road Traffic Injury Prevention : An Assessment of Risk Exposure and Intervention Cost-Effectiveness in Different World Regions. Geneva: World Health Organization (2008). p. 1–59. 23. Chauhan A, Ahmed N, Singh JV, Singh VK, Singh A. Kumar S. Disability and mortality following road traffic injury: a follow-up study from a tertiary care centre of India. Int J Communt Med Public Health. (2017) 4:4712– 7. doi: 10.18203/2394-6040.ijcmph20175356 8. WHO. World Report on Road Traffic Injury Prevention. Geneva: World Health Organization. (2004). 9. Hyder PAA, Puvanachandra DP, Allen KA. Road Traffic Injury And Trauma Care: Innovations For Policy (Road Trip) Road Traffic Injury. (2013). Available online at: https://2020.wish.org.qa/app/uploads/2021/01/27425_WISH_Road_Injuries_Report_ web.pdf 24. WHO AR. Road Safety in the WHO African Region the Facts 2013. (2013). p. 1–13. 25. Huang C, Rau C, Chuang J, Kuo P. Characteristics and outcomes of patients injured in road traffic crashes and transported by emergency medical services. Int J Environ Res Public Health. (2016) 13:236. doi: 10.3390/ijerph13020236 10. WHO. Road Traffic Injury Prevention: Training Manual. Geneva: WHO. (2004). p. 1–20. 11. (IRTAD) ITSD and AG. Road Safety Annual Report. Geneva: WHO. (2017). 26. Chalya PL, Mabula JB, Dass RM, Mbelenge N. Ngayomela IH. 30. Singh SK. Road traffic accidents in India: issues and challenges road traffic accidents in India: issues and challenges. Transp Res Proc. (2017) 25:4708– 19. doi: 10.1016/j.trpro.2017.05.484 References Injury characteristics and outcome of road traffic crash victims at Bugando Medical Centre in Northwestern Tanzania. J Trauma Manag Outcomes. (2012) 6:1. doi: 10.1186/1752-2897-6-1 12. WHO. Road-Traffic-Injuries @. (2018). Available online at: www.who.int (accessed December 21, 2018). 13. UN. General Assembly on Transforming our World: the 2030 Agenda for Sustainable Development. (2015). p. 1–35. 27. Hamzeh B, Naja F, Karamimatin B, Ahmadijouybari T. Salari A. Epidemiology of traffic crash mortality in west of Iran in a 9 year period. Chin J Traumatol. (2016) 19:70–4. doi: 10.1016/j.cjtee.2015.12.007 14. WHO. The Global Burden of Disease 2004. Geneva: WHO. (2004). 28. Rao BB, Sudharani G. Prevalence of Road Traffic Injury and Associated Factors Among Patients Visiting the Emergency Department of Osmania General Hospital, Hyderabad, Telangana, India Abstract: Introduction. (2015). p. 146–9. 15. Bhalla K, Harrison J, Abraham J, Bartels D, Naghavi M, Lozano R, et al. Burden of Road Injuries in Sub-Saharan Africa. (2014). p. 1–112. 16. ECSA. ETHIOPIA Demographic and Health Survey 2016. (2016). p. 1–551. 29. Bewket TT, Dachew AB, Bifftu BB. Incidence of road traffic injury and associated factors among patients visiting the emergency department of Tikur Anbessa specialized teaching hospital, Addis Ababa, Ethiopia. Emerg. Med. Int. (2014) 2014:0–5. doi: 10.1155/2014/ 439818 17. St.Luke. Index. (2019). Available online at: www.stlukecatholichospital.org. (accessed September 10, 2019). 18. Negesa L, Egziabher G, Mohammed J. Assessment of Magnitude and Treatment Outcome of Road Traffic Accident from January 2013-January 2015 in Dilchora Referral Hospital, Diredawa Eastern Ethiopia. (2017). 30. Singh SK. Road traffic accidents in India: issues and challenges road traffic accidents in India: issues and challenges. Transp Res Proc. (2017) 25:4708– 19. doi: 10.1016/j.trpro.2017.05.484 19. WHO. Guidelines for Conducting Community Surveys on Injuries and Violence. Geneva: WHO. (2004). p. 1–151. Frontiers in Disaster and Emergency Medicine frontiersin.org 10
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The use of creative and humorous designs as vehicles for health education and infection control
Antimicrobial resistance and infection control
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cc-by
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Objectives The concept was to incorporate humour and creative designs to give a glimpse of the world of microbes and their impacts in human health and disease. Introduction For viewing of the characters designed, please go to http://www.ilovemicrobes.com Ilovemicrobes.comTM was started in late 2014 by a microbiologist cum ex-lecturer at a Singapore institute of higher learning. The concept was to incorporate humour and creative designs to give a glimpse of the world of microbes and their impacts in human health and disease. SWL Soh From 3rd International Conference on Prevention and Infection Control (ICPIC 2015) Geneva, Switzerland. 16-19 June 2015 Conclusion It is the vision of ilovemicrobes.comTM to promote Health Education and Infection Control in a fun and exciting way! Soh Antimicrobial Resistance and Infection Control 2015, 4(Suppl 1):I12 http://www.aricjournal.com/content/4/S1/I12 Soh Antimicrobial Resistance and Infection Control 2015, 4(Suppl 1):I12 http://www.aricjournal.com/content/4/S1/I12 Soh Antimicrobial Resistance and Infection Control 2015, 4(Suppl 1):I12 http://www.aricjournal.com/content/4/S1/I12 Open Access © 2015 Soh; 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 cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Methods Published: 16 June 2015 Published: 16 June 2015 Published: 16 June 2015 Each design is scientifically-based on microscopic or macroscopic attributes of the real image of the actual microbes magnified up thousands if not millions times over. doi:10.1186/2047-2994-4-S1-I12 Cite this article as: Soh: The use of creative and humorous designs as vehicles for health education and infection control. Antimicrobial Resistance and Infection Control 2015 4(Suppl 1):I12. doi:10.1186/2047-2994-4-S1-I12 Cite this article as: Soh: The use of creative and humorous designs as vehicles for health education and infection control. Antimicrobial Resistance and Infection Control 2015 4(Suppl 1):I12. The uniqueness of these microbes’ designs are not only to generate curiosity but also to demystify the dis- eases caused in a fun and approachable manner. Microbes are known to cause disease. However, there is still a sense of complacency and resignations when it comes to infectious disease. The failure of people to exercise appropriate precaution and infection control processes are most likely due to ignorance and the lack of awareness. 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 s an Open Access article distributed under the terms of the Creative Commons /licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in perly cited. The Creative Commons Public Domain Dedication waiver (http:// pplies to the data made available in this article, unless otherwise stated. Thus, the importance of a vehicle, which can capture the attention of people, especially children to be aware of the existence of these infectious agents, and to under- stand the way they caused infections; and finally as to how we can performance interventions to reduce the risk of infections are of significance. K Ventures, Singapore © 2015 Soh; 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 cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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Thesis Supervisory Panels as the New Design Studio Paradigm in UTM Master of Architecture Programme
Journal of design and the built environment
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48 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et. Published:31 August 2022 The UTM’s M.Arch programme revitalized its Design Studio programme, also known as the Architectural Design Thesis (ADT), when it introduced the Thesis Supervisory Panel system in 2013. Developed as an expansion from the preceding 5-year B.Arch and 3+3 Integration programme, the ADT expanded the previous 2-stage final year Design Studio spanning over two semesters into four, introducing the TSP, implementing the vertical studio and allowing students to choose their own paths. The programme was developed from the Workbase system, a hands-on, master-led Design Studio approach influenced by Ecole des Beaux Arts and Bauhaus. The authors intend to tell the story of the programme and its changes throughout the years. This paper records the growth and development of the TSPs in the M.Arch programme, tracing its origin in the Workbase practiced since the 1980s with data collected from the Thesis Coordinator’s files, departmental publications and documentations and have been organized in a simple narrative. By framing the narrative on the back story of the TSPs, the paper offer better understanding on the inner workings of the UTM M.Arch studio programme and encourages improving and strengthening the established system and opens up new studies that could enrich and diversify it further. Keywords: Thesis Supervisory Panels as the New Design Studio Paradigm in UTM Master of Architecture Programme Azari Mat Yasir*1, Syed Ahmad Iskandar Syed Ariffin2, Dilshan Remaz Ossen3 1Faculty of Built Environment and Surveying, Universiti Teknologi Malaysia, Malaysia (PhD Candidate); 2Faculty of Built Environment and Surveying, Universiti Teknologi Malaysia, Malaysia; 3College of Architecture Engineering and Design, Kingdom University, Bahrain. 2Faculty of Built Environment and Surveying, Universiti Teknologi Malaysia, Malaysia; 3College of Architecture Engineering and Design, Kingdom University, Bahrain. Corresponding author: azarimy@gmail.com*1 Published:31 August 2022 Corresponding author: azarimy@gmail.com*1 Corresponding author: azarimy@gmail.com*1 Corresponding author: azarimy@gmail.com*1 1. INTRODUCTION masters programme, awarding the Bachelor of Science in Architecture (B.Sc.Arch) and the Master of Architecture (M.Arch) and still currently running in UTM Johor Bahru campus. In all its iterations, the Design Studio plays a key role especially towards the end of the studies. Prospective graduates are expected to achieve specific criteria and skills in line with the stipulated Programme Educational Outcomes. The final year was marked by a major design studio event known as the Architectural Design Thesis (ADT), celebrating the students’ skills and knowledge in architecture by tackling a major design project for a year. 1. The Design Studio in the architecture programme of Universiti Teknologi Malaysia could trace its routes back to the early days of Sekolah Teknik in Jalan Gurney, Kuala Lumpur in 1904. Sitting at the core of architecture education, the Design Studio is integral in the training of an architect. Each school is an architecture education provider, and they are responsible of formulating their own programme, curriculum and syllabus, creating their own mold to shape and form the next generation of architects. The UTM Architecture Programme (previously known as The Department of Architecture, and in this paper will refer to as The School) embraces this and have been responsible in designing the programmes to suit the needs of the industry as well as the country as a whole. Initially it was handled by supervision, where each student is assigned to a lecturer to supervise him in developing his project. This relationship is not unlike the research supervision commonly practiced in graduating projects in other programmes, and both student and supervisor will work hand-in-hand to produce a formidable outcome to be presented to a panel of assessors in a viva-like assessment at the end of the semester. Later, the one-on-one supervision was replaced by the panel system in the year 2000, where like- minded lecturers would band together to form a supervision group that would guide a number of final year architecture students to develop and complete their works. This panel system is known as the Thesis Panel, and would later evolve further into the Thesis Supervisory Panel in 2013. Looking back at the history of the UTM architecture programme during the days of the Technical College, the Design Studio began with the introduction of the architecture programme in 1962 (Kosman, 2011). 1. INTRODUCTION It went through a review in 1969 which introduced a more structured system with allocation of credit hours and courseworks. Later the curriculum was further updated when the Technical College was upgraded into Institut Teknologi Kebangsaan in 1972, and for the first time the Bachelor of Architecture degree was offered (Jabatan Senibina, 1986). It was further upgraded with a the 3+3 year integration programme, which offered a three year diploma followed by another three year degree in 1975 (Kosman, 2011). Throughout the years, the studio remained integral to the training of the architecture student, led by tutors and lecturers with their own specific approach and design agendas. The 3+3 year integration ran for 27 years before being replaced by the 5 year degree programme in 2003, which saw significant restructuring to the studios and the content of the curriculum to better suite the contemporary needs. The single Bachelor of Architecture programme was supposed to be a temporary measure, a means to standardize the UTM architecture programme with the rest of Malaysia (Jabatan Senibina, 2005). However, despite the records and paperworks, the progress and development behind the Thesis Supervisory Panels were not well documented. A significant amount of the ideas, intent and decisions were left unsorted over the semesters and there were little attempt to assemble them together. This paper traces back the journey of these panels and its aspirations, from its inception to its current form. Keywords: INTRODUCTION DISCUSSION 3. The Design Studio is both an academic system of teaching and learning, as well as a physical space where the design teaching and learning takes place (Majlis Akreditasi dan Pengajian Senibina Malaysia, 2013). The Design Studio is critical to the training of an architect as it constitutes the main pillar where all other topics or subjects are propagate around to support it (Zairul, 2018) (Mat Yasir, 2021).As it sits at the core of the architectural programme, the Design Studio became the subject of all sorts of innovative evolutions, mainly to keep up with the current industry, technological achievements as well as societal needs. Its dependence on integrative syllabus is essential to assure optimal process of learning amongst architectural students (Ibrahim & Utaberta, 2012). 2. The intent of this paper is to build a narrative that explains the process and reasonings behind the running of the Thesis Supervisory Panels currently conducted in the M.Arch programme in UTM. The authors were involved in the Architectural Design Thesis programme since However the programme ran for 10 years before being replaced by the 3+2 year degree and 49 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al 2002 and took charge of the programme together as Thesis Coordinator beginning September 2010. Being one of the earliest school of architecture in Malaysia, UTM aspired to provide a wholesome programme in order to produce graduates who’re able to be address all the needs of the industry. So writing a programme in an open ended way, allowing for flexibility in the Design Studio was crucial, at the same time minimizing the need for revisions and amendments to the curriculum. Therefore, a systematic structure was needed in order to provide the scaffold to both allow integration of peripheral courses that enriches the programme and flexibility of the Design Studio. 2.1. The narration of this paper is based on the perspective of the authors as the Thesis Coordinators, managing the programme from the 5-year B.Arch and later through the transition into the 3+2 year M.Arch programme. It will follow the evolution of the programme, detailing the rationale behind some of the changes that leads to the current programme. To address this, the UTM Architecture Programme has embraced Design Studio approaches with heavy influences from Bauhaus and Ecole des Beaux Arts (Kosman, 2011), where one of the characteristic features include utilizing critique as a method to develop students’ design works (Alagbe et al., 2017). The Ateliers as it is known at the Ecole, centers around learning by doing that is appended on the input lectures inspired the Design Studio as a pedagogy. Bauhaus focuses learning by creating a functional, ideal community where students became apprentices to the master (Broadfoot & Bennett, 2003). Throughout the year when the school was led by Julius Posener in 1956, the first Head of Architecture Department of the Technical College, followed by Arthur Bumbury in 1964 and D. R. Pritchard in 1967 (Kosman, 2011), the master-led Design Studio gradually took form into what is known as the Workbase system. This paper also discusses the reasons and causes of certain key decisions were made that has affected the development of the TSPs. Ten years of growth and development of the TSPs in the M.Arch programme will also be recorded, tracing its origin in the Workbase system practiced since the 1970s, up until the end of the 2021/2022 academic session. Data is collected from the Thesis Coordinator’s files, departmental publications, course documentations and portfolio meeting reports have been organized in a simple narrative to establish the process in chronological order. 50 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et. 3.1. The Workbase is a teacher-centric pedagogical approach for teaching and learning in the Design Studio, first introduced to UTM in 1982 (Syed Ariffin, 2008) where the teacher (also known as Workbase Tutor or Master) will be responsible of the entire teaching and learning throughout the semester. They are given ample flexibility in determining the heading, theme, content and style of the design tasks tackled in the studio, so long as it is within the acceptable parameters set by The School (Mat Yasir, 2021). Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al Figure 1 Studio Structure for Dip.Arch (Jabatan Senibina, 2002) Head of Panel Head of Panel Head of Panel Head of Panel Head of Panel Head of Panel Internal Panel Members Internal Panel Members Internal Panel Members Internal Panel Members Internal Panel Members Internal Panel Members M.Arch Programme Coordinator Thesis (Course) Coordinator Head of Socio-Culture Unit Head of Urbanism Unit Head of Sustainable Technologies Unit Head of Department Figure 1 Studio Structure for Dip.Arch (Jabatan Senibina, 2002) studio (Majlis Akreditasi dan Pengajian Senibina Malaysia, 2013). It allows the Tutors to offer students opportunity to learn creatively, widens their horizons with diversity with a degree of freedom in doing so (Syed Ariffin, 2008). In a Workbase, the Tutor will be incharge of a small group of students, usually between six to a maximum of 15 students, the maximum size in accordance to the Board of Architects Malaysia’s (LAM) requirements of staff:student ratio in the Figure 2 Workbases within the Undergraduate Design Studio (Program Senibina, 2022b) Design Studio Year Coordinator Studio Master Studio Master Students Students Workbase Workbase Figure 2 Workbases within the Undergraduate Design Studio (Program Senibina, 2022b) architecture, it seems to have the opposite effect when it comes to coopration and peer-learning amongst students in the same cohort (Syed Ariffin, 2008). This was observed at 2nd year of the diploma where students were allowed to chose their own path based on a selection of individual Workbases. Each Workbase Tutor is responsible in shaping and moulding the design studio brief and parameters according to specific needs and preference. For example Theory and Philosophy (TAP) Workbase lets the students roleplay as one of the proponents of an established architectural theory or philosophy. Urban Context Workbase encourages design Practically, the Workbase system splits the larger studio into smaller groups for ease of management (Figure 2). 51 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al 3.1. Tutors are occasionally shared between Workbases, but most often they are allowed to run their own programmes. Lecture inputs, critique sessions and tutorials are usually run together due to the common parameters. Critique sessions occur frequently, both formal and informally, typical of any Design Studios (Zairul, 2018). The Workbase system was later utilized in the 3+3 Integration programme in 1986 (Jabatan Senibina, 1986). Although it was formed to encourage healthy sense of individualism and devotion in 51 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al building type and scale from each other. Gradually over the years, this nudges the Workbases further and further apart, creating disparity between the Workbases, as illustrated in following Table 1. within tight urban settings; Vernacular Workbase inculcates awareness of regional and heritage architecture; and Enviro Workbase focuses on practical environmental approaches in architectural design (Jabatan Senibina, 2001). This would mean two Workbases of the same level might be doing entirely different projects, Table 1 Projects by Workbases for 2nd year, Dip.Arch 2001/2002 semester 1 (Jabatan Senibina, 2001) Workbase Project Urban Context (Wan Hashimah) Project 1: 2 weeks 15% Project 2: 4 weeks 25% Project 3: 7 weeks 45% Pet Project: 2 weeks 15% Enviro (Hong Lim Foo) Project 1: 3 weeks 20% Project 2: 4 weeks 25% Project 3: 6 weeks 40% Pet Project: 2 weeks 15% Attendance: 10% of each project Vernacular (Abdul Razak) Project 1: 1 week 10% Project 2: 7 weeks 45% Project 3: 6 weeks 40% Vernacular on CAD (Abdul Halim) Project 1: 1 week 0% Project 2: 2 weeks 10% Project 3: 3 weeks 15% Project 4: 4 weeks 20% Project 5: 4 weeks 40% Table 1 Projects by Workbases for 2nd year, Dip.Arch 2001/2002 semester 1 (Jabatan Senibina, 2001) Although this encourages students to develop their own unique style, it eventually became so divergent that students could hardly compare their works with their peers. It was a sudden jump from the Fundamental Unit (1st year), where everyone in the same cohort attempted similar projects of scale and size, only differing on client or site (Jabatan Senibina, 2001). To address this issue, the School introduced Unit-Workbase in 1989, a bigger group that comprises of Workbases with similar ideas or approaches (Syed Ariffin, 2008). 3.1. It was an attempt to consolidate the Workbases from being too diverse from each other, which will form the basis of the Thesis Studio Units which will be discussed later in this paper. the first semester, followed by high-density housing in the second semester. This is subsequently followed by urbanism studio at fifth year, before going into their practical training with the industry, and later rejoin for their final year. 3.1.1. Workbases in Architectural Design Thesis 3.1.1. Workbases in Architectural Design Thesis The final year of the B.Arch programme is known as the Architectural Design Thesis (ADT) (Jabatan Senibina, 2002). It is a comprehensive programme that encompasses a wide range of aspects pertaining to architectural design from architectural theory to practical application into design. Each aspect will be analyzed and put under scrutiny during the critique sessions (Lawson, 1997) and eventually the final assessment (evaluation) at the end of the semester. It is divided into two stages, one per semester: Stage 1 (Pre-Thesis) and Stage 2 (Thesis) spanning over the final year. Prior to Yet the individual nature of the Workbases apparent in the diploma programme was suddenly replaced with a tight, regimented programme when they continue for their B.Arch. At 4th year, the students went through thematic studio focusing on highrise building complex in 52 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al 1998, students were paired up with Thesis Supervisors elected from one of the school members. The Supervisors were responsible in guiding the students throughout the final two semesters (Syed Ariffin, 2008). was introduced to replace the 3+3 Integration in 2002, it assimilated both the Dip.Arch and B.Arch into a single degree programme. The duration of the studies was reduced to five years, where a significant restructuring of the course structures was implemented. Yet the matter of the Workbase focus remains. The 2nd and 3rd year of studies were too individualised in their own approaches and philosophies, again a creating similar situation where students became too specialised before they get to their upper years. The Workbase system reappeared in the back into the programme with the introduction of the Thesis Panels in 1998 (Syed Ariffin, 2008). It is a a concept similar to the ones practiced in the Unit-Workbase of the lower years, but instead of one on one relationship with the Thesis Supervisors, students are grouped together under the same panel with Tutors who share similar interests. The panels provide unique opportunities for students to pursue their own interests in architecture through the medium of a building or complex of buildings (Jabatan Senibina, 2002). Each panel has its own specialisation, expertise and individual approach which will guide the students prepare and develop their briefs, design proposals, documentations, solutions and conclusions. Thesis Studio Units (TSU) Thesis Studio Units (TSU) members of the School. And this created a conflict as all design studio documentations at the time still have strong technical requirements written, such as the Studio Rubric (Senibina, 2007). Initially the School regarded the 5-year programme as a short term implementation to align with other architectural programmes in Malaysia. However, the 5-year programme ran for about 10 years before being replaced, amplifying the confusion on the identity of the programme. One of the most important question during the writing of the 3+2 programme was “What is the heading of the School?”. UTM used to be a very technical-centric programme, where a large emphasis was given on the buildability, practicality, construction details and drawing conventions (Jabatan Senibina, 2001) to the point that it had become the primary characteristic that the graduates are known for up to the early 2000s. However, with the internet accessibility became more affordable and available on and near campus, students had access to wider range of references, particularly on esthetically and visually pleasing architecture. At the same time, philosophical and conceptual theoretical design approaches such as Deconstructivism or Critical Regionalism, as well as movements put forward by popular schools at the time such as The Bartlett, Architectural Association, Carnegie Mellon University and M.I.T, became convenient read, gradually creating another end on the stick. One of the most important question during the writing of the 3+2 programme was “What is the heading of the School?”. UTM used to be a very technical-centric programme, where a large emphasis was given on the buildability, practicality, construction details and drawing conventions (Jabatan Senibina, 2001) to the point that it had become the primary characteristic that the graduates are known for up to the early 2000s. However, with the internet accessibility became more affordable and available on and near campus, students had access to wider range of references, particularly on esthetically and visually pleasing architecture. At the same time, philosophical and conceptual theoretical design approaches such as Deconstructivism or Critical Regionalism, as well as movements put forward by popular schools at the time such as The Bartlett, Architectural Association, Carnegie Mellon University and M.I.T, became convenient read, gradually creating another end on the stick. 3.2.1. Workbases in the M.Arch 72 Credit Programme 3.1.1. Workbases in Architectural Design Thesis This calls for another review of the curriculum, conducted in 2007, to address the issue and potentially revamp how the Workbases were run. The School recognised the need for lower year students to have a common objectives that are shared amongst the Workbases, in order to have a more comparable outcomes and encourage peer-learning. The individualistic, highly unique approaches to studios should be left for the upper end of the studies in the Architectural Design Thesis. This realignment is further strengthen with the introduction of the 3+2 year programme (B.Sc.Architecture and Master of Architecture) in 2013, which saw the implementation of the revised Studio Rubric, later renamed as the Design Matrix. This forms the basis of the Thesis Supervisory Panel and the core approach of the entire Design Studio currently being practised in the M.Arch programme, which will be discussed further in section 3.3. When the 5-year B.Arch programme Figure 3 Overview of the Workbase Focus after the realignment process (Mat Yasir, 2021) Figure 3 Overview of the Workbase Focus after the realignment process (Mat Yasir, 2021) approach in 5th year in the 3+2 programme. This approach affirms the continued existence of the Thesis Panels in the new programme. approach in 5th year in the 3+2 programme. This approach affirms the continued existence of the Thesis Panels in the new programme. Figure 3 above shows how the focus of the studios gradually shifted from being broadbased in the 1st year to highly focused on individual 53 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al Figure 4 Studio Structure for B.Sc.Architecture (Mat Yasir, 2021) Figure 4 Studio Structure for B.Sc.Architecture (Mat Yasir, 2021) 3.2. 3.2.1. Workbases in the M.Arch 72 Credit Programme A series of curriculum review workshops held between 2010 to 2011 design and develop the replacement for the 5-year B.Arch programme. Amongst others it seeked to remedy the issue by establishing a clear heading of The UTM Architectural Programme. One of the steps taken is to relook at how the Design Studios were run and to create a more reasonable learning curve to bridge between the B.Sc.Arch degree and the subsequent M.Arch degree. The School resolved to create a series of studios with a diversity of specialized focus at the upper end of the architectural studies (the 5th year), as well as celebrate the assortment of members of the School as well.This gave birth to the Thesis Studio Units, formed based on the shared topics of expertise or areas of interests amongst the faculty members. The Thesis Studio Units inherited elements from the previous 3rd year and 4th year studios, rearranged into three semesters: The theoretical approaches became highly popular amongst the students at them, which saw imitative works throughout the cross-section of the school (Senibina, 2007). This was viewed favourably by many members of the school as an outcome of globalization, which was a very popular catchphrase at the turn of the century. The introduction of the 5-year programme in 2002, combined with the rapid digitalization of the architectural programme, created a dilemma where the traditional technical-centric approach was rapidly falling out of favour amongst the Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al Table 2 Conversion from Workbase into Thesis Supervisory Units (author) Workbase M.Arch TSU Social-Housing, Vernacular, and Theory and Philosophy Socio-Culture Urban Design and Urban Context Urbanism Tectonic (Highrise), Environmental & Enviro Sustainable Technologies Table 2 Conversion from Workbase into Thesis Supervisory Units (author) qualification. The structure of the TSUs and the TSPs contained within it is illustrated in Figure 4 below. 3.2.1. Workbases in the M.Arch 72 Credit Programme The companion subjects requisitioned components that used to be a part of the Design Pre-Thesis studio: i. Manuscript Writing (report, journal article) ii. Seminar (group studies and presentation) iii. Generic Skills Development (activities) i. Manuscript Writing (report, journal article) i. Manuscript Writing (report, journal article) ii. Seminar (group studies and presentation) iii. Generic Skills Development (activities) ii. Seminar (group studies and presentation) iii. Generic Skills Development (activities) Series of thematic lecture inputs that used to be a part of the Design Pre-Thesis studio are now offered in this companion subject to allow more studio time to be dedicated at design development. At the same time, it also introduced a new component that was previously assessed as Course Learning Outcome (CLO) 3 in the Design Pre-Thesis Studio into an assignment called the Argument Board, to be assessed integratively at the end of the semester. As more and more architectural schools started their own M.Arch (LAM Part 2) programme, it became clear to the School that the 72 Credit programme is cumbersome and overburdening for both the student and the lecturers. Originally, the design of the M.Arch 72 Credit programme were meant to better reflect the actual amount of work done based on the Student Learning Time (SLT). Although it did achieve that outcome to a certain extent, it also caused the programme to be viewed unfavourably heavy when compared to other M.Arch programmes. As illustrated in Figure 6, the companion subjects now fell under the jurisdiction of the Cultural Context and Technology and Environment subject clusters. The subjects were given names reflecting their specific area of focus, and this became the defining characteristics of a particular Design Pre-Thesis studio when taken together. The companion subjects provide flexibility in running the masters studios as an additional layer of complexity for students to explore and develop their ideas on. The focus on topics need not to be embedded in the studio outline itself, allowing for future changes to address new issues or topics. 3.2.2. Upgrading into the M.Arch 60 Credit Programme 3.2.1. Workbases in the M.Arch 72 Credit Programme Within these Thesis Studio Units, smaller studio groups similar to the Workbases called Thesis Supervisory Panel would be formed and headed by any member of the School with a LAM Part 2 Within these Thesis Studio Units, smaller studio groups similar to the Workbases called Thesis Supervisory Panel would be formed and headed by any member of the School with a LAM Part 2 Figure 4 Studio Structure for M.Architecture 72 Credits (Mat Yasir, 2021) Figure 4 Studio Structure for M.Architecture 72 Credits (Mat Yasir, 2021) Programme, therefore forming the basis of its identity. This is inline with the Design Studio focus illustrated in Figure 3 earlier. During the establishment of the Thesis Studio Unit, it was understood that the three themes will form the core pillars of the UTM Architectural Figure 5 Studio Structure for M.Arch 72 Credits (Program Senibina, 2018) Figure 5 Studio Structure for M.Arch 72 Credits (Program Senibina, 2018) The Architectural Design Thesis still maintains the two-stage format inherited from the 3+3 Integration programme, but then 1st stage expanded into three semesters (Figure 5). This effectively occupies the two years of the M.Arch studios. Then each Thesis Supervisory Unit is assigned to one of the 1st stages, with the course names and themes as illustrated in Table 3 below. 55 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al Table 3 Assignment of TSU to relevant studio courses Stage Course TSU 1A Design Pre-Thesis I Socio-Culture 1B Design Pre-Thesis II Urbanism 1C Design Pre-Thesis III Sustainable Technologies 2 Design Thesis - Table 3 Assignment of TSU to relevant studio courses into two smaller courses: The pre-thesis studio course (carrying 6 credits) and the companion subject course (3 credits). But the themes of the TSUs were not specifically written into the title of the course, so it is often overlooked when one were to look only at the course structure. It is only available in the Thesis Coordinator’s documents and the Design Matrix (Jabatan Senibina, 2016). The intention for this was to keep the conversion to the 3+2 programme as minimum as possible, therefore most of the courses retain similar names, outlines, outcomes and contents from the 5-year B.Arch programme. Eventually, the M.Arch 72 credits programme was launched and took its first cohort of students in 2012. 56 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et. 56 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al 3.3. ten students per panel. Since the beginning of the M.Arch programme, it has inherited ten Thesis Supervisory Panels from the older Bachelor of Architecture programme, and ten new ones were formed especially for M.Arch. Details of the panel names can be found in Table 4 below: 3.2.2. Upgrading into the M.Arch 60 Credit Programme Another curriculum review was held 2018 with the objective of reducing the number of credits from 72 to 60, in-line with other architectural programmes offered in Malaysia. The School took this opportunity to further refine the M.Arch programme by restructuring some of the courses (Figure 6). The Stage 1 Design Studio course (Design Pre-Thesis) originally 9 credits is split Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al Figure 6 Studio Structure for M.Architecture 60 Credits (Mat Yasir, 2021) Head of Design Cluster Head of Panel Head of Panel Head of Panel Head of Panel Head of Panel Head of Panel Internal Panel Members Internal Panel Members Internal Panel Members Internal Panel Members Internal Panel Members Internal Panel Members Head of Cultural Context Cluster Head of Technology & Environment Cluster Companion Subject Course Owner Companion Subject Course Owner Companion Subject Course Owner Architecture Programme Director M.Arch Programme Coordinator Thesis (Course) Coordinator Head of Socio-Culture Unit Head of Urbanism Unit Head of Sustainable Technologies Unit Head of Cultural Context Cluster Figure 6 Studio Structure for M.Architecture 60 Credits (Mat Yasir, 2021) 57 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al 3.4. Formation of Thesis Supervisory Panels (TSP) the programme accordingly. Each semester, the Thesis Coordinator with the M.Arch Programme Coordinator would decide on the number of panels to offer, taking into consideration the aspects mentioned earlier. Candidates for Head of Panels along with Internal Panel Members within a TSU would be suggested to the Programme Director, whom will be incharge to appoint them to form the TSP. Supervising and scrutinizing through the students works meticulously four to five times a semester became more taxing to the lecturers as problems became more complex, deeper issues to tackle, and solutions subjected to a barrage of tests and experiments. Students are also required to present their works covering the objectives, studies and theoretical framework on top of the design project. This is critical in order to demonstrate understanding of a complete scheme from ideation to execution (Tafahomi, 2021). As the school aspires to expand its size, more panels need to be opened to accommodate larger student population. Following the maximum allowable lecturer-student ratio of 1:10 set by LAM (Majlis Akreditasi dan Pengajian Senibina Malaysia, 2013), each new panel would open up 10 spots for M.Arch studies. 3.3. Thesis Supervisory Panels ten students per panel. Since the beginning of the M.Arch programme, it has inherited ten Thesis Supervisory Panels from the older Bachelor of Architecture programme, and ten new ones were formed especially for M.Arch. Details of the panel names can be found in Table 4 below: In essence, the TSP is expanded from Workbase system used in the lower year studios, housed within one of the Thesis Studio Units. It is the smallest group in the studio with a maximum of Table 4 M.Arch Thesis Supervisory Panel Past and Present (Program Senibina, 2022b) Inherited Panels New Panels HOME, Vector (previously Gen-X), Regionalism, Urban, New Spirit, Environmental, EnviroTectonic (previously Fundamentalism), Reform, Paramatrix (previously X-Factor) and Technovation. SoCult, Catalyst, Citra, ENVI, Re.Urban (previously Urban Renaissance), Praxis, EnviroArc, Recursive Design Studio, Lestari and Enviro. Table 4 M.Arch Thesis Supervisory Panel Past and Present (Program Senibina, 2022b) number of students in the sixth year of the 3+3 programme became large enough that the one-on- one supervision previously used became cumbersome, particularly during the final year assessment (Syed Ariffin, 2008). It is critical to note that the these panels are the embodiment of what the members of the School aspire to explore, based on their own field of expertise or area of interests. The three Thesis Studio Units provide a larger sandbox to play in, but it isn’t explicitly limited to it. It provides freedom for students to explore their own interests in architecture through the project of their choice (Syed Ariffin, 2008). Similarly with the Workbase, a TSP is led by a Tutor called the Head of Panel appointed by the School. In addition to that, a TSP is also made of least two other Internal Panel Members, consisting of academicians, professional architects or experts. The three-member panel started in 1998 when the Grouping the members of the School together encourages cross-learning between the students as well as promoting them to get second or third opinion during the development of the thesis. This format was maintained since then, with panel members being shuffled every now and then to keep the group dynamics fresh and challenging. New panels can be formed based on configuration of expertise in the School at the time, aspiration towards new or alternative topic 57 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. / URBANISM Figure 7 M.Arch Thesis Supervisory Panel Line-up for 2021/2022 semester 2 (Program Senibina, 2022b) 3.4. Formation of Thesis Supervisory Panels (TSP) 3.3. Thesis Supervisory Panels et.al of interests, or to fill in spaces left by members who have left the School. Combined, they would enrich the students with a plethora of ideas and perspectives, more in-line with what is commonly practiced in the industry. As Head of New Spirit, Dr. Sharifah is still incharge of the project, brief and heading of the TSP, and the panel members are always encourage to engage the students in discussions and conversations about their works to stretch their horizons rather than being too focused in one particular idea. The following diagram is the M.Arch Architectural Design Thesis teaching staff composition, with two TSPs making up a Thesis Studio Unit, accompanied by a companion subject for each unit (Program Senibina, 2022a). Although some TSPs are firmly embedded under its respective units, it does not limit the lecturers from transitioning to a different unit, even if the unit is outside their field of expertise. It is quite often that a TSP would consist of members from different backgrounds. For example, under the New Spirit panel (Figure 7), Dr. Sharifah Salwa is an expert in urban design and Space Syntax; Dr. Muhammad Faizal on interior design; while Dr. Lim Yaik Wah is an environmental design expert. Figure 7 M.Arch Thesis Supervisory Panel Line-up for 2021/2022 semester 2 (Program Senibina, 2022b) VECTOR CATALYST NEW SPIRIT URBAN RENAISSANCE RECURSIVE DESIGN ENVIRO Head PM Dr. Khairul Anwar b Khaidzir PM Dr. Alice Sabrina bt Ismail 2 Dr. Sharifah Salwa bt Syed Mahdzar 2 Ar. Chan Wai Lai 2 Azari b Mat Yasir 2 Dr Roshida bt Abdul Majid 2 Internal 01 Dr. Iziq Eafifi b Ismail 1 Ts. Dr. Shahariah Norain bt Shaharudin Dr. Muhammad Faizal b Abdul Rani Dr. Aiman b Mohd Rashid 1 Dr. Leng Pau Chung 2 Dr. Tengku Intan Suraya bt Tengku Aziz Internal 02 Dr. Lim Yong Long 2 Dr. Azalilah Ramdani bt Musa PM Dr. Ar. Lim Yaik Wah 1 Dr. Doris Toe 1 Ar. Hanis Nazurah bt Abu Hassan TS. Ar. Noraslinda bt Abdul Rahman 1 Companion M.ARCH THESIS 2021/22-2 PANEL LINE-UP SUSTAINABLE TECHNOLOGIES URBANISM SOCIO-CULTURE Dr. Izura bt Tukiman Dr. Sharifah / Dr. Wan Mohd Zakri / Ar. Chan Wai Lai PM Dr. Khairul Anwar b Khaidzir 3.5. Swapping and Closing down of Thesis Supervisory Panels Keeping up with the dynamics of the School, the TSP line-up is often changed to allow experts with different interests, experience and ideas to take up the mantle as head of a TSP. Handling a group of masters students who are more mature and often already exposed to some industry experience opens up possibilities of handling deeper, more complex and more challenging subjects. Therefore it offers a mutually beneficial relationship between the student and the lecturer, as well as the School and the University as a whole. A common example is for the Head of Panel to engage the students in his or her research or projects, such as New Spirit panel with Dewan Despite allowing each TSP to flourish in its own field of expertise, this requirement limits the ability for the UTM Architecture Programme to expand rapidly, as The School would have to ensure it has sufficient man-power in order to run 58 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al 58 Bandaraya Kuala Lumpur on Ampang Great Street studies(Program Senibina, 2021), and Recursive Design Studio’s facade prototyping project under the Living Lab initiative . Regionalism panel headed by Dr. Alice Sabrina Ismail. At the time, there was a strong urge to focus on regional and heritage architecture, which used to be one of the strength of the School. Regionalism panel last took students in 2012 during 5-year B.Arch, and was dormant throughout the M.Arch years. As more opportunities arise, members of the Schools should be given the chance to head a TSP and encourage students to be a part of something bigger than just the studio. The New Spirit Head was rotated to 3rd year studios to make way for Dr. Hazrina Haja Bava Mohidin to run her Praxis studio in 2018/2019 semester 1 (Figure8). Similar arrangement occured a year earlier in 2017/2018 semester 1 when Vector was rested to establish Later Regionalism was taken over by Dr. Raja Nafida with support of Pusat KALAM (Centre for the Study of Built Environment in the Malay World) research center, while Dr. Alice Sabrina proceeded to form her own Socio-Culture panel called Catalyst. 59 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al 3.5. Swapping and Closing down of Thesis Supervisory Panels This effectively reduces the size of the student population in order to keep the staff:student ratio within the LAM’s stipulated numbers. various niche areas. After all, the School has experienced a situation when Workbases became too individualized that the students felt lost and too different from each other. This paper amongst others would highlight the heading of the School from the perspective of the Architectural Design Thesis. Finally, it is hoped that this paper would provide an understanding of what goes on behind the programme, particularly with regards of the Thesis Supervisory Panels. The uniqueness of the programme has been acknowledged by the visiting External Examiners as well as Accreditation Panels from MAPS Malaysia, and the School strives to maintain and further expand it in the future. Although this measure was meant to be temporary, but it was followed by the COVID- 19 pandemic that saw schools closed as a precautionary measure. The student numbers remained low (under 50 students) for the next 18 months. In a proactive measure to keep the diversity of the M.Arch panels open, some panels are kept open at half capacity. Keeping them open allows more lecturers to be involved at the post-graduate studio levels, which was particularly important when cross-exposure of the younger lecturers are needed before more senior members leave the School. 6. REFERENCES 1. Alagbe, O., Aderonmu, P., Alagbe, T., Sonola, O., Olagunju, O., & Erebor, M. (2017). Students' Perception of Design Studio Jury in Schools of Architecture in Nigeria. Paper presented at the 11th International Technology, Education and Development Conference, Valencia, Spain. 3.5. Swapping and Closing down of Thesis Supervisory Panels Figure 8 M.Arch Thesis Supervisory Panels offered according to session/semester (Program Senibina, 2022b) Head of Panel Sem 1 Sem 2 Sem 1 Sem 2 Sem 1 Sem 2 Sem 1 Sem 2 Sem 1 Sem 2 Sem 1 Sem 2 Sem 1 Sem 2 Sem 1 Sem 2 Sem 1 Sem 2 Tareef HOME HOME HOME HOME HOME HOME HOME HOME Halim SoCult SoCult SoCult Samsiah SoCult SoCult SoCult SoCult SoCult SoCult SoCult Khairul Vector Vector Vector Vector Vector Vector Vector Vector Vector Vector Vector Vector Vector Raja Nafida Regionalism Alice RegionalismRegionalism Catalyst Catalyst Catalyst Catalyst Catalyst Catalyst Mahmud Citra Roshida ENVI ENVI Wan Zakri Urban I Urban I Wan Hashimah Urban II Urban II Urb.United Urb.United Urb.United Urb.United Urb.United Urb.United Sharifah New Spirit New Spirit New Spirit New Spirit New Spirit New Spirit New Spirit New Spirit New Spirit New Spirit New Spirit New Spirit New Spirit New Spirit New Spirit New Spirit Chan WL Urb.Ren Urb.Ren Urb.Ren Urb.Ren Urb.Ren Re.Urban Hazrina Praxis Zin K Environme ntal Environme ntal Environme ntal Environme ntal Environme ntal Environme ntal Remaz Env.Tec Env.Tec Env.Tec Env.Tec Env.Tec Env.Tec Lim YW Env.Tec Env.Tec Amin Env.Arc Env.Arc Env.Arc Rashid Reform Azari Recursive Recursive Recursive Recursive Recursive Recursive Doris Lestari Lestari Lestari Roshida Enviro Enviro Enviro Enviro Razin TRD Group TRD Group SOCIO-CULTURE URBANISM SUSTAINABLE TECHNOLOGIES 2021/22 2014/15 2015/16 2013/14 2016/17 2017/18 2018/19 2019/20 2020/21 Figure 8 M.Arch Thesis Supervisory Panels offered according to session/semester (Program Senibina, 2022b) TSP can also be suspended or closed down for various reasons or needs. Typically, the number of available TSP is determined by the contemporary enrolement of pre-thesis students, where bigger student population warrants new panels to be opened while smaller population would suspend one or two panels. Table 5 TSP abbreviation and full name Abbreviation Full Name HOME SoCult ENVI Citra Urb.Ren Recursive Env.Tec Env.Arc TRD Group Housing and Community Socio-Culture Environmental Behaviour Citra Regionalisme Urban Renaissance Recursive Design Studio Environmental Tectonic Environmental Architecture The Responsible Designer Group Table 5 TSP abbreviation and full name 59 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al Since the beginning of M.Arch, the smallest number of operating TSP was four. This happened in 2019 and continued for three semesters due to the reduction of teaching staff where the retiring members were not replaced quickly enough by newer members. 5. The authors sincerely acknowledge Architecture Programme of Universiti Teknologi Malaysia (UTM) for allowing access to data and information relevant for the production of this paper. Throughout the years, three long-term TSP have closed indefinitely: HOME, SoCult, Urban (Urban I, Urban II and Urban United) and Enviro-Tectonic. As discussed earlier in this paper, the TSPs are a manifestation of the undergraduate studio workbases, and are in essence led by the individual lecturers. Some TSPs are so intertwined with the lecturer incharge that the entire aspiration and movement inspired by the panel ends when the lecturer left the School. Throughout the years, three long-term TSP have closed indefinitely: HOME, SoCult, Urban (Urban I, Urban II and Urban United) and Enviro-Tectonic. As discussed earlier in this paper, the TSPs are a manifestation of the undergraduate studio workbases, and are in essence led by the individual lecturers. Some TSPs are so intertwined with the lecturer incharge that the entire aspiration and movement inspired by the panel ends when the lecturer left the School. CONCLUSION 4. 2. Broadfoot, O., & Bennett, R. (2003). Design studios: online. Paper presented at the Apple University Consortium Academic and Developers Conference Proceedings 2003. 2. Broadfoot, O., & Bennett, R. (2003). Design studios: online. Paper presented at the Apple University Consortium Academic and Developers Conference Proceedings 2003. As initially outlined earlier, this paper provides a narrative on the Thesis Supervisory Panels in UTM’s Architectural Design Thesis programme. The Design Thesis used to be just the final major design exercise before the students graduate undertaken during the final year of B.Arch, but it has evolved into a complex, manifesto driven exploratory studies centered around the students’ own interests. Their accumulated knowledge, skills and abilities will be tested to ensure they fulfill the requirements of LAM’s Graduate Attribute. 3. Ibrahim, N. L. N., & Utaberta, N. (2012). Learning in Architecture Design Studio. Procedia - Social and Behavioral Sciences, 60, 30-35. doi: https://doi.org/10.1016/j.sbspro.2012. 09.342 4. Jabatan Senibina. (1986). Memorandum Akreditasi Program Sarjana Muda Senibina UTM. Skudai, Johor. Keeping track of the growth and development of the TSPs is critical for the future of the school, as the School ventures deeper into 60 60 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al 13. Program Senibina. (2018). Thesis Briefing 2018/2019. Skudai, Johor. 5. Jabatan Senibina. (2001). Workbase Informatik Sesi 2001/2002 (A. H. Hussein Ed.). Skudai, Johor: Fakulti Alam Bina, UTM. 14. Program Senibina. (2021). 2020/2021 Programme of Architecture UTM Continuing Accreditation by Lembaga Arkitek Malaysia. 2022, from https://sites.google.com/utm.my/utm- architecture/intro-mapsm 6. Jabatan Senibina. (2002). Unit Workbase Booklet 2002/2003. Skudai, Johor: Jabatan Senibina. 7. Jabatan Senibina. (2005). Memorandum Akreditasi Program Sarjana Muda Senibina. Skudai, Johor. 15. Program Senibina. (2022a). M.Arch Studio Brief 2021/2022. The Architecture Programme, Universiti Teknologi Malaysia, Skudai, Johor. 8. Jabatan Senibina. (2016). Design Matrix for M.Architecture. Jabatan Senibina, UTM, Johor, Malaysia. 16. Program Senibina. (2022b). M.Arch Thesis Coordination File. UTM Johor Bahru: Universiti Teknologi Malaysia. 9. Kosman, K. A. (2011). Sejarah perkembangan pendidikan senibina di Universiti Teknologi Malaysia dari 1955 hingga 2005. (Doctoral Dissertation), Universiti Teknologi Malaysia, Skudai, Johor. 17. Senibina, J. (2007). Unit Workbase Booklet Semester 2007/2008. Skudai, Johor: Fakulti Alam Bina, UTM. 18. Syed Ariffin, S. A. I. (2008). Consultant Editor's Note. In C. S. Ang (Ed.), An Alternative Book: Design Thesis Folio 2007 (pp. 9-10). Skudai, Johor: Penerbit UTM Press. 10. Lawson, B. (1997). How Designers Think: The Design Process Demystified (Third Edition ed.). 61 Journal of Design and Built Environment, Vol22(2) 2022, 48-61, August 2022 Azari M.Y. et.al CONCLUSION Oxford: Architectural Press. 19. Tafahomi, R. (2021). An Attempt to Fill the Gap between the Architectural Studies and Conceptualization in Architectural Thesis Design Studio. Journal of Design Studio, 3(2), 175- 190. doi: 10.46474/jds.1012778 11. Majlis Akreditasi dan Pengajian Senibina Malaysia. (2013). The Manual of Accreditation for Architecture Programme: Institute Sultan Iskandar of Urban Habitat and Highrise, Universiti Teknologi Malaysia. 20. Zairul, M. (2018). Introducing Studio Oriented Learning Environment (SOLE) in UPM, Serdang: Accessing Student-Centered Learning (SCL) in the Architectural Studio. Archnet- IJAR: International Journal of Architectural Research, 12(1), 241- 250. 12. Mat Yasir, A., Syed Ariffin, S.A.I., Ahmad, A.S., Mohd Jusan, M., Ismail, A.S., Abd Majid, R., Lim, Y.L. (Eds.) (Ed.). (2021). Architecture Studio Guide Book (Vol. 1): Universiti Teknologi Malaysia.
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https://figshare.com/articles/journal_contribution/Figure_S3_from_CREBBP_EP300_Bromodomain_Inhibition_Affects_the_Proliferation_of_AR-Positive_Breast_Cancer_Cell_Lines/22515885/1/files/39978417.pdf
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Figure S2 from CREBBP/EP300 Bromodomain Inhibition Affects the Proliferation of AR-Positive Breast Cancer Cell Lines
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Figure S3 Figure S3 EGR1 IFI6 HLA-B IRF9 ISG15 IRF7 HLA-A HLA-C IFIT1 IP6K2 IRF1 IFI27 OASL HLA-F JAK1 IRF3 IRF6 PTPN6 OAS1 SOCS1 RNASEL NES = 2.23 FDR = 0.003 P = 0 ORC3 CDK2 CDT1 DBF4 RPA1 RPA2 PRIM1 MCM7 CDC6 MCM4 MCM6 POLA1 MCM3 PRIM2 POLA2 MCM5 CDC45 POLE2 MCM8 MCM2 MCM10 ORC1 NES = -2.29 FDR < 0.001 P = 0DMSOJQ1CBP30DMSOJQ1CBP30 INTERFERON_ALPHA_BETA _SIGNALLING ACTIVATION _OF_THE PREREPLICATIVE_COMPLEX CBP30 vs. DMSO CBP30 vs. DMSO B A Downregulated genes MDA-MB-231 SKBR3 MDA-MB-453 REACTOME PATHWAY CBP30 JQ1 CBP30 JQ1 CBP30 JQ1 CLASS_A1_RHODOPSIN_LIKE_RECEPTORS INTERFERON_ALPHA_BETA_SIGNALING ACTIVATION_OF_THE_PRE_REPLICATIVE_COMPLEX DNA_REPLICATION COLLAGEN_FORMATION NCAM1_INTERACTIONS Upregulated genes MDA-MB-231 SKBR3 MDA-MB-453 REACTOME PATHWAY CBP30 JQ1 CBP30 JQ1 CBP30 JQ1 MRNA_PROCESSING ACTIVATION_OF_THE_PRE_REPLICATIVE_COMPLEX GLYCOLYSIS INTERFERON_ALPHA_BETA_SIGNALING TRANSCRIPTION ES 0.7 0.0 0.0 -0.8 min max A Upregulated genes MDA-MB-231 SKBR3 MDA-MB-453 REACTOME PATHWAY CBP30 JQ1 CBP30 JQ1 CBP30 JQ1 MRNA_PROCESSING ACTIVATION_OF_THE_PRE_REPLICATIVE_COMPLEX GLYCOLYSIS INTERFERON_ALPHA_BETA_SIGNALING TRANSCRIPTION Upregulated genes MDA-MB-231 SKBR3 MDA-MB-453 REACTOME PATHWAY CBP30 JQ1 CBP30 JQ1 CBP30 JQ1 MRNA_PROCESSING ACTIVATION_OF_THE_PRE_REPLICATIVE_COMPLEX GLYCOLYSIS INTERFERON_ALPHA_BETA_SIGNALING TRANSCRIPTION Downregulated genes MDA-MB-231 SKBR3 MDA-MB-453 REACTOME PATHWAY CBP30 JQ1 CBP30 JQ1 CBP30 JQ1 CLASS_A1_RHODOPSIN_LIKE_RECEPTORS INTERFERON_ALPHA_BETA_SIGNALING ACTIVATION_OF_THE_PRE_REPLICATIVE_COMPLEX DNA_REPLICATION COLLAGEN_FORMATION NCAM1_INTERACTIONS B EGR1 IFI6 HLA-B IRF9 ISG15 IRF7 HLA-A HLA-C IFIT1 IP6K2 IRF1 IFI27 OASL HLA-F JAK1 IRF3 IRF6 PTPN6 OAS1 SOCS1 RNASEL NES = 2.23 FDR = 0.003 P = 0 ORC3 CDK2 CDT1 DBF4 RPA1 RPA2 PRIM1 MCM7 CDC6 MCM4 MCM6 POLA1 MCM3 PRIM2 POLA2 MCM5 CDC45 POLE2 MCM8 MCM2 MCM10 ORC1 NES = -2.29 FDR < 0.001 P = 0DMSOJQ1CBP30DMSOJQ1CBP30 INTERFERON_ALPHA_BETA _SIGNALLING ACTIVATION _OF_THE PREREPLICATIVE_COMPLEX CBP30 vs. DMSO CBP30 vs. DMSO B ES 0.7 0.0 0.0 -0.8 min max EGR1 IFI6 HLA-B IRF9 ISG15 IRF7 HLA-A HLA-C IFIT1 IP6K2 IRF1 IFI27 OASL HLA-F JAK1 IRF3 IRF6 PTPN6 OAS1 SOCS1 RNASEL NES = 2.23 FDR = 0.003 P = 0 INTERFERON_ALPHA_BETA _SIGNALLING CBP30 vs. DMSO B ES 0.7 0.0 mi
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https://zenodo.org/records/7889054/files/IJISRT23APR1646.pdf
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Utilizing the Monte Carlo Simulation for Estimating Construction Costs of Residential Buildings
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Keywords:- Monte Carlo Simulation; Residential Building; Cost Overrun. Volume 8, Issue 4, April – 2023 Volume 8, Issue 4, April – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 I. Risks in construction projects are unavoidable. Cost/budget risk is one of the risks that residential building developers have to deal with [1]. It is not uncommon that the developer allocated an unrealistic budget for its residential project which could hinder the completion of the project [2]. The Monte Carlo Simulation method is frequently used in construction project schedule [2]–[5] and cost [6]–[9] risk analysis. Monte Carlo simulation is particularly advantageous in project management because it is a highly effective method for comprehending and measuring the possible impacts of project risks in both cost and schedule [10]. The objective of this study is to offer a feasible approach for utilizing the Monte Carlo Simulation method to estimate the cost of a residential building project by using the Microsoft Excel spreadsheet software. Utilizing the Monte Carlo Simulation for Estimating Construction Costs of Residential Buildings 1Adnan Fadjar Civil Engineering Department Tadulako University Palu, Indonesia Abstract:- Cost risk in a residential construction project is one of the risks developers have to cope with. It is not unusual that the developer allocated a budget lower than what is needed to complete the project. This study aims to provide a practical way to estimate a more realistic cost of a construction project using the Monte Carlo Simulation method. A typical house type 36 construction activities and their respective costs were simulated with 5000 iterations to obtain the probability density function and the cumulative density function of the project cost. The study found that to avoid cost underestimation, which is a primary reason for cost overrun, the budget should be increased by 8.38% to attain a 95% probability of the project's successful completion. Cost overrun is a common risk in a construction project. According to Flyvbjerg [12] nine out of ten megaprojects had experienced cost overruns and concluded that overruns of up to 50 percent are common, and overruns of more than 50 percent are not unusual. According to Flyvbjerg et al. [13], the primary reason for cost overruns is the underestimation of costs. A. Data Data of typical house type 36 construction activities and their respective costs, as shown in Table 1, were collected from residential building developers in Palu City, the capital of Central Sulawesi Province, Indonesia. The activity cost in Table 1 is the cost of labor and material and is in Indonesian Rupiah currency (IDR). Table 1 Construction Activity and Cost Activity Cost (In thousands of IDR) Expected Maximum 1. Site clearance 100 200 2. Stake out and footing excavation 250 400 3. Footing 2650 3000 4. Plumbing 800 1000 5. Footing backfills 600 800 6. Steel fabrication 5100 6100 7. Lower tie beam 700 900 8. Wall, column, window, and door frame 8700 9500 9. Upper tie beam 650 900 10. Brick rafter 2000 2400 11. Steel Roof 7200 7700 12. Electrical rough-in 150 300 13. Wall plaster 4200 4600 14. Bathroom and toilet fixtures 1250 1400 15. Floor 4000 4400 16. Septic tank 620 750 17. Carport 420 550 18. Ceiling 3300 3800 19. Doors and windows installation 3780 4200 20. Painting 4850 5150 21. Electrical fitting 550 700 22. Cleaning 100 150 Total 51970 58900 Table 1 Construction Activity and Cost www.ijisrt.com 1187  Analyze the Results The Monte Carlo Simulation method assumed that the construction cost of house type 36 as in Table 1 was a random variable with a value between the expected and the maximum project cost i.e., between 51970 and 58900. Since the project cost is the sum of other random variables i.e., the cost of each activity, the project cost would be normally distributed. Since random variables of the project cost were normally distributed, the difference between the mean and the median should be small which was 0.016%. The true error of the simulation results was 50.260, with a percentage of only 0.09%, as shown in Table 2. This indicates that the simulation results were sufficiently accurate. The random cost of each activity in Table 1 was calculated using a Microsoft Excel function called RAND. For example, the random cost of the site clearance activity in Table 1 was computed by =RAND()*(200-100)+100. This computation generated a random number between 100 and 200. The probability density function and the cumulative density function of the simulation results after 5000 iterations are shown in Table 3 and Fig. 1. II. Risk and uncertainty are two different situations that a decision-maker may face. Risk refers to a situation where the decision-maker knows the probabilities of potential outcomes, while uncertainty is a situation where the decision-maker lacks information about the probabilities of potential outcomes [11]. IJISRT23APR1646 1187 www.ijisrt.com Volume 8, Issue 4, April – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 The true error (ε) was calculated using (2). C. Iteration Number Table 3 Probability and Cumulative Percentages Table 3 Probability and Cumulative Percentages Upper bin Frequency Probability % Cumulative % 54000 10 0.20% 0.10% 54250 43 0.86% 1.00% 54500 138 2.76% 3.10% 54750 314 6.28% 8.60% 55000 551 11.02% 19.50% 55250 828 16.56% 36.10% 55500 890 17.80% 52.80% 55750 858 17.16% 70.80% 56000 654 13.08% 85.10% 56250 401 8.02% 93.60% 56500 203 4.06% 98.20% 56750 76 1.52% 99.60% 57000 31 0.62% 99.90% More 3 0.06% 100.00% There is no definite answer on how many iterations should be run on Monte Carlo Simulation. Heijungs [16] states that 1000 or 10,000 iterations are typically needed. Burmaster and Anderson [17], and Xin [18]suggest that the iteration number should be more than 10,000, while Hongxiang and Wei [19] suggest that more than 2000 iterations should be performed. Based on the above suggestions, the number of iterations of each activity cost in this study was set to 5000 iterations which should be sufficient for this study. The % true error was computed using (3). % 𝜀= 𝜀 𝑥̅ (3) % 𝜀= 𝜀 𝑥̅ (3) Monte Carlo Simulation descriptive statistics result of 5000 iterations is shown in Table 2.  The Monte Carlo Simulation Method follows the following Steps [15].  The Monte Carlo Simulation Method follows the following Steps [15].  The Monte Carlo Simulation Method follows the following Steps [15]. Table 2 Simulation Descriptive Statistics  Create a Quantitative Model of Y=F(X1, X2, …,Xn);  Generate Random Variables X1, X2, …,Xn;  Evaluate the Model and Output Stored in Yi;  Repeat Steps 2 and 3 as Needed (for I=1 To K);  Analyze the Results Table 2 Simulation Descriptive Statistics Parameters Results Mean 55428.639 Median 55420.046 % Difference between Median and Mean 0.016% Standard Deviation 529.788 True Error 50.260 % True Error 0.09%  Create a Quantitative Model of Y=F(X1, X2, …,Xn);  Generate Random Variables X1, X2, …,Xn;  Evaluate the Model and Output Stored in Yi;  Repeat Steps 2 and 3 as Needed (for I=1 To K);  Analyze the Results  Create a Quantitative Model of Y=F(X1, X2, …,Xn);  Generate Random Variables X1, X2, …,Xn;  Evaluate the Model and Output Stored in Yi;  Repeat Steps 2 and 3 as Needed (for I=1 To K);  Analyze the Results B. Monte Carlo Simulation Method The Monte Carlo Simulation method involves repeatedly simulating the entire system by randomly selecting values for each variable from their respective probability distributions [10]. This is done numerous times, often thousands, resulting in a probability distribution of the system's overall value, which is calculated based on the iterations of the model [14]. 𝜀= 3𝜎 √𝑁 (2) 𝜀= 3𝜎 √𝑁 (2) The % true error was computed using (3). V. The Monte Carlo Simulation method can be a useful tool for residential construction developers or project managers to anticipate cost-related risk, which is not uncommon in a construction project. The results obtained from the Monte Carlo Simulation method can help residential construction developers to allocate a more realistic budget and successfully deliver the project. [4]. A. Namazian, S. H. Yakhchali, V. Yousefi, and J. Tamošaitienė, “Combining Monte Carlo Simulation and Bayesian Networks Methods for Assessing Completion Time of Projects under Risk,” Int. J. Environ. Res. Public. Health, vol. 16, no. 24, p. 5024, Dec. 2019, doi: 10.3390/ijerph16245024. However, the accuracy of the Monte Carlo Simulation method heavily relies on its input variables, which in this study are cost estimations. It should also be noted that the Monte Carlo Simulation method is not a solution provider, but rather a tool that helps predict system behavior by taking into account the risks inherent in construction project budgeting. [5]. J. Sobieraj and D. Metelski, “Project Risk in the Context of Construction Schedules—Combined Monte Carlo Simulation and Time at Risk (TaR) Approach: Insights from the Fort Bema Housing Estate Complex,” Appl. Sci., vol. 12, no. 3, p. 1044, Jan. 2022, doi: 10.3390/app12031044. A. Simulation Results After 5000 iterations, the standard deviation (σ) was obtained using (1). 𝜎= √ ∑(𝑥−𝑥̅)2 𝑁 (1) 𝜎= √ ∑(𝑥−𝑥̅)2 𝑁 (1) The cumulative density function in Table 3 can be used to find the probability of the project's success based on the available budget. If, for instance, the developer can only provide a budget of IDR 55,5000K then the probability of the project can be executed successfully is only 55.48% as depicted in the solid lines in Fig. 1. Where N = the population number; and 𝑥̅ = population mean. IJISRT23APR1646 www.ijisrt.com IJISRT23APR1646 1188 www.ijisrt.com Volume 8, Issue 4, April – 2023 Volume 8, Issue 4, April – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Likewise, if the developer wants to complete the project successfully with a 95% probability, by interpolating the numbers in Table 3, it was found that the developer needs to provide a budget of IDR 56,328K as depicted in the dotted lines in Fig. 1. In other words, the developer has to increase the project budget by 8.38% over its expected budget of IDR 51,970K to attain 95% probability of the project successful completion. lines in Fig. 1. In other words, the developer has to increase the project budget by 8.38% over its expected budget of IDR 51,970K to attain 95% probability of the project successful completion. Fig 1 Probability and Cumulative Density Function 10 43 138 314 551 828 890 858 654 401 203 76 31 3 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0 100 200 300 400 500 600 700 800 900 1000 54000 54250 54500 54750 55000 55250 55500 55750 56000 56250 56500 56750 57000 More Frequency Cost Frequency Cumulative % Fig 1 Probability and Cumulative Density Function V. [3]. Z. Kong, J. Zhang, C. Li, X. Zheng, and Q. Guan, “Risk Assessment of Plan Schedule by Monte Carlo Simulation,” in Proceedings of the 4th International Conference on Information Technology and Management Innovation, Shenzhen, China: Atlantis Press, 2015. doi: 10.2991/icitmi-15.2015.83. REFERENCES [6]. X. Chen, L. Cheng, G. Deng, S. Guan, and L. Hu, “Project duration-cost-quality prediction model based on Monte Carlo simulation,” J. Phys. Conf. Ser., vol. 1978, no. 1, p. 012048, Jul. 2021, doi: 10.1088/1742- 6596/1978/1/012048. [1]. P. Loizou and N. French, “Risk and uncertainty in development: A critical evaluation of using the Monte Carlo simulation method as a decision tool in real estate development projects,” J. Prop. Invest. Finance, vol. 30, no. 2, pp. 198–210, Mar. 2012, doi: 10.1108/14635781211206922. [7]. K. W. Chau, “Monte Carlo simulation of construction costs using subjective data,” Constr. Manag. Econ., vol. 13, no. 5, pp. 369–383, Sep. 1995, doi: 10.1080/01446199500000042. [2]. N. H. Kadume and H. I. Naji, “Building Schedule Risks Simulation by Using BIM with Monte Carlo Technique,” IOP Conf. Ser. Earth Environ. Sci., vol. 856, no. 1, p. 012059, Sep. 2021, doi: 10.1088/1755- 1315/856/1/012059. [8]. F. Allahi, L. Cassettari, and M. Mosca, “Stochastic Risk Analysis and Cost Contingency Allocation Approach for Construction Projects Applying Monte Carlo Simulation,” 2017. IJISRT23APR1646 www.ijisrt.com 1189 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 4, April – 2023 [9]. B. A. Traynor and M. Mahmoodian, “Time and cost contingency management using Monte Carlo simulation,” Aust. J. Civ. Eng., vol. 17, no. 1, pp. 11– 18, Jan. 2019, doi: 10.1080/14488353.2019.1606499. [10]. Y. H. Kwak and L. Ingall, “Exploring Monte Carlo Simulation Applications for Project Management,” IEEE Eng. Manag. Rev., vol. 37, no. 2, p. 9, 2009. [11]. K. F. Park and Z. Shapira, “Risk and Uncertainty,” in The Palgrave Encyclopedia of Strategic Management, M. Augier and D. J. Teece, Eds., London: Palgrave Macmillan UK, 2017, pp. 1–7. doi: 10.1057/978-1-349-94848-2_250-1. [12]. B. Flyvbjerg, “What you Should Know about Megaprojects and Why: An Overview,” Proj. Manag. J., vol. 45, no. 2, pp. 6–19, Apr. 2014, doi: 10.1002/pmj.21409. [13]. B. Flyvbjerg, M. S. Holm, and S. Buhl, “Underestimating Costs in Public Works Projects: Error or Lie?,” J. Am. Plann. Assoc., vol. 68, no. 3, pp. 279–295, Sep. 2002, doi: 10.1080/01944360208976273. [14]. O. B. Tokdemir, H. Erol, and I. Dikmen, “Delay Risk Assessment of Repetitive Construction Projects Using Line-of-Balance Scheduling and Monte Carlo Simulation,” J. Constr. Eng. Manag., vol. 145, no. 2, p. 04018132, Feb. 2019, doi: 10.1061/(ASCE)CO.1943-7862.0001595. [15]. D. Vose, “Quantitative Risk Analysis: A Guide to Monte Carlo Simulation Modelling. John Wiley & Sons, New York, NY,” 1996. [16]. R. REFERENCES Heijungs, “On the number of Monte Carlo runs in comparative probabilistic LCA,” Int. J. Life Cycle Assess., vol. 25, no. 2, pp. 394–402, Feb. 2020, doi: 10.1007/s11367-019-01698-4. [17]. D. E. Burmaster and P. D. Anderson, “Principles of Good Practice for the Use of Monte Carlo Techniques in Human Health and Ecological Risk Assessments,” Risk Anal., vol. 14, no. 4, pp. 477– 481, Aug. 1994, doi: 10.1111/j.1539- 6924.1994.tb00265.x. [18]. L. Xin, “Uncertainty and Sensitivity Analysis of a simplified ORWARE model for Jakarta,” Stockholm. [Online]. Available: https://www.diva-portal. org/smash/get/diva2:411539/FULLTEXT01.pdf [19]. C. Hongxiang and C. Wei, “Uncertainty Analysis by Monte Carlo Simulation in a Life Cycle Assessment of Water-Saving Project in Green Buildings,” Inf. Technol. J., vol. 12, no. 13, pp. 2593–2598, Jun. 2013, doi: 10.3923/itj.2013.2593.2598. 1190 IJISRT23APR1646 www.ijisrt.com
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Analysis of a new begomovirus unveils a composite element conserved in the CP gene promoters of several Geminiviridae genera: Clues to comprehend the complex regulation of late genes
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RESEARCH ARTICLE OPEN ACCESS Citation: Cantu´-Iris M, Pastor-Palacios G, Mauricio-Castillo JA, Bañuelos-Herna´ndez B, Avalos-Calleros JA, Jua´rez-Reyes A, et al. (2019) Analysis of a new begomovirus unveils a composite element conserved in the CP gene promoters of several Geminiviridae genera: Clues to comprehend the complex regulation of late genes. PLoS ONE 14(1): e0210485. https://doi.org/ 10.1371/journal.pone.0210485 Analysis of a new begomovirus unveils a composite element conserved in the CP gene promoters of several Geminiviridae genera: Clues to comprehend the complex regulation of late genes Mariana Cantu´-Iris1, Guillermo Pastor-Palacios2, Jorge Armando Mauricio-Castillo3, Bernardo Bañuelos-Herna´ndez4, Jesu´s Aaro´n Avalos-Calleros1, Alejandro Jua´rez- ReyesID1, Rafael Rivera-Bustamante5, Gerardo R. Argu¨ello-AstorgaID1* Mariana Cantu´-Iris1, Guillermo Pastor-Palacios2, Jorge Armando Mauricio-Castillo3, Bernardo Bañuelos-Herna´ndez4, Jesu´s Aaro´n Avalos-Calleros1, Alejandro Jua´rez- ReyesID1, Rafael Rivera-Bustamante5, Gerardo R. Argu¨ello-AstorgaID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Divisio´n de Biologı´a Molecular, Instituto Potosino de Investigacio´n Cientı´fica y Tecnolo´gica A. C., San Luis Potosı´, SLP, Me´xico, 2 CONACYT–CIIDZA–Instituto Potosino de Investigacio´n Cientı´fica y Tecnolo´gica A. C., San Luis Potosı´, SLP, Me´xico, 3 Unidad Acade´mica de Agronomı´a, Universidad Auto´noma de Zacatecas, Cieneguillas, Zacatecas, Me´xico, 4 Facultad de Agronomı´a y Veterinaria, Universidad De La Salle Bajio, Avenida Universidad 602, Lomas del campestre, Leo´n Guanajuato, Me´xico, 5 Departamento de Ingenierı´a Gene´tica de Plantas, Centro de Investigacio´n y de Estudios Avanzados del IPN, Irapuato, Gto., Me´xico * grarguel@ipicyt.edu.mx Abstract A novel bipartite begomovirus, Blechum interveinal chlorosis virus (BleICV), was character- ized at the genome level. Comparative analyses revealed that BleICV coat protein (CP) gene promoter is highly divergent from the equivalent region of other begomoviruses (BGVs), with the single exception of Tomato chino La Paz virus (ToChLPV) with which it shares a 23-bp phylogenetic footprint exhibiting dyad symmetry. Systematic examination of the homologous CP promoter segment of 132 New World BGVs revealed the existence of a quasi-palindromic DNA segment displaying a strongly conserved ACTT-(N7)-AAGT core. The spacer sequence between the palindromic motifs is constant in length, but its sequence is highly variable among viral species, presenting a relaxed consensus (TT)GGKCCCY, which is similar to the Conserved Late Element or CLE (GTGGTCCC), a putative TrAP- responsive element. The homologous CP promoter region of Old World BGVs exhibited a distinct organization, with the putative TATA-box overlapping the left half of the ACTT-N7 composite element. Similar CP promoter sequences, dubbed “TATA-associated composite element” or TACE, were found in viruses belonging to different Geminiviridae genera, hence hinting unsuspected evolutionary relationships among those lineages. To get cues about the TACE function, the regulatory function of the CLE was explored in distinct experimental systems. Transgenic tobacco plants harboring a GUS reporter gene driven by a promoter composed by CLE multimers expressed high beta-glucuronidase activity in absence of viral factors, and that expression was increased by begomovirus infection. On the other hand, the TrAP-responsiveness of a truncated CP promoter of Tomato golden mosaic virus (TGMV) was abolished by site-directed mutation of the only CLE present in it, whereas the artificial addition of one CLE to the -125 truncated promoter strongly enhanced the Editor: Hanu R. Pappu, Washington State University, UNITED STATES Received: June 6, 2018 Accepted: December 25, 2018 Published: January 23, 2019 Copyright: © 2019 Cantu´-Iris 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. Introduction The family Geminiviridae is the largest group of plant viruses, with >440 recognized species distributed throughout all land ecosystems with warm and temperate climates around the world [1]. Geminiviruses have small genomes composed of one or two circular single-stranded DNA molecules encapsulated into twinned quasi-icosahedral virions [2]. These viruses infect a broad variety of wild plants and agricultural crops, causing significant shrinkage of staple food supplies and huge economic losses worldwide [3,4]. Geminiviruses are transmitted by sap- sucking insects in the order Hemiptera [5], and the transmission process is highly specific and dependent on the viral coat protein (CP) [6,7]. Accordingly, the insect vector and the virus genome organization are the main criteria to classify the family Geminiviridae into separate taxons. Nine genera are currently recognized: Becurtovirus, Begomovirus, Capulavirus, Curto- virus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus and Turncurtovirus [8,9].The bego- moviruses (BGVs) constitute the largest genus, comprising nearly 88% of all geminivirus species (https://talk.ictvonline.org/taxonomy/). BGVs solely infect dicots, and are transmitted by the cosmopolitan agricultural pest Bemisia tabaci, a complex of cryptic, morphologically indistinguishable whitefly species [10,11]. Two major lineages of BGVs have been largely rec- ognized on the basis of their genome arrangement and geographical distribution: the Old World (OW: Eurasia, Africa, and Oceania) and the New World BGVs (NW: the Americas) [12]. With only two reported exceptions [13,14] the NW BGVs possess a bipartite genome (DNA-A and DNA-B), whereas the OW BGVs include both bipartite and monopartite species. p p p The DNA-A of NW BGVs typically contains five open reading frames (ORFs), one in the virion-sense strand (AV1 or CP) encoding the coat protein, and four in the complementary sense (AC1 or Rep; AC2 or Trap; AC3 or Ren; and AC4) encoding proteins involved in a variety of essential functions for the virus infective cycle: replication, interference of the plant cell cycle, temporal regulation of viral gene expression, and suppression of host antiviral responses [15,16]. The DNA-B, encodes two proteins, BV1 or NSP, and BC1 or MP, which participate in the intra- and intercellular movement of viral DNA, respectively [15,16]. The DNA-A and DNA-B molecules of bipartite BGVs are very different in sequence, with the exception of a shared DNA segment ranging from 140 to 200-nt in length, which contains the virus replica- tion origin. Discovery of a composite element in the CP gene promoter of geminiviruses transactivation level in tobacco protoplasts. These results indicate that the CLE is a TrAP- responsive element, hence providing valuable clues to interpret the recurrent association of the CLE with the TACE. On the basis of the aforesaid direct evidences and the insights afforded by the extensive comparative analysis of BleICV CP promoter, we propose that the TACE might be involved in the TrAP-mediated derepression of CP gene in vascular tissues. collection and analysis, decision to publish, or preparation of the manuscript. collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Editor: Hanu R. Pappu, Washington State University, UNITED STATES Editor: Hanu R. Pappu, Washington State University, UNITED STATES Received: June 6, 2018 Accepted: December 25, 2018 Published: January 23, 2019 Copyright: © 2019 Cantu´-Iris 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: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by the Consejo Nacional de Ciencia y Tecnologı´a (CONACYT) of Mexico (URL- https://www.conacyt. gob.mx/). Grant SEP-CB-2015- 257136 to GRAA. The funders had no role in study design, data 1 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Introduction Lacatus and Sunter [29] demonstrated that the sequences mediating repression and activation of TGMV and Cabbage leaf curl virus (CbLCuV) CP promoters are bound by different nuclear factors common to three plant species, whereas TGMV TrAP can interact with both sequences independently, as it was determined by chromatin immunopre- cipitation assays. These observations indicate that TrAP could interact with diverse plant tran- scription factors, in addition to the Arabidopsis PEAPOD-2 protein [30] to differentially regulate the CP promoter activity in mesophyll and vascular tissues. The former evidence and the lack of virus-specific functions of TrAP naturally lead to the idea that promoters of BGV late genes might contain shared sequences functioning as TrAP-responsive elements. In an early comparative analysis of the intergenic region of bipartite and monopartite BGVs, a sequence motif (i.e., GTGGTCCC) was identified in many CP and BV1 (NSP) promot- ers. This sequence, termed "conserved late element” (CLE), was postulated to be a functional target for TrAP [31]. A subsequent study of the Pepper huasteco yellow vein virus (PHYVV) CP promoter lent experimental support to the CLE hypothesis. Transgenic tobacco plants with a truncated CP promoter, termed -115CP, fused to the GUS gene exhibited barely detectable glu- curonidase activity in all analyzed tissues; this activity was strongly increased with infection by PHYVV, hence indicating that the truncated -115CP promoter is still responsive to the viral transactivator. This short promoter contains three sequences identical to the CLE [32]. Fur- thermore, synthetic promoters including one or two CLEs fused to the CaMV 35S minimal promoter exhibited TrAP-responsiveness in transient expression assays [32]. The actual importance of the CLE in the transactivation of TGMV CP promoter was questioned in a study by Sunter and Bisaro [33] who deleted the only canonical CLE present in a -657 CP pro- moter:GUS construct and did not observe significant effect on the TrAP-responsiveness of the CP promoter. However, in the same study the authors delimited the minimal TGMV CP pro- moter which is responsive to the transactivator, and concluded that an element essential for TrAP-mediated activation lies between -125 and -107 (5´-CGTCTAAGTGGTCCCGCA-3’), an 18-bp long region where the CLE is located [33]. Since the latter element is presumably irrelevant for the transactivation process, and the sequences flanking the CLE in the -125 and -107 region are not conserved in other BGVs, the conclusions of this study seem paradoxical, and deserve a careful analysis. Introduction The Ori, also known as “common region” in bipartite BGVs, is a part of the inter- genic region (IR) that includes bidirectional promoters controlling the expression of the virion-sense and the complementary-sense genes of each genomic component. The CP gene is expressed at the late stage of the infection process, and is regulated at the transcriptional level by a small (~14 kDa) multifunctional protein termed “transcriptional acti- vator protein” (TrAP), encoded by the AC2/C2/Trap gene [16]. TrAP is expressed at the early stage of the infection process and subsequently activates the expression of the late genes (i.e., CP and the two genes encoded in the DNA-B) [17–20]. The transactivator has three discern- able domains: a basic N-terminal domain including a nuclear localization signal, a central region that contains an atypical zinc-finger motif, and a C-terminal acidic activation domain PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 2 / 24 Discovery of a composite element in the CP gene promoter of geminiviruses that is critical for its function as transcriptional activator [21]. The last function is not virus specific as it has been demonstrated in studies of complementation of AC2 mutants [22, 23] and transactivation of heterologous CP promoters fused to a reporter gene [24]. Moreover, TrAP does not bind dsDNA in a sequence-specific manner [25, 26], like other well-character- ized viral transactivators (e,g., herpesvirus VP16 and adenovirus E1A) that recognize their tar- get elements in viral promoters through interaction with host transcription factors that do bind specific DNA sequences [27]. Diverse studies with transgenic plants harboring chimeric genes with the begomovirus CP promoter fused to the GUS reporter gene, have shown that the regulation of CP gene expres- sion is a complex process interweaving the action of different kinds of regulatory elements: i) a transcriptional silencer which suppresses the activity of the CP promoter in vascular tissues in absence of TrAP; this silencer was mapped in Tomato golden mosaic virus (TGMV) into a ~300 bp segment encompassing a part of the AC2/Trap and AC3/Ren genes [28], and ii) one or more cis-acting positive elements that activate the CP promoter in mesophyll cells of infected plants [28]. PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Materials and methods Blechum piramidatum (Acanthaceae) plants with symptoms of interveinal chlorosis were found in an area of wild vegetation located between the town of Nohacal and the archaeolog- ical site of Edzna (19˚ 36´14.78´´N; 90˚ 19´ 10.44´´W), in Campeche, Mexico. This area is out- side the protected area of Edzna, and no specific permission is required to collect. Introduction More recent advances in the knowledge of the complex regula- tion of late promoters of BGVs and other geminiviruses were reviewed by Borah et al. [34]. In this work, we describe a new begomovirus which possesses a CP promoter with an atypi- cal assortment of putative cis-regulatory elements. Comparative analyses of that CP promoter led to discover a conserved complex element with partial dyad symmetry, which is closely asso- ciated to the TATA box. 3 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Discovery of a composite element in the CP gene promoter of geminiviruses Virus source and cloning of genomic components The DNA-B was PCR amplified using two sets of degenerate primers, BC1-290rev/BV1- 310for and BC1-290for/BV1-470-rev, as previously described [37]; the latter primers are com- plementary to conserved sequences in the B genomic component of New World BGVs. The amplicons were cloned into pGEM-T Easy Vector (Promega) and analysed in restriction frag- ment length polymorphism (RFLP) assays using EcoRI and HinfI endonucleases. The digestion products were separated by electrophoresis in 2.5% agarose gels, and PCR clones exhibiting distinct restriction patterns were identified. Independent clones with a similar restriction pat- tern were sequenced and full-length contigs were then assembled. DNA sequencing and sequence analysis Automated sequencing was carried out at LANBAMA (IPICYT, San Luis Potosı´, Mexico) using a 3130 Genetic Analyzer (Applied Biosystems). The overlapping DNA fragments were subsequently assembled using SeqMan of DNAStar software (DNAStar Inc., Madison, WI, USA). The assembled sequences of the DNA-A and DNA-B components were compared with those from the NCBI database using the Nucleotide Basic Local Alignment Search Tool (BLASTn). Sequences with the highest scores were selected for comparison by alignment using MUSCLE [38] and pairwise nucleotide sequence identities were calculated with Sequence Demarcation Tool (SDT v1.2) [39]. Virus source and cloning of genomic components Leaf material from four symptomatic plants of the weed B.pyramidatum were collected (November 2011) in an area of wild vegetation site between the town Nohacal and the archaeo- logical zone of Edzna´, in Campeche, Mexico. Total DNA was obtained from dried leaf samples by a modified Dellaporta method [35]. To increase both the quantity and quality of the DNA for subsequent manipulations, the extracts were subjected to rolling circle amplification (RCA) by using the TempliPhi kit (New England Biolabs) following the recommendations of the manufacturer. The existence of BGVs in the samples was assessed by PCR using several degenerate primers: RepDGR-for/ CpYMAC-rev [36], RepYIDK-rev (5´-CAAGTCCTACA TCGACAAGGAYGGAGA-3) and Cp-EGP70-for (5´-GGTTGTGAAGGNCCNTGTAAGGTYC Leaf material from four symptomatic plants of the weed B.pyramidatum were collected (November 2011) in an area of wild vegetation site between the town Nohacal and the archaeo- logical zone of Edzna´, in Campeche, Mexico. Total DNA was obtained from dried leaf samples by a modified Dellaporta method [35]. To increase both the quantity and quality of the DNA for subsequent manipulations, the extracts were subjected to rolling circle amplification (RCA) by using the TempliPhi kit (New England Biolabs) following the recommendations of the manufacturer. The existence of BGVs in the samples was assessed by PCR using several degenerate primers: RepDGR-for/ CpYMAC-rev [36], RepYIDK-rev (5´-CAAGTCCTACA TCGACAAGGAYGGAGA-3) and Cp-EGP70-for (5´-GGTTGTGAAGGNCCNTGTAAGGTYC A-3´). The DNA-A was amplified by PCR using two pairs of primers that produce amplicons overlapping along a ~520-bp segment, and that jointly encompass the full A genomic compo- nent. The DNA-B was PCR amplified using two sets of degenerate primers, BC1-290rev/BV1- 310for and BC1-290for/BV1-470-rev, as previously described [37]; the latter primers are com- plementary to conserved sequences in the B genomic component of New World BGVs. The amplicons were cloned into pGEM-T Easy Vector (Promega) and analysed in restriction frag- ment length polymorphism (RFLP) assays using EcoRI and HinfI endonucleases. The digestion products were separated by electrophoresis in 2.5% agarose gels, and PCR clones exhibiting distinct restriction patterns were identified. Independent clones with a similar restriction pat- tern were sequenced and full-length contigs were then assembled. p A-3´). The DNA-A was amplified by PCR using two pairs of primers that produce amplicons overlapping along a ~520-bp segment, and that jointly encompass the full A genomic compo- nent. Phylogenetic analyses For phylogenetic analyses of DNA-A and DNA-B of BleICV and representative BGVs, sequences were aligned in MEGA 7 software using Clustal W [40]. Phylogenetic trees were constructed using maximum-likelihood (ML) method based on the Tamura-Nei model. The tree support was tested by bootstrapping with 1000 replicates. For determining the percent nucleotide identity, viral sequences were aligned by MUSCLE in the sequence demarcation tool version 1.2 (SDTv1.2) software. The GenBank accession numbers of BGVs used in DNA-A and DNA-B sequence comparisons and phylogenetic reconstruction are listed in S1 Table. 4 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Discovery of a composite element in the CP gene promoter of geminiviruses Generation of tobacco transgenic plants Selected leaves of Nicotiana tabacum plants were sliced into 0.5 x 0.5 cm squares and placed on solid MS medium in contact to a suspension of Agrobacterium tumefaciens harbouring the plasmids with the promoter constructs. The leaf fragments were transferred to a MS medium with growth regulators (2mg/l 6-benzylaminopurine, BAP), 150mg/l kanamycin, 300mg/l cefotaxime, to select transformed plants using two consecutive rounds of selection. In this media the kanamycin resistance marker which is linked to promoter construct cassettes selects for transformant plants. Plants were removed and placed in MS solid medium without growth regulators. The plants were maintained at 25˚C and 16/8h light-dark cycles for the rest of the experiments. Phylogenetic-structural analysis of non-coding sequences Since conventional computer programs for DNA sequence comparisons usually fail to detect evolutionarily related but structurally variable promoter regions, several alternative approaches, such as the “phylogenetic-structural method” of sequence analysis [41], have been devised. This method is based on the search of “homologous” (rather than only “similar”) non-coding DNA sequences. In this study we identified homologous putative cis-regulatory elements by looking for conserved arrays of specific DNA motifs in geminivirus CP promoters. This was accomplished both by computer-assisted searches and/or by visual inspection of equivalent promoter segments. The criteria to identify the putative TATA box of the CP gene were its similarity to the TATA box consensus (i.e., TATAWWW), its position and distance rela- tive to the start codon of the CP gene (in NW BGVs) or, in the case of geminiviruses with over- lapped genes in the virion sense (e.g., OW BGVs, curtoviruses and topocuviruses), the TA-rich sequence closest to the first ORF in the set of genes overlapping the CP gene. Construction of chimeric promoters with CLE multimers To construct integrative vectors pBI46S-3CLE, pBI90S-3CLE and pBI46S-6CLE which contain multiple copies of the CLE element in the same orientation, a synthetic dsDNA fragment con- taining 3 copies of the CLE with 8bp spacing and HindIII and SpeI restriction sites located at the ends was designed (S3 Table). The oligonucleotides were resuspended at 100ng/μl in a buffer, containing Tris 20mM pH 7.5, MgCl2 10mM and NaCl 250mM, heated to 85˚C in a water bath for 5 minutes and were taken out to reach room temperature. The dsDNA fragment obtained was ligated into HindIII -SpeI sites upstream of truncated -46 and -90 CaMV 35S pro- moter of vectors pBI46S and pBI90S [42]. The truncated -46 promoter represents a minimal promoter which contains the TATA box and an initiator element. In contrast, truncated pro- moter -90 contains in addition an AS-1 transcriptional element. To generate pBI46S-6CLE an additional dsDNA fragment (B) Hind III compatible was cloned upstream the 3CLE copies of pBI46S-3CLE. Constructs for the study of Tomato golden mosaic virus (TGMV) CP promoter A series of progressively shorter versions of CP promoter of TGMV were generated by PCR amplification of CP promoter using designed primers (S3 Table) and cloned into pBSGUS cut with HindIII and XbaI. The generated truncated promoters were -184, -125 and -107. The pBSGUS is a pBlueScript KS-derived vector harbouring the reporter gene uidA (GUS) and an MCS for insert promoters. The constructs generated reproduced a series of promoter deletions used by Sunter and Bisaro [33] to characterize a minimal sequence required for CP gene 5 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Discovery of a composite element in the CP gene promoter of geminiviruses activation in the same virus. As control, a pair of synthetic modified-promoter constructs were generated using the same methodology. The -125 CLEmut construct was generated by using an oligonucleotide which contained a mutated CLE (changing GTGGTCCC to GTAATAAC) at the native CLE position found on the -125 construct; the construct -125(2CLE) adds an extra copy of CLE upstream of the -125 promoter construct. The relevant primers are listed in S3 Table. Transient expression assays in NT1 suspension cell protoplasts The different module-promoter constructs were tested by transient expression assays in proto- plasts prepared from Nicotiana tabacum NT1 suspension cell cultures. Protoplasts were obtained from cells at the logarithmic growth phase after treatment with an enzymatic solution containing a mixture of Trichoderma viride cellulase and Aspergillus japonicum pectolyase (Sigma-Aldrich Co.). Protoplast transfection was performed by electroporation, using 15μg of purified DNA from expression vectors and 500μF and 250v pulses in a Bio-Rad Gene Pulser XcellTM. Treated cells were then incubated at 25˚C for 48hrs. Protoplasts were harvested and total protein was measured by the Bradford method. beta-glucuronidase activity was measured in a GENios TECAN fluorescence reader through the quantification of the coloured MU prod- uct of hydrolysis of MUG catalyzed by the product of the gene uidA (GUS) using wavelengths λex = 355–372 nm and λem = 440–480 nm. PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Isolation and characterization of a novel begomovirus Leaves of Blechum pyramidatum (Acanthaceae) plants with symptoms of interveinal chlorosis (S1 Fig) were collected in Campeche, Mexico. DNA extracts of dried leaf samples (n = 4) were amplified by polymerase chain reaction (PCR) using the RepDGR-for/ CpYMAC-rev primers. Sequencing of the ~1.4 Kb amplicons from four samples showed that they contained the same virus (sequence identity: >99%). The full DNA-A sequence of the infecting BGV was obtained by sequencing a second amplicon (~1.7 Kb) that overlapped the former PCR product along a ~520 bp segment. The sequence of the virus DNA-B was obtained by an analogous procedure (see Methods). The assembled sequences of genomic components A and B (GenBank acces- sion number: JX827487, JX827488) were compared with those from the NCBI database, and pairwise nucleotide sequence identities were calculated with SDTv1.2. The sequence analyses showed that the closest relative of the Blechum-infecting virus was Tomato chino La Paz virus (ToChLPV) at 80% of full DNA-A sequence identity (Fig 1). Based on the current ICTV taxo- nomic criterion for begomoviruses establishing that a full length DNA-A sequence identity lower than 90% with other BGV is indicative of a separate species [10], and the virus was named Blechum interveinal chlorosis virus (BleICV). The BleICV genome exhibited the typical organization of the New World BGVs. The DNA-A (2645 nt) encoded one open reading frame (ORF) in the virion-sense strand (AV1/ CP), and four ORFs in the complementary strand (AC1/Rep, AC2/TrAP, AC3/REn, and AC4). On the other hand, the DNA-B (2640-nt in length) comprised two ORFs: BV1/NSP in the virion-sense strand and BC1/MP in the complementary strand. Both genomic components shared a region of 153 bp displaying a sequence identity of 94.8%; this common region included the stem-loop sequence harboring the invariant nonanucleotide TAATATTAC (the Rep-nicking site), and three GGGGGA iterons (putative Rep-binding sites) with the characteris- tic arrangement of the NW BGVs (i.e., two repeats in tandem adjacent to the TATA box and one inverted copy closer to the Rep gene start codon) [43]. 6 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Discovery of a composite element in the CP gene promoter of geminiviruses Fig 1. Maximum likelihood (ML) phylogenetic tree and colour coded matrix of pairwise sequence identity based on full- length sequences of genome components A and B. (A) DNA-A; (B) DNA-B. Isolation and characterization of a novel begomovirus ML phylogenies were constructed by multiple alignments of complete DNA-A and DNA-B sequences of BleICV and representative begomoviruses. ML method in MEGA 7 was based on the Tamura-Nei model and the tree support was tested by bootstrapping with 1000 replicates. The scale bar represents the genetic distance. The genomic sequence of Tomato pseudo-curly top virus (TPCTV), a topocuvirus, was used as outgrup. For begomovirus acronyms see S1 Table. htt //d i /10 1371/j l 0210485 001 Fig 1. Maximum likelihood (ML) phylogenetic tree and colour coded matrix of pairwise sequence identity based on full- length sequences of genome components A and B. (A) DNA-A; (B) DNA-B. ML phylogenies were constructed by multiple alignments of complete DNA-A and DNA-B sequences of BleICV and representative begomoviruses. ML method in MEGA 7 was based on the Tamura-Nei model and the tree support was tested by bootstrapping with 1000 replicates. The scale bar represents the genetic distance. The genomic sequence of Tomato pseudo-curly top virus (TPCTV), a topocuvirus, was used as outgrup. For begomovirus acronyms see S1 Table. https://doi.org/10.1371/journal.pone.0210485.g001 The phylogeny inferred with the ML method implemented in MEGA 7 showed that BleICV is related to viruses belonging to the Squash leaf curl virus (SLCV) clade, an ancient lineage of NW BGVs. This relationship is clearest for the DNA-B which is grouped in a distinct branch with three members of the former lineage (i.e., ViYMV, BCaMV and EuMV) (Fig 1B). How- ever, BleICV lacks the distinctive signatures of SLCV clade members, such as number and arrangement of iterons and several amino acid motifs in the N-terminal half of Rep, which are absent in other BGVs. Identification of a symmetric phylogenetic footprint in the BleICV CP promoter In the course of extensive BLASTn analyses we noted that the non-coding region comprised between the CP gene start codon and the conserved stem-loop element of BleICV Ori, did not show significant similarity with other begomoviral sequences in public databases, with the sole PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 7 / 24 Discovery of a composite element in the CP gene promoter of geminiviruses exception of ToChLPV, with which it shares a 73-bp promoter segment displaying 74% of sequence identity. Visual examination of BleICV sequences upstream of the latter segment identified two direct repeats of 15 bp including a (G)GGACCAC motif, which is the CLE (GTGGTCCC) in inverted orientation. None other begomovirus available in public databases displays analogous repeats, thus explaining the apparent oddness of BleICV CP promoter. The 73-bp segment shared with ToChLPV was subsequently analyzed to identify discrete “phyloge- netic footprints” (PhyF), which are defined as DNA stretches larger than 6-bp whose sequence, spacing and position relative to other cis-acting elements, is conserved in a phylogenetic pro- moter series of orthologous genes [41, 44]. The evolutionary conservation of a DNA motif in non-coding regions suggests a regulatory function; thus, it is presumed that PhyFs represent binding sites for transcription factors [44]. Three PhyFs were identified in the aforesaid region of BleICV and ToChLPV CP promoters: the putative CCAAT and TATA-box elements, and a 23 bp sequence of unknown function located between the latter cis-acting elements. In the case of ToChLPV-MM4 (DQ347949) promoter, this PhyF exhibited a perfect dyad symmetry (Fig 2). Searching for sequences homologous to the BleICV-ToChLPV symmetric PhyF in New World begomoviruses A BLASTn search for identical or similar sequences to the 23 bp palindromic element of ToChLPV-MM4 produced a single hit at 100% of identity (i.e., Bean yellow mosaic Mexico virus, BYMMV), although sequences exhibiting significant but different levels of identity were found in the CP promoter of the majority of BGVs native to the Americas. A systematic analy- sis of those sequences in the ~130 recognized species of New World BGVs was carried out by means of a phylogenetic-structural approach. This analysis revealed short promoter regions Fig 2. Region of BleICV CP promoter encompassing the three phylogenetic footprints (PhyF) shared with ToChLP. Upper module. Simplified representation of BleICV CP promoter, illustrating the two repeats with canonical CLEs (letters in red). Lower module. Phylogenetic footprints in the CP promoter of BleICV and ToChLPV (spacing nucleotides between PhyFs were omitted for simplicity). CLE = Conserved Late Element; the arrows denote either direct or inverted repeats; the blue diamond indicates the central nucleotide of the palindromic 23-nt sequence of ToChLPV. https://doi.org/10.1371/journal.pone.0210485.g002 Fig 2. Region of BleICV CP promoter encompassing the three phylogenetic footprints (PhyF) shared with ToChLP. Upper module. Simplified representation of BleICV CP promoter, illustrating the two repeats with canonical CLEs (letters in red). Lower module. Phylogenetic footprints in the CP promoter of BleICV and ToChLPV (spacing nucleotides between PhyFs were omitted for simplicity). CLE = Conserved Late Element; the arrows denote either direct or inverted repeats; the blue diamond indicates the central nucleotide of the palindromic 23-nt sequence of ToChLPV. PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 8 / 24 Discovery of a composite element in the CP gene promoter of geminiviruses that are the evolutionary counterpart to the symmetric BleICV-ToChLPV phylogenetic foot- print. Three remarkable common features of the latter regions were discerned: i) they exhibit partial dyad symmetry, with an almost invariant ACTT-N7-AAGT core sequence; ii) the hep- tanucleotide sequence (N7) between the inverted half-sites is GC-rich and highly variable in sequence; iii) they are invariably associated to the TATA-box element. The sequences of the aforesaid 25 bp-promoter region (hereafter named the “TATA box-associated composite ele- ment” or TACE) of 30 New World BGVs are shown in Fig 3. Searching for sequences homologous to the BleICV-ToChLPV symmetric PhyF in New World begomoviruses The inferred primeval sequence of this symmetric region, i.e., CAACAACTT-(N7)-AAGTTGTTG, is utterly conserved in a number of BGV species (e.g., in 10 out 30 viruses illustrated in Fig 3), whereas in other BGVs the 9 bp half-sites (“arms”) of the interrupted palindrome are unevenly conserved, as in BleICV and BDMV, that only maintain the original left and right half-sites, respectively. More- over, in several NW BGVs the dyad symmetry of its TACE is weakly preserved, such as in SiMAV, TGMV and SiYMV (Fig 3). In contrast to the strong conservation of the TACE core (i.e., ACTT-N7-AAGT), the GC- rich heptanucleotide separating the symmetrical parts of this complex element, exhibited great variability, although some specific sequences occur with higher frequency. For example, among the TACEs illustrated in Fig 3 the spacer sequence was GGGCCCT in nine of them, GGTCCCY in eight, GGTGACC in two, and the remaining eleven TACEs contained distinct spacer sequences. The consensus of the heptanucleotide spacer of the said 30 TACEs was determined (i.e., G28G28K29C24C23C28Y29). Since the spacer is preceded by the double T of the ACTT motif, the TACE 9 bp-central consensus (TTGGKCCC) is remarkably similar to the CLE (GTGGTCCC). To establish the global frequency of heptanucleotide spacers with CLE-like sequences (GGTCCCY or variants differing in a single nucleotide, like GGGCCCY, GGTGCCCY, GGTCGCY, GGTCCTY, etc.) the TACE of the 122 species of NW viruses listed in the review of the genus Begomovirus by Brown et al. [10], plus another eight recently described species, were methodically examined. The results of this analysis showed that the variability of the heptanu- cleotide separating the palindromic half-sites is much greater than that observed in the 30 TACEs illustrated in Fig 3. Nonetheless, a high proportion (~50%) of the examined New World BGVs display TACE with spacers identical or alike to the CLE core. In effect, 15 BGV species exhibit a GGTCCCY motif, whereas 50 viral species display sequences differing on a sin- gle nucleotide from the latter motif. Therefore, it can be affirmed that 65 species of NW BGVs have TACEs with "CLE-like" sequences. The names, acronyms, and GenBank accession num- bers of those 65 BGVs are summarized in S2 Table. PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Identification of sequences homologous to the TACE in Old World begomoviruses The begomoviruses of continents other than the Americas are collectively called “Old World” BGVs, which are more ancient and diverse than the NW BGVs [45,46]. An important differ- ence between the NW and OW BGVs is that the latter have a small ORF that precedes and partially overlaps the CP gene, termed AV2/V2 or “precoat” gene. Because the NW BGVs pre- sumably evolved of one or more OW lineages [45,46] it was anticipated that the TACE would be present in all or a majority of Old World BGVs. Therefore, it was somehow surprising to find very few sequences of OW BGVs in BLASTn analyses with either the BleICV-ToChLPV phylogenetic footprint or distinct TACEs of other NW BGVs. This unexpected result pro- mpted us to carefully examine the CP promoter region of the few Asian BGVs exhibiting a rec- ognizable TACE, such as Jatropha leaf crumple India virus (JaCrIV) and Sri Lankan cassava mosaic virus (SLCMV). An interesting observation was that the 3´ end of the putative TACE in these viruses is found 10 bp upstream of the AV2 gene start codon. This position is clearly PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 9 / 24 Discovery of a composite element in the CP gene promoter of geminiviruses PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 10 / 24 Discovery of a composite element in the CP gene promoter of geminiviruses Discovery of a composite element in the CP gene promoter of geminiviruses Fig 3. Symmetric region ACTT-(N7)-AAGT of 30 New World begomoviruses. GenBank accesion numbers: TYLDV (KU232891), BleICV (JX827487), ToChLPV (DQ347949), MaBYMV (KU058856), PSLDV (KT899302), BYMMV (FJ944023), AbMBV (JF694480), ToMHV (KT099130), OYMMV (HM035059), SiYMoV (HE806448), MacYMV (AJ344452), DaLCV (JN848773), ToMoV (AY965900), MacYSV (KJ939895), VEM-5 (KT099138), SiCVV (KX691405), SiYBV (KX640991), JaMV (KJ174333), TLCSiV (KY064014), CoMoV (JQ805781), VEM-3 (KT099127), CoYSV (DQ875868), SiMAV (KX896421), BDMV (M88179), TGMV-cs (JF694488), AbGMYV (KC430935), SicGMV (JX857691), SiYMV (AY090558), MaYMJV (FJ601917), TbMoLCV (FM160943). different from that observed in NW BGVs, in which the TACE is always upstream of the TATA box. Another notable feature of the putative TACEs of JaCrIV and SLCMV is that these are closely associated by its 5’-end with a canonical CLE, an arrangement which was not observed in any NW BGV. Using the TACE of those two Asian viruses as reference, we were able to identify the homologous promoter region in a large number of BGVs native to the Indian subcontinent, Asia, Africa, and Oceania. Identification of sequences homologous to the TACE in Old World begomoviruses The relevant features of the OW BGV TACEs are as follows: 1) they contain the same symmetric core ACTT-N7-AAGT like their New World counterparts, but do not exhibit extensive dyad symmetry; 2) the heptanucleotide spacer (N7) sequences are GC-rich and variable in sequence, like its homologous in NW BGVs, but they exhibit more frequently the CLE core sequence; 3) unlike the TACE of NW BGVs, which is consistently upstream of the TATA box, the TACE of the Old World BGVs is downstream of the latter element, which overlaps the TACE left arm; 4) in a considerable number of viruses from the Indian subcontinent and Asia, a canonical CLE is located near the 5 ’end of the TACE, separated from it by the putative TATA box. The CP promoter region con- taining the TACE of 30 Old World BGVs is illustrated in Fig 4. An extensive (but not all-inclu- sive) list of OW BGVs whose heptanucleotide spacer is identical (43 species) or similar (23 species) to the CLE core, is presented in the S2 Table. PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Identification of begomovirus TACE homologous in the CP promoter of curtoviruses Since the CLE is a putative TrAP-responsive element, and a considerable number of begomo- virus TACEs include CLE-like spacer sequences and/or are closely associated to CLEs, we were interested to know if geminiviruses of other genera encoding TrAP homologs also con- tain TACEs in their CP promoters. Accordingly, we examined the late promoters of curto- viruses, topocuviruses and turncurtoviruses, looking for sequences homologous to begomovirus TACEs. The genus Curtovirus includes three species native to North America, but only one of them, Beet curly top virus (BCTV), encodes a protein (C2) homologous to TrAP. BCTV comprises several strains that few years ago had the status of separate species. The analysis revealed the existence of BGV TACE homologs in all BCTV strains. For example, in BCTV-Cfh and its rel- atives the TACE homologous is a palindromic 21 bp sequence displaying 87% of identity with the equivalent element of two Asian BGVs, Synedrella leaf curl virus (SyLCV) and Ageratum enation virus (AEV). The similarity of the CP promoter regions of BCTV-Cfh and the afore- mentioned BGVs extends upstream of the TACE to include a canonical CLE (Fig 5). The authentic evolutionary relationship of BCTV and begomovirus CP promoter regions is further emphasized by its distance from the TATA box, which is similar to that observed in NW BGVs. An additional finding of this analysis was that in several BCTV isolates, such as BCTV-Logan (AF379637), the CP promoter contains three copies in tandem of the segment encompassing the CLE-TACE array (not shown) upstream of the TATA-box. Close examination of the promoter region of the partially overlapped V3/V2/CP genes of Horseradish curly top virus (HrCTV) [47] and Spinach severe curly top virus (SpiSCTV) [48], PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 11 / 24 Discovery of a composite element in the CP gene promoter of geminiviruses the two curtoviruses that do not encode TrAP homologs, revealed the existence of TACE homologous in both of them. The HrCTV TACE (5'-AAACTTGGTCGGCAAGTTT-3') is a 19-bp sequence partially symmetric, whereas the equivalent element of SpiSCTV is a 25-bp the two curtoviruses that do not encode TrAP homologs, revealed the existence of TACE homologous in both of them. The HrCTV TACE (5'-AAACTTGGTCGGCAAGTTT-3') is a 19-bp sequence partially symmetric, whereas the equivalent element of SpiSCTV is a 25-bp Fig 4. CP promoter region containing the TACE of 30 Old World begomoviruses. PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Fig 5. Alignment of the CLE-TACE region of a curtovirus and four Asian begomoviruses. Note the 17 bp segment of the BCTV, SyLCV and AEV TACEs, which is 100% identical in sequence. The distance between the CLE and the TACE is also conserved in these geminiviruses. GenBank accession numbers: BCTV-Cfh (X97203), SyLCV (KU933258), AEV (HE861940), TbCSV (KU934097), SbYCrV (AB050781). Fig 5. Alignment of the CLE-TACE region of a curtovirus and four Asian begomoviruses. Note the 17 bp segment of the BCTV, SyLCV and AEV TACEs, which is 100% identical in sequence. The distance between the CLE and the TACE is also conserved in these geminiviruses. GenBank accession numbers: BCTV-Cfh (X97203), SyLCV (KU933258), AEV (HE861940), TbCSV (KU934097), SbYCrV (AB050781). sequence displaying strong dyad symmetry (5'-CAATTCCTTGCTGGGCAAGGAATTG-3'), although its central core is CCTT-N7-AAGG, instead the canonical ACTT-N7-AAGT core. Discovery of a composite element in the CP gene promoter of geminiviruses Fig 5. Alignment of the CLE-TACE region of a curtovirus and four Asian begomoviruses. Note the 17 bp segment of the BCTV, SyLCV and AEV TACEs, which is 100% identical in sequence. The distance between the CLE and the TACE is also conserved in these geminiviruses. GenBank accession numbers: BCTV-Cfh (X97203), SyLCV (KU933258), AEV (HE861940), TbCSV (KU934097), SbYCrV (AB050781). https://doi org/10 1371/journal pone 0210485 g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Identification of begomovirus TACE homologous in the CP promoter of curtoviruses GenBank accession numbers: MaBYMV (KU058856), JaLCrIV (KM189818), SLCMV (KP455484), TLCJV (KM383747), ToLCRaV (GQ994095), TYLCVV (EU189150), EgGMV (KU569598), PaLCCNV (KU892674), ChLCVV (HM007121), ICMV (Z24758), StLCV (AJ564742), PepLCBV (JN663853), EYVThV (KY373213), CroYVMV (JX270684), RaMV (KX885030), PaLCV (LT009397), CraYVV (FN401520), AYVV (X74516), EuLCGV (AM411424), ChLCAV (KM880103), TYLCMV (LM651401), TYLCV-IL (EF523478), PepYVMV (FM876849), EACMKV (KJ887946), ToLCBV (KM383762), ToLCNV (AM701761), ToLCoV (AJ865341), EACMMV (KP890350), ToLCAntV (AM701767), SiYVVV (KF990601), AYVCNV (HG003652). Note that the first virus, MaBYMV, is an American BGV. https://doi org/10 1371/journal pone 0210485 g004 12 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 TACE homologous in CP promoters of topocuvirus and geminiviruses unassigned to a genus Careful scrutiny of the CP promoter of the two species of turncurtoviruses currently recog- nized did not reveal sequences with significant identity to the begomovirus TACE. However, the CP promoter of TPCTV, the only topocuvirus described to date, contains an element that by its sequence and proximity to the TATA box can be considered the counterpart of the New World BGV TACE, with a slightly modified 5’-end (Fig 6). The CP promoter of viruses belong- ing to other Geminiviridae genera which do not encode TrAP homologs, i.e., Becurtovirus, Capulavirus, Eragrovirus, Grablovirus and Mastrevirus, were also examined; nonetheless, we were unable to identify homologous to BGV TACEs in the upstream sequences of their late genes. In addition to the currently recognized genera, there are five species of geminiviruses that have not yet been assigned to a genus due to their peculiar genome organization and high divergence in overall genome sequence. Their characteristics might eventually lead to the establishment of new genera once their insect vector is unequivocally identified. Those gemini- viruses include apple [49], grapevine [50], citrus [51], mulberry [52], and tomato [53] patho- gens. The analysis of their CP promoters revealed, in four out of five geminiviruses, the existence of sequences with the distinctive features of TACEs, that is, a partially symmetric pri- mary structure, a conserved ACTT-N7-AAGT core, a GC-rich heptanucleotide spacer, and nearness to the putative TATA box. The TACEs of these four atypical geminiviruses are illus- trated in S2 Fig. 13 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Discovery of a composite element in the CP gene promoter of geminiviruses Fig 6. TACE and associated cis-acting elements in the CP promoter of viruses belonging to three Geminiviridae genera. Coloured boxes: red, CLE; yellow, TACE; green, TATA-box. Nucleotides of TbYLJV and BCTV TACEs which are identical are underlined. Fig 6. TACE and associated cis-acting elements in the CP promoter of viruses belonging to three Geminiviridae genera. Coloured boxes: red, CLE; yellow, TACE; green, TATA-box. Nucleotides of TbYLJV and BCTV TACEs which are identical are underlined. Fig 6. TACE and associated cis-acting elements in the CP promoter of viruses belonging to three Geminiviridae genera. Coloured boxes: red, CLE; yellow, TACE; green, TATA-box. Nucleotides of TbYLJV and BCTV TACEs which are identical are underlined. https://doi.org/10.1371/journal.pone.0210485.g006 https://doi.org/10.1371/journal.pone.0210485.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Synthetic multimers of the CLE fused to a minimal promoter drives high GUS expression in transgenic plants Considering the high proportion of TACEs that exhibit a heptanucleotide spacer identical or similar to the CLE core, as well as the frequent association of the TACE with canonical CLEs, it is really important to clarify the unresolved question of whether this element actually consti- tutes a functional target of TrAP. In a study conducted in 2005 by Cazonelli et al. [54] it was established that the CLE is recognized by host transcriptional factors, since non-infected trans- genic tobacco plants harboring constructs with direct repeats of that element placed upstream from a minimal CaMV 35S promoter-Luc reporter gene cassette, showed luciferase activity in most organs. The level of expression increased directly with the number of CLE copies (4–12). With the aim of establish at a finer level the specific expression pattern directed by CLE multi- mers, tobacco transgenic plants harbouring chimeric genes [3CLE-35Sprom(-46)-GUS], [3CLE- 35Sprom(-90)-GUS] and [6CLE-35Sprom(-46)-GUS] were generated (see description in Methods). Several independent lines of each of the genetic constructs were obtained, and the beta-glucuronidase activity of F1 seedlings was quantified fluorometrically. As expected, the GUS expression correlated with the number of CLEs: high in plants with the construct har- boring 6 copies of the CLE upstream of the minimal CaMV 35S promoter, from high to mod- erate in plants with 3CLEs upstream of the 35S promoter truncated at position -90, and moderate to low in lines transformed with the construct [3CLE-35S prom(-46)-GUS] (Fig 7). Histochemical staining of transgenic seedlings revealed that the CLE multimers fused to the minimal 35S promoter directed the expression of the reporter gene in the photosynthetic tis- sues, but not in the stem and root tissues (Fig 8). In lines harboring the construct [6CLE-35S Considering the high proportion of TACEs that exhibit a heptanucleotide spacer identical or similar to the CLE core, as well as the frequent association of the TACE with canonical CLEs, it is really important to clarify the unresolved question of whether this element actually consti- tutes a functional target of TrAP. In a study conducted in 2005 by Cazonelli et al. [54] it was established that the CLE is recognized by host transcriptional factors, since non-infected trans- genic tobacco plants harboring constructs with direct repeats of that element placed upstream from a minimal CaMV 35S promoter-Luc reporter gene cassette, showed luciferase activity in most organs. The level of expression increased directly with the number of CLE copies (4–12). Discovery of a composite element in the CP gene promoter of geminiviruses Fig 7. Effect of multiple copies of CLE on GUS expression driven by a truncated -46 and -90 CaMV 35S promoter. Several independent lines of transgenic plants harbouring constructs with synthetic multimers of the CLE upstream of truncated 35S promoters were analysed. Total protein was extracted of the different plant sets and assayed for GUS activity. An average expression (dotted line) and standard deviation are shown for each set of plants. (46-3CLE n = 6, 90-3CLE n = 9, 46-6CLE Fig 7. Effect of multiple copies of CLE on GUS expression driven by a truncated -46 and -90 CaMV 35S promoter. Several independent lines of transgenic plants harbouring constructs with synthetic multimers of the CLE upstream of truncated 35S promoters were analysed. Total protein was extracted of the different plant sets and assayed for GUS activity. An average expression (dotted line) and standard deviation are shown for each set of plants. (46-3CLE n = 6, 90-3CLE n = 9, 46-6CLE n = 7). Fig 7. Effect of multiple copies of CLE on GUS expression driven by a truncated -46 and -90 CaMV 35S promoter. Several independent lines of transgenic plants harbouring constructs with synthetic multimers of the CLE upstream of truncated 35S promoters were analysed. Total protein was extracted of the different plant sets and assayed for GUS activity. An average expression (dotted line) and standard deviation are shown for each set of plants. (46-3CLE n = 6, 90-3CLE n = 9, 46-6CLE n = 7). https://doi.org/10.1371/journal.pone.0210485.g007 https://doi.org/10.1371/journal.pone.0210485.g007 prom(-46)-GUS] the expression of GUS was very strong in all cells with chloroplasts as well as in trichomes (Fig 8). prom(-46)-GUS] the expression of GUS was very strong in all cells with chloroplasts as well as in trichomes (Fig 8). PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Synthetic multimers of the CLE fused to a minimal promoter drives high GUS expression in transgenic plants With the aim of establish at a finer level the specific expression pattern directed by CLE multi- mers, tobacco transgenic plants harbouring chimeric genes [3CLE-35Sprom(-46)-GUS], PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 14 / 24 Infection of transgenic plants increased the reporter gene expression As the transgenic plants matured, the GUS activity in the leaves gradually decreased but remained at levels detectable by histochemical techniques in the mesophyll, but not in the leaf veins or in the petiole and stem vascular bundles (not shown). To determine if the transcrip- tional activity driven by the CLEs multimers was modified in the presence of viral factors, we carried out experiments with four different lines of transgenic plants harboring the [3CLE- 35Sprom(-46)-GUS] construct. Plants were inoculated with the bipartite begomovirus Euphor- bia mosaic virus (EuMV) by particle bombardment. The activity of beta-glucuronidase 15 dpi was quantified and compared with non-inoculated control plants. The expression of the reporter gene in the four transgenic lines was significantly higher in the infected plants than in the uninfected controls (Fig 9). The conclusion derived from these results is that the CLE mediates the increase in transcriptional activity of the promoter and this increment is induced by one or more factors encoded by EuMV, most likely TrAP. PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 15 / 24 Discovery of a composite element in the CP gene promoter of geminiviruses Fig 8. Tissue specific-expression directed by a synthetic promoter containing six copies of the CLE. Transgenic tobacco plantlets harboring a [6CLE-35S prom(-46)- GUS] construct displayed high GUS expression in all leaf cells, including trichomes. A seedling harbouring a [35S prom(-46)-GUS] construct did not express the reporter gene in none of the tissues. The name of the binary vector used to transform the plants is indicated: pBI 46S harbors the [35S prom(-46)-GUS] construct; pBI 46S 6CLE harbors the [6CLE-35S prom(-46)-GUS] construct. Fig 8. Tissue specific-expression directed by a synthetic promoter containing six copies of the CLE. Transgenic tobacco plantlets harboring a [6CLE-35S prom(-46)- GUS] construct displayed high GUS expression in all leaf cells, including trichomes. A seedling harbouring a [35S prom(-46)-GUS] construct did not express the reporter gene in none of the tissues. The name of the binary vector used to transform the plants is indicated: pBI 46S harbors the [35S prom(-46)-GUS] construct; pBI 46S 6CLE harbors the [6CLE-35S prom(-46)-GUS] construct. https://doi.org/10.1371/journal.pone.0210485.g008 https://doi.org/10.1371/journal.pone.0210485.g008 https://doi.org/10.1371/journal.pone.0210485.g008 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 The mutation and duplication of the CLE in the -125 CP promoter of TGMV altered its response to viral factors Four independent transgenic plant lines harbouring the [3CLE-35Sprom(-46)-GUS] construct were tested in the absence or presence of viral factors. Total protein was extracted from the plants 15 dpi and d f GUS i i A il d i d h d GUS i diff f li  M d d d d i i h (Li Fig 9. Effect of EuMV infection on GUS expression driven by synthetic promoter containing three copies of the CLE. Four independent transgenic plant lines harbouring the [3CLE-35Sprom(-46)-GUS] construct were tested in the absence or presence of viral factors. Total protein was extracted from the plants 15 dpi and assayed for GUS activity. A two-tailed paired t-test showed GUS expression differences for most lines at p<0.05. Mean and standard deviation are shown. (Line1 n = 4, p = 0.0869, Line2 n = 7, p = 0.0125, Line3 n = 11, p = 0.0010, Line4 n = 17, p = 0.0002). https://doi.org/10.1371/journal.pone.0210485.g009 analogous to those obtained by Sunter and Bisaro [33]. The most interesting results were obtained with the constructs -125 CLEmut and -125 (2CLE). Indeed, the CLE mutation abol- ished the transcriptional response of the -125 promoter to proteins expressed by EuMV-A, hence indicating that the CLE is the element mediating the transactivation of the promoter by viral factors. This conclusion was corroborated with the response of the -125 (2CLE) promoter, that increased the expression of GUS to a similar level to that of the truncated -184 promoter. Interestingly enough, the -125 (2CLE) promoter showed higher basal activity than the -125 promoter, an observation that is consistent with the additive effect of CLE copies on the pro- moter basal activity reported by Cazonelli et al. [54]. Consequently, the ratio between the tran- scriptional activity of the -125 (2CLE) promoter in the presence and absence of viral factors is lower than that determined for the -184 promoter (S3 Fig). The mutation and duplication of the CLE in the -125 CP promoter of TGMV altered its response to viral factors In a pioneering study of the CP promoter of TGMV, Sunter and Bisaro [33] concluded that the CLE is not relevant for its activation by TrAP. To determine whether or not this element is critical for the promoter responsiveness to TrAP, we reproduced a series of truncated CP pro- moters examined in the aforementioned study. For this purpose, we designed primers for the PCR amplification of the promoter regions -184/+1, -125/+1, and -107/+1, including the appropriate restriction sites to clone the amplicons into the vector pBS-GUS, which contains the cassette GUS-3’nos derived from pBI121 (see Methods). Additionally, we designed primers to mutate the CLE (changing GTGGTCCC to GTAATAAC) or to add one CLE to the 5´ end of the native -125 promoter, thus producing modified amplicons to generate the constructs -125 CLEmut. and -125 (2CLE), respectively. This collection of truncated CP promoters and mutant variants were tested in transient expression assays in tobacco protoplasts, transfecting the con- structs individually or in combination with the DNA-A of the begomovirus EuMV, as a source of TrAP. The results of these experiments are shown in Fig 10. As it can be observed, the trun- cated promoters at positions -184 and -125 significantly increased the expression of the h f f d d b h f d A A h l reporter gene in the presence of factors provided by the co-transfected EuMV DNA-A, while the promoter truncated at -107 did not show responsiveness to viral factors. These results are PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 16 / 24 Discovery of a composite element in the CP gene promoter of geminiviruses Fig 9. Effect of EuMV infection on GUS expression driven by synthetic promoter containing three copies of the CLE. Four independent transgenic plant lines harbouring the [3CLE-35Sprom(-46)-GUS] construct were tested in the absence or presence of viral factors. Total protein was extracted from the plants 15 dpi and assayed for GUS activity. A two-tailed paired t-test showed GUS expression differences for most lines at p<0.05. Mean and standard deviation are shown. (Line1 n = 4, p = 0.0869, Line2 n = 7, p = 0.0125, Line3 n = 11, p = 0.0010, Line4 n = 17, p = 0.0002). Fig 9. Effect of EuMV infection on GUS expression driven by synthetic promoter containing three copies of the CLE. Discussion In this study, the complete genome of a novel bipartite begomovirus native to Southeast Mexico, Blechum interveinal chlorosis virus, was characterized. Comparative analyses of its DNA-A intergenic region revealed a 23-bp long PhyF with dyad symmetry in the proximal PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 17 / 24 Discovery of a composite element in the CP gene promoter of geminiviruses Fig 10. The CLE is a TrAP responsive element. Protoplast transient expression assays were performed with different versions of truncated TGMV CP promoter Fig 10. The CLE is a TrAP responsive element. Protoplast transient expression assays were performed with different versions of truncated TGMV CP promoter constructs in presence or absence of EuMV DNA-A factors. Total protein of transfected protoplasts was extracted two days post-transformation and assayed for GUS activity. The average and standard deviation of three independent experiments are shown. The red dotted line represents the average expression of empty (promoter- less) vector pBS-GUS in absence of viral factors. This vector was the backbone used for the generation of the different promoter constructs. For comparison purposes a construction with the uidA gene driven by the complete CaMV 35S promoter was included in the experiments. Fig 10. The CLE is a TrAP responsive element. Protoplast transient expression assays were performed with different versions of truncated TGMV CP promoter constructs in presence or absence of EuMV DNA-A factors. Total protein of transfected protoplasts was extracted two days post-transformation and assayed for GUS activity. The average and standard deviation of three independent experiments are shown. The red dotted line represents the average expression of empty (promoter- less) vector pBS-GUS in absence of viral factors. This vector was the backbone used for the generation of the different promoter constructs. For comparison purposes a construction with the uidA gene driven by the complete CaMV 35S promoter was included in the experiments. https://doi.org/10.1371/journal.pone.0210485.g010 region of the CP promoter. A search for this PhyF homologs in New World BGVs unveiled a collection of related sequences that are composed by two discernible kind of elements. Because of its complex composition and consistent association with the TATA-box we dubbed it “TATA-associated composite element” (TACE). The equivalent element in the CP promoter of Old World BGVs has a different position relative to the TATA-box, which is placed upstream of the TACE and partially overlaps its left arm (Figs 4 and 5). PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Discussion Begomovirus TACE homologs were also identified in members of other two Geminiviridae genera, and four atypi- cal geminiviruses currently unassigned to a genus. Several questions naturally arise from the present study findings: What is the TACE func- tion? Why does the heptanucleotide spacer varies in sequence, not only between viral species, but even between isolates of the same species? Which host and/or virus factors interact with that complex element? Searches in databases specialized in cis-regulatory elements and tran- scriptional factors of plants, such as PlantCare [55], PLACE [56], and PlantPAN [57] did not provide significant data to ascertain the probable function of the palindromic arms of New World BGV TACEs (i.e., CAACAACTT-(N7)-AAGTTGTTG). In the case of the GC-rich spacer sequence, our data indicate that it is related to the CLE, without being identical or even similar to that element in a considerable number of BGVs. Indeed, a comprehensive analysis PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 18 / 24 Discovery of a composite element in the CP gene promoter of geminiviruses showed that in (at least) 66 species of Old World BGVs the TACE spacer is similar to the CLE core, whereas ~50% of the New World BGVs exhibit CLE-like spacer sequences in their TACEs. Furthermore, canonic CLEs are closely associated with the TACE in a significant number of viruses native to the Indian subcontinent and Asia (Figs 4 and 5). This association of the TACE with CLE-like sequences is particularly clear in some divergent lineages of BGVs, such as the New World SLCV clade, the Asian lineage of Tomato leaf curl New Delhi virus (ToLCNDV), and the lineage of the so-called “sweepoviruses”, probably originated in China. The CP promoters of BGVs belonging to the SLCV lineage (~30 species) commonly contain two or three copies of the CLE (in direct or inverse orientation) one of which is placed between the TACE and the TATA box (S4 Fig). A single member of this lineage (i.e., Cabbage leaf curl virus) exhibits an incomplete TACE but retains the CLE adjacent to the TATA box (S4 Fig). On the other hand, eleven BGVs related to ToLCNDV display a CLE adjacent to the TATA box, like many other OW BGVs, but that CLE is in reverse orientation (S5 Fig). Discussion Finally, the 13 recognized species of sweepoviruses, that have the smallest CP promoters among all BGVs (i.e., 89 to 126 bp in length), exhibit a very short TACE which is only composed of the ACTT (N7) AAGT core; this element is not associated with the TATA box, but it is immediately adja- cent to the V2 gene, a unique position observed among geminiviruses. The heptanucleotide spacer of sweepovirus TACEs differs in one or two nucleotides of the CLE core. Moreover, the CP promoter of all sweepoviruses contains a canonical CLE downstream the conserved stem- loop element, and three species exhibit a second CLE in reverse orientation, which together with the first CLE forms a long interrupted palindrome (S6 Fig). PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 The CLE is an element of response to TrAP As mentioned in the introductory section, the experimental evidence obtained by different lab- oratories has been contradictory. So, for example, Ruiz-Medrano et al. [32] that studied the PHYVV CP promoter, reported experimental data consistent with the CLE hypothesis, whereas Sunter and Bisaro [33] concluded that this conserved element is not involved in the TrAP-mediated activation of TGMV CP promoter, because the deletion of the single CLE present in it did not affect its TrAP-responsiveness (in the context of the entire intergenic region). In the present study, we have shown that the CLE is really necessary for the transacti- vation of the TGMV CP promoter truncated at position -125. What is more, the artificial addi- tion of one CLE upstream of the -125 CP promoter significantly enhanced its transactivation by TrAP. A plausible explanation of the results obtained by Sunter and Bisaro in their study of 2003 [33] is that there is one, or more, TrAP responsive element different to the CLE upstream of the -125 nucleotide of TGMV CP promoter. This alternative interpretation is consistent with data from the same study, which showed that the level of transactivation of the truncated promoter at position -184 practically doubled the -125 promoter activity. The comparative analysis of the smallest truncated CP promoters that have shown respon- siveness to TrAP, like -115 PHYVV [32], -125 TGMV [33] and -151 CbLCV [24] promoters, also led to the conclusion that the CLE is plausibly the cis-acting element mediating the response to the transactivator, because those truncated promoters only have an element in common besides the TATA-box, i.e., the CLE. An important corollary of the validation of the CLE as a TrAP-responsive element is that this opens new prospects to identify plant genes functioning as primary targets of the transactivator. Indeed, TrAP is a key factor of begomo- viruses ability to reprogram the host to backing viral infection and to evade plant defense responses, as suggested by the observation that the expression of the AC2 gene of ACMV and CbLCV strongly altered the transcriptomes of tobacco and Arabidopsis, respectively [58, 59]. The CLE is an element of response to TrAP It is very plausible that the promoters of some of the host genes responding directly to the PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 19 / 24 Discovery of a composite element in the CP gene promoter of geminiviruses presence of TrAP contain CLEs, and therefore could be identified by bioinformatics approaches in the genome of model plants. Recently, Babu et al [60] undertook a genome- wide mapping of CLEs in Arabidopsis thaliana and identified 122 promoters containing exact GTGGTCCC motifs. Interestingly enough, several of the identified promoters correspond to genes encoding transcription factors of the MYB, WRKY and BHLH DNA binding families, thus suggesting the indirect regulation by TrAP of multiple plant genes involved in plant anti- viral defenses and enhanced support of virus infection [60]. What regulatory function does the TACE have? In this study, it was showed that transgenic tobacco plants harboring synthetic promoters with either 3 or 6 copies of the CLE upstream of the 35S minimal promoter expressed moderate (i.e., 3CLEs) or strong (6CLEs) beta-glucuronidase activity in mesophyll cells and trichomes, but not in mature vascular tissues (Figs 7 and 8). It has been established that TrAP interacts with diverse DNA-binding proteins, including PEAPOD2 (PPD2) of Arabidopsis [30]. Puri- fied PPD2 bound to the so-called CP promoter activator region of TGMV (-166 to -59) in elec- trophoretic mobility shift assays (EMSA), but the PPD2 binding site could not be identified with precision. Recently, Gonzalez et al. [61] carried out a genome-wide determination of PPD2 target sites, and they found among 2042 peak sequences identified, that two specific motifs were highly represented. The first motif, present in 726 peak sequences, was GmCACGTGkC. The second motif was yctCACGCGCyt, which was present in 275 peak sequences. The PPD2 factor is a repressor, that downregulates the expression of its target genes [61]. So, it is possible that PPD2 could be involved in the repression of CP and other late promoters in the absence of TrAP. Indeed, is currently no clear understanding of the molecu- lar mechanisms involved in the transcriptional processes mediated by TrAP; however, the con- sistent association of the TACE with the TATA box of CP promoters, in addition to the similarity of the heptanucleotide spacer with either the CLE or the PPD2 target sequences, sug- gests that this composite element could be involved in the TrAP-mediated derepression of the CP gene in the phloem. Acknowledgments We thank to Dr. Roberto Ruiz-Medrano, Dr. Jose´ Trinidad Ascencio-Ibañez and MSc. Nadia Martı´nez-Marrero, for critical reading of the manuscript and thoughtful comments and sug- gestions. MCI and AAC were supported by a fellowship from the Consejo Nacional de Ciencia y Tecnologı´a (CONACYT, Mexico). We dedicate this work to the memory of Rosa Marı´a Rangel-Cano, an excellent technician and an exceptional human being. Author Contributions Conceptualization: Mariana Cantu´-Iris, Gerardo R. Argu¨ello-Astorga. Conceptualization: Mariana Cantu´-Iris, Gerardo R. Argu¨ello-Astorga. Formal analysis: Jesu´s Aaro´n Avalos-Calleros, Gerardo R. Argu¨ello-Astorga. Investigation: Mariana Cantu´-Iris, Guillermo Pastor-Palacios, Jorge Armando Mauricio-Cas- tillo, Bernardo Bañuelos-Herna´ndez, Alejandro Jua´rez-Reyes, Gerardo R. Argu¨ello- Astorga. Methodology: Mariana Cantu´-Iris, Guillermo Pastor-Palacios, Jorge Armando Mauricio-Cas- tillo, Bernardo Bañuelos-Herna´ndez, Jesu´s Aaro´n Avalos-Calleros, Alejandro Jua´rez-Reyes, Rafael Rivera-Bustamante, Gerardo R. Argu¨ello-Astorga. Project administration: Gerardo R. Argu¨ello-Astorga. Resources: Rafael Rivera-Bustamante. Resources: Rafael Rivera-Bustamante. Supervision: Rafael Rivera-Bustamante, Gerardo R. Argu¨ello-Astorga. Validation: Gerardo R. Argu¨ello-Astorga. Writing – original draft: Mariana Cantu´-Iris, Guillermo Pastor-Palacios, Jesu´s Aaro´n Avalos- Calleros. Writing – review & editing: Rafael Rivera-Bustamante, Gerardo R. Argu¨ello-Astorga. References S3 Fig. Expression of different CLE-containing CP promoters in presence of EuMV factors. (PDF) S3 Fig. Expression of different CLE-containing CP promoters in presence of EuMV factors. (PDF) S4 Fig. CP promoter region with the TACE-CLE-TATA box conserved arrangement in members of the SLCV lineage. (PDF) S4 Fig. CP promoter region with the TACE-CLE-TATA box conserved arrangement in members of the SLCV lineage. (PDF) S5 Fig. CP promoter region containing the inverted CLE-TATA box-TACE arrangement in members of the ToLCNDV lineage. (PDF) S6 Fig. Complete V2/CP promoter of selected Sweepoviruses, a divergent lineage of Old World begomoviruses. S5 Fig. CP promoter region containing the inverted CLE-TATA box-TACE arrangement in members of the ToLCNDV lineage. (PDF) Supporting information S1 Table. Names, acronyms and GenBank accession numbers of geminiviruses compared in Fig 1. (PDF) S1 Table. Names, acronyms and GenBank accession numbers of geminiviruses compared in Fig 1. (PDF) S2 Table. Begomoviruses exhibiting TACE spacer sequences identical or similar to the CLE core. (PDF) S3 Table. Oligonucleotides used in this study. (PDF) S4 Table. Experimental data of GUS activity presented in Figs 7, 9 and 10, and S3 Fig. (XLSX) S1 Fig. Symptoms caused by BleICV in Blechum pyramidatum plants. (PDF) S2 Fig. TACE and associated putative cis-acting elements in four geminiviruses unassigne to a genus. (PDF) S2 Table. Begomoviruses exhibiting TACE spacer sequences identical or similar to the CLE core. (PDF) S4 Table. Experimental data of GUS activity presented in Figs 7, 9 and 10, and S3 Fig. (XLSX) S1 Fig. Symptoms caused by BleICV in Blechum pyramidatum plants. (PDF) S2 Fig. TACE and associated putative cis-acting elements in four geminiviruses unassigned to a genus. (PDF) 20 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 Discovery of a composite element in the CP gene promoter of geminiviruses S3 Fig. Expression of different CLE-containing CP promoters in presence of EuMV factors (PDF) S4 Fig. CP promoter region with the TACE-CLE-TATA box conserved arrangement in members of the SLCV lineage. (PDF) S5 Fig. CP promoter region containing the inverted CLE-TATA box-TACE arrangement in members of the ToLCNDV lineage. (PDF) S6 Fig. Complete V2/CP promoter of selected Sweepoviruses, a divergent lineage of Old World begomoviruses. (PDF) References 1. Zerbini FM, Briddon RW, Idris A, Martin DP, Moriones E, Navas-Castillo et al. ICTV virus taxonomy pro- file: Geminiviridae. Journal of General Virology. 2017 Mar 13; 98(2):131–3. https://doi.org/10.1099/jgv. 0.000738 PMID: 28284245 2. Jeske H. Geminiviruses. In: TT Viruses 2009 (pp. 185–226). 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Babu KSD, Manoharan P, Pandi G. Computational studies on Begomoviral AC2/C2 proteins 2018. Bioinformation 14(6): 294–303. https://doi.org/10.6026/97320630014294 PMID: 30237675 61. PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019 References Gonzalez N, Pauwels L, Baekelandt A, De Milde L, Van Leene J, Besbrugge et al. A repressor protein complex regulates leaf growth in Arabidopsis. The Plant Cell. 2015 Aug 1; 27(8):2273–87. https://doi. org/10.1105/tpc.15.00006 PMID: 26232487 24 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0210485 January 23, 2019
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Persistent mammalian orthoreovirus, coxsackievirus and adenovirus co-infection in a child with a primary immunodeficiency detected by metagenomic sequencing: a case report
BMC infectious diseases
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© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lewandowska et al. BMC Infectious Diseases (2018) 18:33 DOI 10.1186/s12879-018-2946-7 Lewandowska et al. BMC Infectious Diseases (2018) 18:33 DOI 10.1186/s12879-018-2946-7 Open Access Persistent mammalian orthoreovirus, coxsackievirus and adenovirus co-infection in a child with a primary immunodeficiency detected by metagenomic sequencing: a case report Dagmara W. Lewandowska1†, Riccarda Capaul1†, Seraina Prader2, Osvaldo Zagordi1, Fabienne-Desirée Geissberger1, Martin Kügler1,5, Marcus Knorr1, Christoph Berger3, Tayfun Güngör4, Janine Reichenbach2, Cyril Shah1, Jürg Böni1, Andrea Zbinden1, Alexandra Trkola1, Jana Pachlopnik Schmid2† and Michael Huber1*† Abstract Background: We report a rare case of Mammalian orthoreovirus (MRV) infection in a child with a primary immunodeficiency (PID). Infections with Mammalian orthoreovirus are very rare and probably of zoonotic origin. Only a few cases have been described so far, including one with similar pathogenesis as in our case. Case presentation: The patient, age 11, presented with flu-like symptoms and persistent severe diarrhea. Enterovirus has been detected over several months, however, exact typing of a positive cell culture remained inconclusive. Unbiased metagenomic sequencing then detected MRV in stool samples from several time points. The sequencing approach further revealed co-infection with a recombinant Coxsackievirus and Adenovirus. MRV-specific antibodies detected by immunofluorescence proved that the patient seroconverted. Conclusion: This case highlights the potential of unbiased metagenomic sequencing in supplementing routine diagnostic methods, especially in situations of chronic infection with multiple viruses as seen here in an immunocompromised host. The origin, transmission routes and implications of MRV infection in humans merit further investigation. Keywords: Orthoreovirus, Coxsackievirus, Adenovirus, Primary immunodeficiency, Metagenomic se Rapid diagnosis of viral infections is crucial in immuno- compromised patients. While a range of established mo- lecular tests can detect specific viruses, high-throughput metagenomic sequencing is based on virus-sequence inde- pendent amplification of nucleic acids isolated directly from clinical samples; as such, it has the potential to identify any virus in an “open diagnostics” approach [2, 3]. Hence, this approach can detect rare viruses that are not included in routine diagnostic panels and viruses with sequence variations that would otherwise be missed [4]. Here we used metagenomic sequencing to complement routine diagnostic methods in a child with a PID suffering from persistent diarrhea. * Correspondence: huber.michael@virology.uzh.ch †Equal contributors 1Institute of Medical Virology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland Full list of author information is available at the end of the article Case presentation sequenced both the Caco-2 culture supernatant and the original stool suspension. We detected many reads of Mammalian orthoreovirus 3 (MRV-3) in the cell culture supernatant and reads of Coxsackievirus A (CV-A) in the original stool suspension (Table 1). No virus reads were detected in the supernatant of a non-infected Caco-2 cell culture used as a negative control. p We report on a female child living in Switzerland with a combined B- and T-cell immunodeficiency, hypogamma- globulinaemia and autoimmunity (diabetes mellitus) under immunoglobulin replacement therapy. In early February 2014, the patient, aged 11 at that time, pre- sented with flu-like symptoms with cough, headache, fever and severe diarrhea. Although the other symptoms resolved, the diarrhea persisted. In view of low initial calprotectin levels (100–400 μg/g), a fecal marker for in- testinal inflammation, the diarrhea was considered as caused by the underlying PID and not by an infectious agent. However, a rise in calprotectin level to 4000 μg/g in March 2015 prompted us to initiate virological investi- gations. Enterovirus had been detected over several months using a specific RT-PCR (Additional file 1: Table S1) and was thus considered as causative agent although other viruses were not tested for during this time period. To trace the timing of these two virus infections we con- ducted a retrospective analysis of the available stool sam- ples by both cell culture and metagenomic sequencing (Table 1). In cell cultures, a CPE was visible after about 14 days of culturing. By sequencing the cell culture superna- tants, we found numerous reads for Human adenovirus C (HAdV-C) in these earlier time points, but none for MRV- 3. By sequencing original stool suspensions, we identified reads corresponding to the previously identified CV-A. We verified all the metagenomics analyses by specific PCRs designed for the isolates in this study (Table 1). In addition to confirming MRV-3 in the three cell cultures supernatants with MRV-3 reads, we also detected MRV- 3 in two corresponding stool suspensions by specific PCR. CV-A was confirmed in all stool suspensions and in one of two colon biopsies; HAdV-C was confirmed in all tested cell cultures and stool suspensions (Table 1). As symptoms continued, additional testing was per- formed. A stool sample (November 3rd 2015) was positive in Caco-2 cell culture showing a cytopathic effect (CPE) after 9 days of incubation. Background Individuals suffering from primary immunodeficiencies (PIDs) are prone to a variety of infections, and some types of PIDs can predispose the affected individuals to particu- lar pathogens. Infections that are usually controlled and asymptomatic in immunocompetent individuals often cause chronic, active disease in immunocompromised individuals [1]. * Correspondence: huber.michael@virology.uzh.ch †Equal contributors 1Institute of Medical Virology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland Full list of author information is available at the end of the article Lewandowska et al. BMC Infectious Diseases (2018) 18:33 Page 2 of 5 Page 2 of 5 na not done or not available, undet undetermined threshold cycle (> 45), cell culture (CPE) was tested on Caco-2 cells Case presentation The culture sample stained positive with a pool of anti-enterovirus monoclonal anti- bodies, but we did not succeed to further subtype the sus- pected enterovirus using routine immunostaining methods. In order to identify the virus amplified in the Caco-2 cell culture, we applied unbiased metagenomic sequencing as previously described [4]. For three sampling time points in September and November 2015, we To define the MRV-3 infection in more detail, we combined sequencing reads from all time points and re- constructed a consensus sequence that covered the full coding sequences in all 10 segments of the MRV-3 iso- late (GenBank KX932029 - KX932038). Phylogenetic Table 1 Retrospective viral diagnostics using cell culture, metagenomic sequencing and specific PCRs Cell culture (CPE) Metagenomic sequencing (number of reads) Specific PCR (threshold cycle) Date Sample Original material Original material Cell culture supernatant MRV-3 original material MRV-3 cell culture supernatant CV-A original material HAdV original material HAdV cell culture supernatant 25.09.2014 colon biopsy na na na undet na 37.3 undet na 02.01.2015 stool negative CV-A (9′618) na undet na 20.1 33.0 na 29.04.2015 colon biopsy na na na undet na undet undet na 30.04.2015 stool positive CV-A (555) HAdV-C (328′173) undet undet 23.6 35.3 12.0 26.05.2015 stool positive CV-A (746′862) HAdV-C (6′131) HAdV-C (228′718) undet undet 28.0 40.5 13.8 30.06.2015 stool positive na HAdV-C (439′345) undet undet 24.0 37.0 14.6 14.07.2015 stool positive na HAdV-C (452′131) undet undet 23.5 33.3 12.2 10.09.2015 stool positive CV-A (195′249) MRV-3 (6′580) undet 21.6 20.1 undet na 3.11.2015 stool positive CV-A (414) MRV-3 (2′742) 35.8 22.2 27.3 undet na 25.11.2015 stool positive CV-A (1′549’977) HAdV-C (102) MRV-3 (850) 37.3 24.0 21.2 undet undet 09.02.2016 stool na na na undet na 14.5 undet na 18.03.2016 stool negative na na undet na undet undet na 04.05.2016 stool negative na na undet na 23.5 38.24 na na not done or not available, undet undetermined threshold cycle (> 45), cell culture (CPE) was tested on Caco-2 cells Lewandowska et al. BMC Infectious Diseases (2018) 18:33 Page 3 of 5 Page 3 of 5 The most unexpected finding was the infection with MRV-3, proven by metagenomic sequencing and ser- ology of samples from several time points. MRVs belong to the Reoviridae family, a group of non-enveloped dsRNA viruses with 10 genome segments and have been isolated from a wide range of mammalian hosts and in a variety of clinical contexts [5–7]. Case presentation To date, the diseases associated with MRV infections in humans include respiratory disease [8], meningitis [9–11], acute necrotiz- ing encephalopathy [12] and (in a similar case involving a child living in Slovenia) acute gastroenteritis [5]. Zoo- notic transmission is often suspected [6, 13, 14]. Our patient lives close to a farm and might have come in contact with pets, farm animals and animal feces making a zoonotic infection conceivable, however, due to the lack of data on MRV distribution and appropriate samples we can only hypothesize on the transmission route [7]. Stool samples from 16 children with suspicion of gastrointes- tinal infection treated at the same hospital during 2015 all tested negative with qPCR (data not shown). analyses showed that the MRV-3 sequence isolated in this study was most related in all 10 segments to an iso- late from a child in Slovenia [5] and further clustered with isolates from bats in Germany [6] and pigs in Italy [7] (Fig. 1a and Additional file 1: Figures S1-S9). In order to proof active replication and diagnosis of MRV infection, we performed immunofluorescence for the detection of MRV-specific antibodies of the patient. Patient plasma from time points after MRV detection were positive for anti-MRV IgG antibodies showing that the patient seroconverted (Fig. 1b). We were able to reconstruct the full-length coxsackie- virus genome present in this patient using additional sequence information from sequencing with CV-A22 serotype consensus primers. Genotyping and bootscan analysis revealed that the isolate (GenBank KX932039) probably resulted from recombination between CV-A19 and CV-A22 (Additional file 1: Figures S10 and S11). The detected HAdV-C was type 2, strain human/ARG/ A15932/2002/2[P2H2F2] (JX173079.1). The virus growing in the initial Caco-2 cell culture was therefore MRV-3 and not an enterovirus as suggested by the serotyping assay. In fact, the manufac- turer’s datasheet for the reagent used states that there is potential for cross-reaction with Hepatitis A, Reovirus 3, and some Rhinovirus and Astrovirus strains. The latter highlights the genuine difficulty in accurate detection of highly diverse virus families with numerous genotypes such as Enteroviruses where typing by specific PCR can be challenging due to the high susceptibility to recom- bination and the emergence of novel strains [15]. Competing interests h h d l h Competing interests The authors declare that they have no competing interests. Competing interests h h d l h Abbreviations CPE: Cytopathic effect; CV-A: Coxsackievirus A; HAdV-C: Human adenovirus C; MRV: Mammalian orthoreovirus; PCR: Polymerase chain reaction; PID: Primary immunodeficiency 6. Kohl C, Lesnik R, Brinkmann A, Ebinger A, Radonic A, Nitsche A, Muhldorfer K, Wibbelt G, Kurth A. Isolation and characterization of three mammalian orthoreoviruses from European bats. PLoS One. 2012;7(8):e43106. 6. Kohl C, Lesnik R, Brinkmann A, Ebinger A, Radonic A, Nitsche A, Muhldorfer K, Wibbelt G, Kurth A. Isolation and characterization of three mammalian orthoreoviruses from European bats. PLoS One. 2012;7(8):e43106. References l 1. Dropulic L, Cohen J. Severe viral infections and primary immunodeficiencies. Clin Infect Dis. 2011;53(9):897–909. 1. Dropulic L, Cohen J. Severe viral infections and primary immunodeficiencies. Clin Infect Dis. 2011;53(9):897–909. Funding g Funding was provided by the Clinical Research Priority Program “Viral Infectious Diseases” of the University of Zurich. The funding body did not Funding was provided by the Clinical Research Priority Program “Viral Infectious Diseases” of the University of Zurich. The funding body did not have any role in the design of the study, in the collection, analysis, and interpretation of data and in writing the manuscript. 8. Chua KB, Crameri G, Hyatt A, Yu M, Tompang MR, Rosli J, McEachern J, Crameri S, Kumarasamy V, Eaton BT, et al. A previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans. Proc Natl Acad Sci U S A. 2007;104(27):11424–9. have any role in the design of the study, in the collection, analysis, and interpretation of data and in writing the manuscript. 9. Johansson PJ, Sveger T, Ahlfors K, Ekstrand J, Svensson L. Reovirus type 1 associated with meningitis. Scand J Infect Dis. 1996;28(2):117–20. 10. Hermann L, Embree J, Hazelton P, Wells B, Coombs RTK. Reovirus type 2 isolated from cerebrospinal fluid. Pediatr Infect Dis J. 2004;23(4):373–5. 9. Johansson PJ, Sveger T, Ahlfors K, Ekstrand J, Svensson L. Reovirus type 1 associated with meningitis. Scand J Infect Dis. 1996;28(2):117–20. 9. Johansson PJ, Sveger T, Ahlfors K, Ekstrand J, Svensson L. Reovirus type 1 associated with meningitis. Scand J Infect Dis. 1996;28(2):117–20. Additional file 2. Delwart EL. Viral metagenomics. Rev Med Virol. 2007;17(2):115–31. 3. Mokili JL, Rohwer F, Dutilh BE. Metagenomics and future perspectives in virus discovery. Current Opinion in Virology. 2012;2(1):63–77. Additional file 1: Portable Document Format. Table S1. Routine screening results for Enterovirus. Figures S1 to S9. Phylogenetic analyses of all Mammalian orthoreovirus segments isolated in this study. Figure S10. Coxsackievirus BLAST genotyping. Figure S11. Coxsackievirus Bootscan Analysis. Supplementary Methods. Supplementary References. (PDF 456 kb) 4. Lewandowska DW, Zagordi O, Zbinden A, Schuurmans MM, Schreiber P, Geissberger FD, Huder JB, Böni J, Benden C, Mueller NJ, et al. Unbiased metagenomic sequencing complements specific routine diagnostic methods and increases chances to detect rare viral strains. Diagn Microbio Infect Dis. 2015;83(2):133–8. 4. Lewandowska DW, Zagordi O, Zbinden A, Schuurmans MM, Schreiber P, Geissberger FD, Huder JB, Böni J, Benden C, Mueller NJ, et al. Unbiased metagenomic sequencing complements specific routine diagnostic methods and increases chances to detect rare viral strains. Diagn Microbiol Infect Dis. 2015;83(2):133–8. 5. Steyer A, Gutierrez-Aguire I, Kolenc M, Koren S, Kutnjak D, Pokorn M, Poljsak- Prijatelj M, Racki N, Ravnikar M, Sagadin M, et al. High similarity of novel orthoreovirus detected in a child hospitalized with acute gastroenteritis to mammalian orthoreoviruses found in bats in Europe. J Clin Microbiol. 2013; 51(11):3818–25. 5. Steyer A, Gutierrez-Aguire I, Kolenc M, Koren S, Kutnjak D, Pokorn M, Poljsak- Prijatelj M, Racki N, Ravnikar M, Sagadin M, et al. High similarity of novel orthoreovirus detected in a child hospitalized with acute gastroenteritis to mammalian orthoreoviruses found in bats in Europe. J Clin Microbiol. 2013; 51(11):3818–25. Received: 12 April 2017 Accepted: 4 January 2018 Received: 12 April 2017 Accepted: 4 January 2018 Received: 12 April 2017 Accepted: 4 January 2018 Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. g 10. Hermann L, Embree J, Hazelton P, Wells B, Coombs RTK. Reovirus type 2 isolated from cerebrospinal fluid. Pediatr Infect Dis J. 2004;23(4):373–5. 11. Tyler KL, Barton ES, Ibach ML, Robinson C, Campbell JA, O’Donnell SM, Valyi- Nagy T, Clarke P, Wetzel JD, Dermody TS. Isolation and molecular characterization of a novel type 3 reovirus from a child with meningitis. J Infect Dis 2004;189(9):1664-1675. Publisher’s Note In summary, this case highlights the complexity of infec- tions in immunocompromised hosts and reveals limita- tions of routine diagnostic methods. A combination of traditional cell culture, metagenomic sequencing, verifica- tion by specific PCRs and serology proved key to detect a persistent co-infection with three clinically relevant viruses. While the presence of these viruses in stool specimen suggests a link with the observed pathogenesis, it cannot be defined to what extent each of the viruses contributed to the initial flu-like symptoms and prolonged diarrhea. Based on the timing of virus detection, the earlier detected CV-A and HAdV-C are more likely to be involved in the child’s condition than the later emerging MRV-3. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements Not applicable 7. Lelli D, Beato MS, Cavicchio L, Lavazza A, Chiapponi C, Leopardi S, Baioni L, De Benedictis P, Moreno A. First identification of mammalian orthoreovirus type 3 in diarrheic pigs in Europe. Virol J. 2016;13(1):139. 7. Lelli D, Beato MS, Cavicchio L, Lavazza A, Chiapponi C, Leopardi S, Baioni L, De Benedictis P, Moreno A. First identification of mammalian orthoreovirus type 3 in diarrheic pigs in Europe. Virol J. 2016;13(1):139. Discussion and conclusions Using a metagenomic sequencing approach, we detected multiple virus infections in a child with PID and persis- tant diarrhea. Although routine PCR methods detected Enterovirus in stool samples for a prolonged period of time, an attempt to subtype the virus after cell culturing was not successful. In order to resolve this, we per- formed metagenomic sequencing of cell culture superna- tants and stool suspensions. Fig. 1 Mammalian orthoreovirus infection confirmed by phylogenetic analysis and immunofluorescence staining. a Phylogenetic analysis of Mammalian orthoreovirus segment S1 isolated in this study (circle, mew716_S1_type_3_human) reveals a close relationship with previously described isolates identified in a child in Slovenia (triangle, KF154730), bats in Germany (JQ412761), and pigs in Italy (KX343206). Phylogenetic trees were constructed in MEGA7 using the Maximum Likelihood method based on the Kimura 2-parameter model. Bootstrap values from 1000 tries are shown. MRV-type and host species are depicted if available. b Anti-MRV-3 immunofluorescence of patient plasma before and after seroconversion on MRV-3-infected and uninfected Caco-2 cells Fig. 1 Mammalian orthoreovirus infection confirmed by phylogenetic analysis and immunofluorescence staining. a Phylogenetic analysis of Mammalian orthoreovirus segment S1 isolated in this study (circle, mew716_S1_type_3_human) reveals a close relationship with previously described isolates identified in a child in Slovenia (triangle, KF154730), bats in Germany (JQ412761), and pigs in Italy (KX343206). Phylogenetic trees were constructed in MEGA7 using the Maximum Likelihood method based on the Kimura 2-parameter model. Bootstrap values from 1000 tries are shown. MRV-type and host species are depicted if available. b Anti-MRV-3 immunofluorescence of patient plasma before and after seroconversion on MRV-3-infected and uninfected Caco-2 cells Page 4 of 5 Page 4 of 5 Lewandowska et al. BMC Infectious Diseases (2018) 18:33 Notably, CV-A was detected in stool suspensions but not in cell culture supernatants. While cell culturing was critical for MRV-3 detection, CV-A, in line with a general difficulty to culture coxsackieviruses [16], did not infect Caco-2 cells. The reason that MRV-3 was not detected by metagenomic sequencing in the original stool suspension, but only after amplification in cell culture, is likely because it was at levels too low to be detected with the applied depth of sequencing. canton of Zurich approved the study and written informed consent was obtained. canton of Zurich approved the study and written informed consent was obtained. Authors’ contributions SP, CB, TG, JR and JPS cared for the patient and provided clinical data and materials. RC, JB and AZ analyzed and interpreted routine diagnostic results. DWL, FDG and CS performed sequencing experiments and PCR assays. DWL, OZ and MH analyzed the sequencing data. MK and MK performed cell culture and immunostaining. DWL, AT and MH wrote the manuscript. All authors read and approved the final manuscript. 12. Ouattara LA, Barin F, Barthez MA, Bonnaud B, Roingeard P, Goudeau A, Castelnau P, Vernet G, Paranhos-Baccala G, Komurian-Pradel F. Novel human reovirus isolated from children with acute necrotizing encephalopathy. Emerg Infect Dis. 2011;17(8):1436–44. 13. Lelli D, Moreno A, Lavazza A, Bresaola M, Canelli E, Boniotti MB, Cordioli P. Identification of Mammalian orthoreovirus type 3 in Italian bats. Zoonoses Public Health. 2013;60(1):84–92. 13. Lelli D, Moreno A, Lavazza A, Bresaola M, Canelli E, Boniotti MB, Cordioli P. Identification of Mammalian orthoreovirus type 3 in Italian bats. Zoonoses Public Health. 2013;60(1):84–92. Author details 1 1Institute of Medical Virology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. 2Division of Immunology, University Children’s Hospital Zurich, Steinwiesstrasse 75, 8032 Zurich, Switzerland. 3Division of Infectious Diseases and Hospital Epidemiology, University Children’s Hospital Zurich, Steinwiesstrasse 75, 8032 Zurich, Switzerland. 4Division of Stem Cell Transplantation, University Children’s Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland. 5Present address: Unilabs, Ringstrasse 12, 8600 Dübendorf, Switzerland. Consent for publication Written informed consent for the publication of this case report was obtained from both parents of the child. Competing interests The authors declare that they have no competing interests. Lewandowska et al. BMC Infectious Diseases (2018) 18:33 associated with acute influenza-like illness in an adult patient. PLoS One. 2011;6(10):e25434. 15. Lukashev AN. Role of recombination in evolution of enteroviruses. Rev Med Virol. 2005;15(3):157–67. 16. Wenner HA, Lenahan MF. Propagation of group a Coxsackie viruses in tissue cultures. II. Some interactions between virus and mammalian cells. Yale J Biol Med. 1961;34:421–38. 16. Wenner HA, Lenahan MF. Propagation of group a Coxsackie viruses in tissue cultures. II. Some interactions between virus and mammalian cells. Yale J Biol Med. 1961;34:421–38. Ethics approval and consent to participate f Non-invasive samples were obtained from patient mew716 in the frame of the Viral Metagenome Study of the Clinical Research Priority Program ‘Viral Infectious Diseases’ of the University of Zurich. The ethics committee of the 14. Chua KB, Voon K, Yu M, Keniscope C, Abdul Rasid K, Wang L-F. Investigation of a potential zoonotic transmission of orthoreovirus 14. Chua KB, Voon K, Yu M, Keniscope C, Abdul Rasid K, Wang L-F. Investigation of a potential zoonotic transmission of orthoreovirus Page 5 of 5 Lewandowska et al. BMC Infectious Diseases (2018) 18:33 • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step:
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Longitudinal changes in functional connectivity in speech motor networks in apraxia of speech after stroke
Frontiers in neurology
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Longitudinal changes in functional connectivity in speech motor networks in apraxia of speech after stroke OPEN ACCESS EDITED BY Veena A. Nair, University of Wisconsin-Madison, United States REVIEWED BY Jolien Schaeverbeke, KU Leuven, Belgium Donald A. Robin, University of New Hampshire, United States *CORRESPONDENCE Helena Hybbinette helena.hybbinette@ki.se SPECIALTY SECTION This article was submitted to Stroke, a section of the journal Frontiers in Neurology RECEIVED 07 August 2022 ACCEPTED 10 November 2022 PUBLISHED 30 November 2022 CITATION Hybbinette H, Östberg P, Schalling E, Deboussard C, Plantin J, Borg J and Lindberg PG (2022) Longitudinal changes in functional connectivity in speech motor networks in apraxia of speech after stroke. Front. Neurol. 13:1013652. doi: 10 3389/fneur 2022 1013652 Helena Hybbinette1,2,3*, Per Östberg1,4, Ellika Schalling1,5, Catharina Deboussard2,3, Jeanette Plantin2,3, Jörgen Borg2,3 and Påvel G. Lindberg2,6 1Division of Speech and Language Pathology, Department of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden, 2Division of Rehabilitation Medicine, Department of Clinical Sciences, Danderyd Hospital, Karolinska Institute, Stockholm, Sweden, 3Department of Rehabilitation Medicine, Danderyd University Hospital, Stockholm, Sweden, 4Medical Unit Speech and Language Pathology, Karolinska University Hospital, Stockholm, Sweden 5Department of Public Health and Caring Sciences, Speech-Language Pathology, Uppsala University, Uppsala, Sweden, 6Institut de Psychiatrie et Neurosciences Paris, INSERM U1266, Université de Paris, Paris, France RECEIVED 07 August 2022 ACCEPTED 10 November 2022 PUBLISHED 30 November 2022 Objective: The cerebral substrates of apraxia of speech (AOS) recovery remain unclear. Resting state fMRI post stroke can inform on altered functional connectivity (FC) within cortical language networks. Some initial studies report reduced FC between bilateral premotor cortices in patients with AOS, with lowest FC in patients with the most severe AOS. However, longitudinal FC studies in stroke are lacking. The aims of the present longitudinal study in early post stroke patients with AOS were (i) to compare connectivity strength in AOS patients to that in left hemisphere (LH) lesioned stroke patients without a speech-language impairment, (ii) to investigate the relation between FC and severity of AOS, aphasia and non-verbal oral apraxia (NVOA) and (iii) to investigate longitudinal changes in FC, from the subacute phase to the chronic phase to identify predictors of AOS recovery. CITATION Hybbinette H, Östberg P, Schalling E, Deboussard C, Plantin J, Borg J and Lindberg PG (2022) Longitudinal changes in functional connectivity in speech motor networks in apraxia of speech after stroke. Front. Neurol. 13:1013652. doi: 10.3389/fneur.2022.1013652 Front. Neurol. 13:1013652. doi: 10.3389/fneur.2022.1013652 COPYRIGHT © 2022 Hybbinette, Östberg, Schalling, Deboussard, Plantin, Borg and Lindberg. Longitudinal changes in functional connectivity in speech motor networks in apraxia of speech after stroke 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. TYPE Original Research PUBLISHED 30 November 2022 DOI 10.3389/fneur.2022.1013652 TYPE Original Research PUBLISHED 30 November 2022 DOI 10.3389/fneur.2022.1013652 TYPE Original Research PUBLISHED 30 November 2022 DOI 10.3389/fneur.2022.1013652 Introduction that the association between insular damage and AOS might primarily relate to the vulnerability of the insula to large MCA strokes. That there is an association between left anterior insular lesions and AOS was not disputed, but its precise role in speech production was viewed as unclear. Eickhoffet al. (19) suggested that the insula could serve as a relay between cognitive aspects of language and the preparation of speech motor movements. Based on a meta-analysis of published functional neuroimaging studies and own fMRI data, they proposed that the phonetic concept of an intended speech act, presumably deriving from Broca’s region, would be received in the insula where the information is encoded into articulatory motor patterns. The insula forwards the plan to the cerebellum and the basal ganglia, which both project to the premotor cortex. The premotor cortex transforms the intended actions into specific patterns for muscle activation, that then are forwarded to primary motor cortex and on to lower motor neurons and the final execution phase (19). It has been shown that in patients with stroke-induced acute AOS, the premotor cortex was the region predominantly associated with AOS symptoms (8). The importance of the premotor cortex in speech production has also been highlighted in neurocomputational models of speech production, such as the Directions Into Velocities of Articulators (DIVA) model (20) and its expanded version, the Gradient Order DIVA (GODIVA) model (21, 22). According to these models, a lesion in the left premotor cortex would impair the conversion of well-formed phonological messages into previously learned speech motor movements and thereby account for the speech programming impairment associated with AOS. Also in patients with PPAOS, abnormalities in premotor and supplementary motor regions are considered as highly plausible candidates responsible for AOS symptoms (23). Apraxia of speech (AOS) is traditionally defined as a disorder of speech motor planning and programming. The disturbance is regarded to be located at the interface between the linguistic formulation phase and speech motor execution, reflecting “inefficiencies in the translation of well- formed and -filled phonological frames into previously learned kinematic information” (1). The core symptoms of AOS include articulatory errors that are perceived as sound and/or syllable distortions, slow speech rate with extended segment and inter- segment durations and prosodic deficits (2, 3). A stroke in the language-dominant (typically left) hemisphere within the territory of the middle cerebral artery (MCA) is considered the most frequent cause of AOS. Methods: Methods: Functional connectivity measures and comprehensive speech-language assessments were obtained at 4 weeks and 6 months after stroke in nine patients with AOS after a LH stroke and in six LH lesioned stroke patients without speech-language impairment. Functional connectivity was investigated in a network for speech production: inferior frontal gyrus (IFG), anterior insula (aINS), and ventral premotor cortex (vPMC), all bilaterally to investigate signs of adaptive or maladaptive changes in both hemispheres. Results: Interhemispheric vPMC connectivity was significantly reduced in patients with AOS compared to LH lesioned patients without speech-language impairment. At 6 months, the AOS severity was associated with interhemispheric aINS and vPMC connectivity. Longitudinal changes in FC were found in individuals, whereas no significant longitudinal change in FC was found at the group level. Degree of longitudinal AOS recovery was strongly associated with interhemispheric IFG connectivity strength at 4 weeks. Frontiers in Neurology 01 Frontiers in Neurology 01 frontiersin.org Hybbinette et al. Hybbinette et al. 10.3389/fneur.2022.1013652 10.3389/fneur.2022.1013652 Conclusion: Early interhemispheric IFG connectivity may be a strong predictor of AOS recovery. The results support the importance of interhemispheric vPMC connection in speech motor planning and severity of AOS and suggest that also bilateral aINS connectivity may have an impact on AOS severity. These findings need to be validated in larger cohorts. apraxia of speech, functional connectivity, resting-state fMRI, stroke, recovery Hybbinette et al. Introduction Less frequent etiologies are traumatic brain injury, tumors, and neurosurgery (3). Apraxia of speech can also be caused by neurodegenerative disease, often as part of the non-fluent/agrammatic variant of primary progressive aphasia (PPA) (4). When progressive AOS is the only or the primary neurological deficit, it is referred to as primary progressive apraxia of speech (PPAOS) (5, 6). AOS after stroke rarely occurs in isolation; rather, it is most often accompanied by aphasia and/or dysarthria (3, 7, 8). Many patients with AOS also have non-verbal oral apraxia (NVOA) (9), especially severe AOS tends to co-occur with NVOA (3). The frequent association indicates that the mechanisms for oromotor and speech motor control to some degree depend upon shared substrates (10). There is a consensus today that the pathogenesis of AOS is associated with disturbances to a network of brain regions. Several different regions have been proposed to be involved, but the exact constituents and the role and relationship between the sites and pathways in this network remain unclear (11–13). Early post-mortem studies identified an area in the posterior inferior frontal gyrus of the dominant hemisphere (“Broca’s area”) as a region associated with speech articulation difficulties (14), a localization that since then has gained rich support in AOS research, for example by Richardson et al. (15), Trupe et al. (16) and Wertz et al. (17). Dronkers (18) instead concluded that the left insula is the most crucial area for motor speech planning/programming and its lesion contributes to AOS. This was later questioned by other researchers. Hillis et al. (11) argued The role of the right (or non-dominant) hemisphere in speech-language function and its ability to compensate after a left hemisphere (LH) stroke has been long debated. It is well established that areas in the right hemisphere (RH), homologous to LH speech-language areas, are activated during speech- language activities in healthy adults. This activation has been shown to increase depending on task difficulty and degree of cognitive load (24, 25). However, it is still not clear whether additional activation of RH regions is beneficial or maladaptive Frontiers in Neurology frontiersin.org 02 10.3389/fneur.2022.1013652 Hybbinette et al. supplementary motor area (SMA) and left posterior temporal lobe to the rest of the speech and language ICN network, with the degree of reduced connectivity negatively correlated with an articulatory error score (38). for speech-language recovery after a stroke or an acquired brain injury. Introduction Some studies argue that good language recovery can only be obtained by a reactivation in the LH, while others describe a positive recovery by activation in the RH. The activation pattern may also change during the different stages after the stroke and factors such as lesion size and site are assumed to be involved (26–28). Functional brain reorganization with an increased activity in homologous areas in the RH has also been observed in individuals with PPA and PPAOS (29, 30). In addition to the limited number on FC and its relation to AOS, few studies have investigated AOS in early stroke patients and how the symptoms evolve over time. Most studies on AOS are focused on patients in a chronic stage, often with milder impairments than those that may occur at an early stage, and factors that may predict recovery are largely unknown (7, 40). The overall aim of this study was thus to address the recognized knowledge gap regarding cortical connectivity within a speech production network related to AOS. Based on previous findings, the specific aims were: Resting state functional magnetic resonance imaging (rs- fMRI) enables study of functional network connectivity (31). Low frequency (<0.1 Hz) BOLD signals acquired during rest show correlated activation within anatomically separated brain regions believed to reflect networks that typically are engaged in shared functions (32–34). While conventional fMRI compares changes in the BOLD signal during a task period and a baseline state, rs-fMRI is obtained in the absence of a stimulus or a task with the patient simply “resting” in the scanner. The technique is therefore considered particularly suitable for individuals who may have difficulty to perform a certain task, for example stroke patients with a speech-language impairment (27). There are several methods to analyze functional connectivity (FC) data. In a seed-based analysis approach, FC is evaluated by computing cross-correlations between different regions of interest (ROI). This method requires a priori selection of ROIs and is often based on a hypothesis or prior results (35). 1) To compare FC strength in patients with AOS after a LH stroke to that in LH lesioned stroke patients without a speech-language impairment, at a subacute phase at 4 weeks and at a chronic phase at 6 months. 2) To investigate the relation between FC and degree of severity in AOS, aphasia and NVOA. Introduction 3) To investigate longitudinal changes in FC in post stroke patients with AOS with concomitant aphasia and NVOA, from the subacute to the chronic phase, to identify predictors of AOS recovery. To the best of our knowledge, only one published study has applied rs-fMRI to investigate FC in patients with AOS after stroke. New et al. (36) studied thirty-two post stroke aphasia patients, fifteen of which had concomitant AOS. The time post stroke onset among the participants with AOS varied between 1 to 156 months, with the majority being in a chronic phase. Functional connectivity was examined based on the DIVA model, in a network of regions proposed as key areas in speech production: the inferior frontal gyrus (IFG), anterior insula (aINS), and ventral premotor cortex (vPMC) (37). To study the relation between the affected and unaffected hemisphere, both hemispheres were included in the analyses. When comparing the whole patient group with eighteen healthy controls, reduced FC between each interhemispheric homotopic seed region was found in the patient group. Comparisons between patients with and without AOS showed that patients with AOS had reduced FC between bilateral vPMC and between left vPMC and right aINS. The reduced FC between interhemispheric homotopic vPMC regions was related to AOS severity, while a negative FC between the left vPMC and right aINS related to NVOA severity. Participants To enable comparison of FC patterns between LH lesioned patients with AOS to the FC in LH patients without AOS symptoms, a control group including six LH lesioned patients with no signs of AOS, aphasia or NVOA was recruited from the same longitudinal cohort. The AOS and the non-AOS comparison group did not differ regarding age or sex but differed significantly in level of hand motor impairment. For descriptives and clinical characteristics, see section “Characterization of lesion data” and Table 3. et al. (45) and Staiger et al. (46), and the total score has been found to be a reliable measure of AOS severity after stroke (47). However, some of the items on the ASRS require a certain level of speech production to be ratable in accordance with the formulations of level descriptors. Problems to confidently score patients with signs of severe AOS with the ASRS have therefore been noted (48). To enable the use of the ASRS in this study and ratings that reflected the observed severity levels also for participants with a limited speech production, adapted strategies for ratings in four of the thirteen items on the ASRS were used. These modifications are presented in Table 1. The complete ASRS can be seen in Utianski et al. (49). The AOS symptoms were rated based on the participants’ performance during conversation, a picture description task, and by use of a Swedish translation of a motor speech protocol developed at the Mayo Clinic, Supplemental Tasks for Assessing Motor Speech Abilities, described in Duffy et al. (5). These supplemental tasks included measurements of vowel prolongation, repetition of words and sentences of varying length and complexity, as well as production of alternating motion rates and sequential motion rates. Speech production was elicited by the first author or by another speech-language pathologist (SLP) at the same clinic and all sessions were video recorded. The ratings were made by the first author and confirmed by an external SLP with long clinical and research experience of both AOS and aphasia. When discrepancies in scoring occurred, they were discussed and resolved by consensus. Since this study was carried out in a clinical setting, the ratings were also discussed with the clinical SLP, who in some cases also had completed the ASRS for the participant. Between the two assessments, all participants received team- based rehabilitation. Participants The participants were recruited from a group of post stroke patients participating in a longitudinal study of hand motor function, the ProHand Study (ClinicalTrials.gov Identifier: NCT02878304). The study was approved by the Regional Ethical Review Board in Stockholm and informed consent was obtained from all participants. To enable the inclusion of patients with impaired language abilities, both oral and written information was adapted and presented in an aphasia-friendly manner. Inclusion criteria were: 1) Patients aged ≥18 years admitted to inpatient care after first ever-stroke, 2) Swedish as first language, 3) Time of enrollment: between 2 and 6 weeks after stroke onset, 4) Clinical evidence of hand motor deficits based on neurological examination and medical records, 5) Awake, alert and capable of participating in assessment procedures. Exclusion criteria were: 1) Inability to understand and comply with instructions (presented in an adapted format for patients with aphasia), 2) Cerebellar lesions, 3) Report of claustrophobia or metal objects in the body, 4) Presence of other neurological, psychiatric, or medical conditions that preclude active participation. Brain imaging examination with sequences for resting state fMRI and behavioral assessments were conducted at two time points: the first (A1) on average at 4 weeks after stroke onset (mean 33 days, Beside the study of stroke patients by New and colleagues, Botha et al. (38) studied FC in twenty-two patients with PPAOS. For the connectivity analyses, a hybrid method was applied by choosing a set of hypothesis-driven, predefined Intrinsic Connectivity Networks (ICN) from the Mayo Clinic Study of Aging (MCSA) functional connectivity atlas (39). The main reported finding was a reduced connectivity between the right Frontiers in Neurology frontiersin.org 03 10.3389/fneur.2022.1013652 Hybbinette et al. Hybbinette et al. range 24– 42 days), and the follow-up (A2) at 6 months post stroke onset (range ± 9 days). Nine patients with AOS at A1 with complete data from both assessments were included in the longitudinal study. All in this group also had aphasia and NVOA at A1, all also had mild to severe upper limb motor impairment. Four of the participants also had unilateral upper motor neuron dysarthria, but to such a mild degree that it did not interfere the other speech-language measurements. Participants During the stay at the inpatient clinic, which lasted between ten to twelve weeks for all the participants in the AOS group, speech-language treatment was provided according to guidelines by the National Board of Health and Welfare in Sweden with four to five sessions a week of individual SLP training. These sessions were in general 30–45 min long and included interventions targeting both language and speech outcomes on a functional and structural level, based on the individual participant impairment profile. Interventions that addressed communication activities and participation based on personal goals were also included in the sessions. After discharge from the inpatient clinic, all participants received speech- language interventions provided by their regional rehabilitation team. These interventions were mainly homebased, with SLP training two to three times a week. No participant received highly intensive speech-language therapy in any form, such as Intensive Language Action Therapy (ILAT) (41). Presence, degree, and type of aphasia was assessed by use of the Swedish standardized assessment instrument Neurolinguistic Aphasia Examination (A-ning) (50). A-ning comprises evaluation of seven linguistic modalities: “oral expression abilities,” “repetition,” “auditory comprehension,” “reading comprehension,” “reading aloud,” “dictation” and “informative writing.” The maximum score is 220 points (= no language impairment) and the cut-offfor an aphasia diagnosis is < 208. Visual confrontation naming ability was assessed by use of the Boston Naming Test (BNT) (51). The maximum result is 60, and scoring was done according to Swedish target words by Tallberg (52). To assess the presence and degree of NVOA, a screening protocol created at the Mayo Clinic was applied. Total maximum score on this screening test is 32 and the recommended cut-offfor an NVOA diagnosis is <29 (6, 53). Behavioral assessment Presence and degree of AOS was investigated using the Apraxia of Speech Rating Scale 2.0 (ASRS 2.0) (42, 43). The ASRS 2.0 includes ratings of thirteen characteristics on a 5- point scale. Maximum total score is 52 and the recommended cut-offvalue for an AOS diagnosis is ≥8 points (42). The descriptors for each level of rating are: (0) not observed in any task/no more than one occurrence”; (1) “infrequent/noted more than once”; (2) “frequent but not pervasive/noted in 20– 50% of all utterances, but not on most tasks or utterances”; (3) “nearly always evident but not marked in severity/noted on many utterances on most tasks but not enough to decrease overall intelligibility” and (4) “nearly always evident and marked in severity/noted on most utterances on most tasks and severe enough to impact intelligibility.” The ASRS is increasingly used in studies of AOS, as for example by Clark et al. (44), Mailend Magnetic resonance imaging 3.1 Rate only for SMRs: Deliberate, slowly sequenced, segmented (gaps between sequences), and/or distorted (including distorted substitutions) speech SMRs in comparison to AMRs. Rate the best effortScore on severity only: 0 = not noted, SMRs normal; 1 = slow, 2 = mildly segmented and/or distorted; 3 = moderately segmented and/or distorted, 4 = severely segmented and/or distorted neurologist who was blinded to all clinical data. Lesion location was verified on FLAIR images, and lesion maps were binarized. Normalization parameters for T1 images were applied to lesion maps using the SPM12 tool Old Normalize. echo-based sequence): field of view, 250×250×181 mm; matrix, 228×227; slice thickness, 1.2 mm; slice spacing, 0.6 mm; and number of slices, 301 (echo time [TE] = 3.456 ms; repetition time [TR] = 7.464 ms). T2 fluid-attenuated inversion recovery (FLAIR) images were also acquired. Resting-state fMRI consisted of a gradient echo-planar sequence (echo time [TE] = 35 ms, flip angle = 90◦, voxel size of 1.8 × 1.8 × 4 mm, repetition time [TR] = 3000 ms) sensitive to BOLD contrast. The resting state fMRI sequence lasted 6 min. Patients were instructed to keep eyes closed, to think about nothing in particular, and to not fall asleep. Anatomical T1-images were normalized to Montreal Neurological Institute template using SPM12 (www.fil.ion.ucl.ac.uk/spm/software/spm12/) Clinical toolbox unified segment-normalize procedure (non- linear enantiomorphic normalization, 3-tissue, “old segment,” optimizing the normalization of clinical data with focal brain lesions by exploiting information from homologous regions of the non-lesioned hemisphere). fMRI images were realigned and coregistered to T1 and then normalized using cost function masking to avoid distortion of lesion by normalization procedure (54). The images were inspected visually to ensure adequate normalization. Lesion maps were manually drawn on all axial slices of native space T1 weighted anatomical images using MRIcron (https://people.cas.sc.edu/rorden/mricron/ index.html) by a researcher (J.P.) and verified by an experienced ROI selection The selection of 10 mm diameter ROIs was based on current knowledge on speech motor networks and included regions hypothesized to have an important role in AOS. These ROIs were also applied in the study by New et al. (36): inferior frontal gyrus (IFG), anterior insula (aINS) and ventral premotor cortex (vPMC). Intra- and interhemispheric connectivity between ROIs was studied between similar (homotopic) and different (heterotopic) regions. Montreal Neurological Institute (MNI) coordinates for the selected regions according to Eickhoffet al. (19) are presented in Table 2. Magnetic resonance imaging Brain imaging at both assessment occasions was performed with an Ingenia 3.0T MR system (www.usa.philips.com) with an 8 HR head coil. High-resolution T1-weighted anatomical images were acquired using TFE 3D (3-dimensional gradient Frontiers in Neurology 04 frontiersin.org Hybbinette et al. 10.3389/fneur.2022.1013652 TABLE 1 Modified ratings for participants with limited speech production on the ASRS 2.0. TABLE 1 Modified ratings for participants with limited speech production on the ASRS 2.0. 0 1 2 3 4 Not observed in any task Infrequent Frequent but not pervasive Nearly always evident but not marked in severity Nearly always evident and marked in severity No more than one occurrence Noted more than once Noted 20–50% of all utterances, but not on most tasks or utterances Noted on many utterances on most tasks but not enough to decrease overall intelligibility Noted on most utterances on most tasks and severe enough to impact intelligibility Items on the ASRS 2.0 Applied rating strategies 1.3 Increased sound distortions or distorted sound substitutions with increased utterance length or increased syllable/word articulatory complexity For individuals who cannot produce phrases or multisyllabic words, but these symptoms are noticed in monosyllabic words and in isolated speech sounds, a score of 4 is applied. 2.1 Syllable segmentation within words > 1 syllable (Brief silent interval between syllables and/or inappropriate equalized stress across syllables) For individuals who cannot produce multisyllabic words but shows apraxic symptoms that are judged be the underlying course of the impairment, a score of 1–4 is applied* 2.2 Syllable segmentation across words in phrases/sentences (Increased inter-word intervals and/or inappropriate equalized stress across words) For individuals who cannot produce phrases/sentences but shows apraxic symptoms that are judged be the underlying course of the impairment, a score of 1–4 is applied* 3.1 Rate only for SMRs: Deliberate, slowly sequenced, segmented (gaps between sequences), and/or distorted (including distorted substitutions) speech SMRs in comparison to AMRs. Rate the best effortScore on severity only: 0 = not noted, SMRs normal; 1 = slow, 2 = mildly segmented and/or distorted; 3 = moderately segmented and/or distorted, 4 = severely segmented and/or distorted For individuals who have major problems producing both AMR and SMRs and show apraxic symptoms that are judged be the underlying course of the impairment, a score of 4 is applied. *Rating value corresponding to degree of observed AOS symptoms being the cause of the impairment, rated in consensus between 2–3 raters/ SLPs. Functional connectivity analyses BOLD time series from the selected ROIs were extracted as the first principal component of all voxel time series within a spherical (radius 5 mm) volume of interest centered on the Frontiers in Neurology 05 frontiersin.org 10.3389/fneur.2022.1013652 Hybbinette et al. TABLE 2 Coordinates for the selected regions of interest. Statistical methods TABLE 2 Coordinates for the selected regions of interest. Regions of interest (ROI) MNI coordinates (x, y, z) (left–right) Inferior frontal gyrus (IFG) −50 10 5 50 10 5 Anterior insula (aINS) −32 15 2 32 15 2 Ventral premotor cortex (vPMC) −58 1 23 58 1 23 Normal distribution was tested by use of Shapiro-Wilk test and visual inspection. The variables were not substantially skewed, but owing to the limited sample size, non-parametric methods were applied. Between-group differences were tested using the Mann-Whitney U test and Pearson’s chi-squared test, whereas within-group differences were assessed with the related- samples Wilcoxon signed-rank test. The degree of recovery was defined as the percentage that a participant improved over time, expressed as a percentage of the maximal possible change: A2- A1/ Max score–A1. Spearman’s correlation coefficients were calculated to test the strength of association between FC strength and behavioral measures at four weeks (A1) and 6 months (A2) and recovery (dynamic change over time) in the different speech-language domains. A p-value below 0.05 was considered statistically significant. False discovery rate (FDR) (58) was applied to control for multiple comparisons in all univariate correlation analyses with a q-value set at < 0.003. Statistical analysis was performed by use of the IBM SPSS Statistics for Windows, Version 27. individual peak coordinates for each maximum. Image pre- processing included (i) head movement and correction, (ii) co- registration of resting state fMRI (EPI) images to T1-weigted anatomical images, (iii) segmentation (gray matter/white matter/CSF), (iv) normalization using the SPM12 Clinical Toolbox, and (v) smoothing (8 mm). Seed-based FC was calculated using the Connectivity toolbox (55). It incorporates the CompCorr strategy for reduction of noise from physiological and other sources, that takes into account the non-homogeneous distribution of noise signals in the brain. For example, voxels close to white matter or large blood vessels show greater BOLD signal noise. Principal components (PCA) were derived from these noise regions and later included as nuisance parameters within the general linear model. EPI images were inspected visually to identify signal drop-out (due to e.g., the presence of meta-hemoglobin and hemosiderin, i.e., breakdown products from haemorrhagic stroke). Realignment parameters were modeled as regressors of no interest. Estimation of head motion parameters and the presence of image outliers (Artifact Detection toolbox: https://www.nitrc.org/projects/ artifact_detect) were included as regressors since it has been shown that this strategy improves motion artifact correction when studying FC (56). Statistical methods The principal components of signal from white matter and CSF masks were regressed out during the analysis. A temporal band pass filter (0.01–0.08 Hz) was applied covering approximately the range between 10 and 100 sec which is standard for resting-state connectivity analyses (57). The Conn toolbox computed the average BOLD time series across all the voxels within each ROI. The beta value reflecting interhemispheric and intrahemispheric FC between each pair of ROIs was extracted for each participant. The beta values were then transformed into Fishers- Z values, where each score represents the functional connectivity strength for each connection in each participant. Statistical methods Activation threshold of z-normalized global brain signal was set to 3 SD and threshold for rotational and translational head motion was set to 2 mm. This resulted in mean (±SD) = 10 (±11) excluded volumes (out of 160 = 6%). White matter and CSF masks were used for partial volume correction. The principal components of signal from white matter and CSF masks were regressed out during the analysis. A temporal band pass filter (0.01–0.08 Hz) was applied covering approximately the range between 10 and 100 sec which is standard for resting-state connectivity analyses (57). The Conn toolbox computed the average BOLD time series across all the voxels within each ROI. The beta value reflecting interhemispheric and intrahemispheric FC between each pair of ROIs was extracted for each participant. The beta values were then transformed into Fishers- Z values, where each score represents the functional connectivity strength for each connection in each participant. individual peak coordinates for each maximum. Image pre- processing included (i) head movement and correction, (ii) co- registration of resting state fMRI (EPI) images to T1-weigted anatomical images, (iii) segmentation (gray matter/white matter/CSF), (iv) normalization using the SPM12 Clinical Toolbox, and (v) smoothing (8 mm). Seed-based FC was calculated using the Connectivity toolbox (55). It incorporates the CompCorr strategy for reduction of noise from physiological and other sources, that takes into account the non-homogeneous distribution of noise signals in the brain. For example, voxels close to white matter or large blood vessels show greater BOLD signal noise. Principal components (PCA) were derived from these noise regions and later included as nuisance parameters within the general linear model. EPI images were inspected visually to identify signal drop-out (due to e.g., the presence of meta-hemoglobin and hemosiderin, i.e., breakdown products from haemorrhagic stroke). Realignment parameters were modeled as regressors of no interest. Estimation of head motion parameters and the presence of image outliers (Artifact Detection toolbox: https://www.nitrc.org/projects/ artifact_detect) were included as regressors since it has been shown that this strategy improves motion artifact correction when studying FC (56). Activation threshold of z-normalized global brain signal was set to 3 SD and threshold for rotational and translational head motion was set to 2 mm. This resulted in mean (±SD) = 10 (±11) excluded volumes (out of 160 = 6%). White matter and CSF masks were used for partial volume correction. Characterization of lesion data Dark blue indicates low lesion overlap, light green indicates high overlap. (A) Participants with apraxia of speech, aphasia, and non-verbal oral apraxia (n = 9). (B) Participants with no speech-language impairment (n = 6). All participants with apraxia of speech, aphasia, and non-verbal oral apraxia had cortical lesions in the middle cerebral artery territory with extensions into deep subcortical white matter where the largest lesion overlap was found. SMA was not lesioned in any of the participants in the AOS group. The participants without speech-language impairment had in general smaller lesions spread in mainly subcortical areas. TABLE 3 Demographic and clinical characteristics at the first assessment. TABLE 3 Demographic and clinical characteristics at the first assessment. Variables AOS group No SLI group Group diff.a,b (n = 9) (n = 6) Age Mean (SD) 49.6 (11.3) 56.7 (5.1) 0.237a Median 52 56.5 Sex N (%) Females 2 (22) 1 (17) 0.792b Males 7 (78) 5 (83) Lesion volume cm3 Mean (SD) 133.0 (88.3) 14.4 (16.8) 0.002a* Median 121.9 8.5 Stroke type N (%) Ischemic 8 (89) 4 (67) 0.292b Haemorrhagic 1 (11) 2 (33) FM-UE Mean (SD) 11.1 (19.2) 44.8 (16.2) 0.013a* Median 2 51.5 AOS group, participants with apraxia of speech, aphasia, and non-verbal oral apraxia; SLI, Speech-Language Impairment; FM-UE, Fugl-Meyer assessment for the upper extremity, max 60 p, cut-offlevel severe-moderate impairment 19 ± 2 points, moderate–mild impairment 47 ± 2 points. aMann-Whitney U test, bPearson Chi-Square, p < 0.05. AOS group, participants with apraxia of speech, aphasia, and non-verbal oral apraxia; SLI, Speech-Language Impairment; FM-UE, Fugl-Meyer assessment for the upper extremity, max 60 p, cut-offlevel severe-moderate impairment 19 ± 2 points, moderate–mild impairment 47 ± 2 points. aMann-Whitney U test, bPearson Chi-Square, p < 0.05. Characterization of lesion data Characterization of lesion data All participants with AOS, aphasia, and NVOA (hereafter called the AOS group) had a left MCA stroke with cortical lesions extending into subcortical white matter. The majority were caused by ischemic strokes; one participant had a hemorrhagic stroke. In the group without a speech-language impairment (No SLI group), the lesions were in general smaller and subcortically distributed (Figure 1). Two in this group had hemorrhagic strokes, whereas all others had ischemic strokes. A Mann-Whitney U test indicated that the lesion volume was significantly larger in the AOS group (Mdn = 121.9 cm3) than in the No SLI group (Mdn = 8.5 cm3), U = 2.0, p = 0.002 (Table 3). No significant correlation existed between lesion volume and any of the behavioral scores in the AOS group (at A1; ASRS rho = −0.35; A-ning rho = 0.18; BNT rho = 0.24; NVOA rho = −0.35). None of the participants in the No SLI group had a lesion overlap with any of the selected ROIs. In the AOS group, three (33%) of the participants had over 25% damage to the left vPMC, seven (78%) had over 25% damage to the left aINS, and seven (78%) to the left IFG. As shown in Table 4, several had over 25% damage to more than one ROI. There was no significant correlation between lesion volume and measures of FC in the selected network. A non-significant trend was found for the left aINS and left vPMC at the 6-months follow-up (rho = 0.65, p = 0.06). Frontiers in Neurology frontiersin.org 06 Hybbinette et al. 10.3389/fneur.2022.1013652 FIGURE 1 Lesion overlap map at ≈4 weeks post stroke onset shown on five axial slices (z coordinates provided). Dark blue indicates low lesion overlap, light green indicates high overlap. (A) Participants with apraxia of speech, aphasia, and non-verbal oral apraxia (n = 9). (B) Participants with no speech-language impairment (n = 6). All participants with apraxia of speech, aphasia, and non-verbal oral apraxia had cortical lesions in the middle cerebral artery territory with extensions into deep subcortical white matter where the largest lesion overlap was found. SMA was not lesioned in any of the participants in the AOS group. The participants without speech-language impairment had in general smaller lesions spread in mainly subcortical areas. FIGURE 1 Lesion overlap map at ≈4 weeks post stroke onset shown on five axial slices (z coordinates provided). Functional connectivity comparison between groups At the first assessment, no significant correlations between FC and any behavioral score results were found. A non- significant trend for an association was present between interhemispheric IFG connectivity and A-ning, ASRS, and BNT results (rho = 0.79, p = 0.010; rho = −0.61, p = 0.081; rho = 0.63, p = 0.068 respectively), but none of these survived an FDR correction for multiple comparisons set at q < 0.003. The relation between interhemispheric vPMC connectivity and the ASRS total score was only moderate (rho −0.49, p = 0.177) (Table 7). At A1, reduced FC was observed between all three interhemispheric homotopic ROIs in the AOS group compared to the No SLI group. The difference was significant for interhemispheric vPMC connectivity (Fisher’s z-score 0.08 vs. 0.78, Mann-Whitney U test p = 0.001) while the reduced interhemispheric IFG connectivity in the AOS group showed a non-significant trend (Fisher’s z-score 0.13 vs. 0.31, Mann- Whitney U test p = 0.08) and the interhemispheric aINS difference was the least prominent (Fisher’s z-score 0.19 vs. 0.44, Mann-Whitney U test p = 0.16). In the non- lesioned hemisphere, participants in the AOS group displayed a significantly stronger intrahemispheric connectivity between the right aINS and right vPMC compared to the No SLI group (Fisher’s z-score 0.32 vs. 0.06, Mann-Whitney U test p = 0.025). The strongest connectivity in the AOS group was found between the right IFG and right aINS (Fisher’s z-score 0.57) (Table 6, Figure 2). At six months follow-up (A2), a significant correlation between ASRS result and interhemispheric aINS connectivity was found (rho = −0.88, p = 0.002, q < 0.003). A strong but non-significant correlation was found between ASRS total score and FC strength between left IFG and right aINS (rho = −0.80, p = 0.009) and with interhemispheric homotopic vPMC FC (rho = −0.79, p = 0.012). There were no significant correlations between FC strength and A-ning (covering all language domains), BNT (naming ability), or NVOA (Table 8). At A2, the interhemispheric vPMC connectivity was still stronger in the No SLI group in comparison to the AOS group, but this did not reach significance (Fisher’s z-score 0.46 vs. 0.12, Mann-Whitney U test p = 0.059). The earlier significantly stronger RH connection between aINS and vPMC in the AOS group was slightly reduced, while it had increased among participants with no SLI. Characterization of behavioral data and recovery in participants with AOS, aphasia and NVOA At six months follow-up (A2), total scores from all assessment instruments demonstrated statistically significant improvements (Wilcoxon signed-rank test ASRS Z = −2.67, p = 0.007; A-ning Z = −2.66, p = 0.008; BNT Z = −2.52, p = 0.012; NVOA Z = −2.52, p = 0.012). 1 participant recovered 78% and showed an almost complete AOS recovery with an ASRS score of 2, that is, below the recommended cut-offvalue at 8 points, while a majority of the remaining still had a moderate AOS. Two participants no longer had NVOA according to the result of the assessment. At the early assessment (A1), six participants in the AOS group had very severe (global) aphasia, one a severe Broca’s aphasia, and two a moderately severe Broca’s aphasia. The mean A-ning score was 57.4 which corresponds to severe aphasia. The majority showed almost no naming ability; the median BNT score was 0, and the mean score 8.8. The ASRS total score ranged between 9 (indicating mild AOS) up to 44 (severe AOS). The mean ASRS score was 24.2 which indicates moderate to marked AOS (23). All had NVOA at this time point, several to a severe degree (Table 5). The individual degrees of recovery covered a wide range in all behavioral assessments. The AOS recovery varied between 4 and 78%, with a mean value of 33%. The mean values for the other clinical recovery percentages were similar, with NVOA Frontiers in Neurology 07 frontiersin.org 10.3389/fneur.2022.1013652 Hybbinette et al. TABLE 4 Percentage lesion overlap with the selected ROIs in participants with AOS, aphasia, and NVOA. TABLE 4 Percentage lesion overlap with the selected ROIs in participants with AOS, aphasia, and NVOA. ID Lesion overlap (%) to ROIs Number of ROIs with >25% overlap vPMC aINS IFG 1 5 31 30 2 2 0 100 100 2 3 0 10 0 0 4 16 100 100 2 5 0 28 0 1 6 94 96 100 3 7 0 0 44 1 8 85 33 100 3 9 100 100 100 3 N (%) with lesion overlap >25% 3 (33%) 7 (78%) 7 (78%) All regions refer to left hemisphere. IFG, inferior frontal gyrus; aINS, anterior insula; vPMC, ventral premotor cortex. ID Lesion overlap (%) to ROIs Number of ROIs w decreased since A1 among AOS participants and increased in the No SLI group (Fisher’s z-score 0.09 vs. 0.37, Mann-Whitney U test p = 0.034). Characterization of behavioral data and recovery in participants with AOS, aphasia and NVOA recovery of 34% and A-ning recovery of 42%. All speech and language results for each participant are presented in Table 5. The results from all seven domains in A-ning Neurolinguistic Aphasia Examination for each participant are provided in Supplementary Table S1. Functional connectivity comparison between groups A significant group difference was found between the right IFG and left aINS; this connection had F, female; M, male; I, ischemic stroke; H, hemorrhagic stroke; ASRS, Apraxia of Speech Rating Scale, max 52, cut-offvalue severity classification. Very severe/Global ≤42, Severe ≤82, Moderate ≤170, Moderate/mild ≤192, Mild ≤208, No Aphasi This study provides one of the first data sets presenting FC changes in a bilateral speech production network in patients with AOS in an early phase after stroke. The main finding was a strong correlation between the interhemispheric IFG connectivity and the individual AOS recovery. Participants with low FC between these seeds at 4 weeks post-stroke showed less recovery at 5 months than participants with higher FC (Figure 4). In line with findings by New et al. (36), participants with AOS in the present study also had a significantly reduced interhemispheric vPMC seed connectivity in comparison with LH lesioned participants without a speech- language impairment. The interhemispheric vPMC connectivity strength was however only moderately related to AOS severity at the early assessment, but nearly reached an FDR-corrected significant correlation at 6 months follow-up (rho = −0.79, p = 0.012). The strongest correlation between AOS severity and FC strength was found between interhemispheric aINS connectivity and the ASRS score result at 6 months (rho = −0.88, p = 0.002). Longitudinal changes in FC in patients with AOS At a group level in the AOS group, no significant changes in FC were found between the two assessment occasions. All three interhemispheric homotopic seed connections remained Frontiers in Neurology 08 frontiersin.org Hybbinette et al. 10.3389/fneur.2022.1013652 ale; I, ischemic stroke; H, hemorrhagic stroke; ASRS, Apraxia of Speech Rating Scale, max 52, cut-offvalue for AOS ≥8 points; BNT, Boston Naming Test, max 60; A-ning, Neurolinguistic Aphasia Examination, total score results and ation. Very severe/Global ≤42, Severe ≤82, Moderate ≤170, Moderate/mild ≤192, Mild ≤208, No Aphasia > 208, maximum 220 points. NVOA, Non-verbal oral apraxia protocol, max 32, cut-offvalue for NVOA diagnosis < 29 points. TABLE 5 Clinical data and behavioral results for participants with AOS, aphasia, and NVOA. ID Sex Stroke Age Les. vol. cm3 ASRS BNT A-ning NVOA A1 A2 Rec.% A1 A2 Rec.% A1 A2 Rec.% A1 A2 Rec.% 1 F I 52 30.6 44 35 20 0 21 35 35 95 32 16 17 6 2 M I 65 121.9 18 15 17 1 2 2 36 46 5 6 7 4 3 M I 57 32.9 23 15 35 0 5 8 26 86 31 2 9 23 4 F I 39 117.1 22 14 36 12 38 54 70 120 33 17 20 20 5 M H 31 167.7 17 9 47 49 51 18 134 202 79 18 30 86 6 M I 39 175.6 28 18 36 0 44 73 38 173 74 5 21 59 7 M I 61 160.2 9 2 78 17 43 60 150 190 57 12 20 40 8 M I 54 73.1 32 23 28 0 49 82 18 133 57 26 30 67 9 M I 48 317.8 25 24 4 0 0 0 10 25 7 0 0 0 Mean 49.6 133.0 24.2 17.2 33.4 8.8 28.1 36.9 57.4 118.9 41.7 11.3 17.1 33.9 (SD) (11.3) (88.3) (9.9) (9.4) (21.0) (16.3) (21.2) (31.5) (50.9) (62.1) (26.7) (8.6) (10.1) (30.8) Median 52 121.9 23 15 35 0 38 35 36 120 33 12 20 23 F, female; M, male; I, ischemic stroke; H, hemorrhagic stroke; ASRS, Apraxia of Speech Rating Scale, max 52, cut-offvalue for AOS ≥8 points; BNT, Boston Naming Test, max 60; A-ning, Neurolinguistic Aphasia Examination, total score results and severity classification. Very severe/Global ≤42, Severe ≤82, Moderate ≤170, Moderate/mild ≤192, Mild ≤208, No Aphasia > 208, maximum 220 points. NVOA, Non-verbal oral apraxia protocol, max 32, cut-offvalue for NVOA diagnosis < 29 points. low. Qualitatively, the largest increase was found between the left IFG and left aINS (Fisher’s z-score 0.21 vs. 0.39, p = 0.11). Although slightly reduced at A2, the strongest connection was still found between right IFG and right aINS (Fisher’s z-score 0.50) (Table 9, Figure 3). Relation between behavioral recovery and FC The interhemispheric homotopic IFG connectivity at the early assessment correlated strongly and positively with AOS recovery (rho = 0.92, p = 0.001). Thus, participants with higher FC here at 4 weeks after stroke showed more favorable AOS recovery to 6 months (Figure 4). This result remained significant when adding number of unaffected gray matter voxels in the IFG ROI as a covariate in the correlation analyses (partial r = 0.91, p = 0.002). The intrahemispheric connection between left aINS and left IFG was approaching a significant correlation with AOS recovery (rho = 0.81, p = 0.007), but did not reach the FDR corrected significance level set at q < 0.003. A-ning, BNT, and NVOA recovery did not correlate with any early FC result (Table 10). Intra–and interhemispheric IFG functional connectivity and AOS recovery The importance of the IFG in our results is in line with Hillis et al. (11), who reported that in patients in an acute stage after stroke, the presence of AOS was associated with Frontiers in Neurology 09 frontiersin.org Hybbinette et al. 10.3389/fneur.2022.1013652 FIGURE 2 Functional connectivity strength between ROIs at A1, group comparison. IFG, inferior frontal gyrus; aINS, anterior insula; vPMC, ventral premotor cortex. Significant group diferences marked with *(p < 0.05) and **(p < 0.01) in the figure. Line thickness illustrates degree of FC strength. FIGURE 2 Functional connectivity strength between ROIs at A1, group comparison. IFG, inferior frontal gyrus; aINS, anterior insula; vPMC, ventral premotor cortex. Significant group diferences marked with *(p < 0.05) and **(p < 0.01) in the figure. Line thickness illustrates degree of FC strength. TABLE 6 Functional connectivity at A1, comparison between groups. Functional connectivity strength (Fisher’s Z) AOS group (n = 9) No SLI group (n = 6) Mean (SD) Median Mean (SD) Median aGroup diff. Effect size (r) Interhemispheric homotopic IFG L–IFG R 0.130 (0.174) 0.132 0.314 (0.179) 0.332 0.077 0.46 aINS L–aINS R 0.187 (0.280) 0.170 0.435 (0.268) 0.372 0.157 0.36 vPMC L–vPMC R 0.079 (0.154) 0.039 0.777 (0.185) 0.756 0.001** 0.82 Intrahemispheric Left IFG L–aINS L 0.211 (0.200) 0.202 0.343 (0.233) 0.333 0.409 0. 21 IFG L–vPMC L 0.109 (0.180) 0.183 0.189 (0.118) 0.164 0.637 0.12 aINS L–vPMC L −0.007 (0.223) 0.025 0.067 (0.089) 0.099 0.479 0.18 Intrahemispheric Right IFG R–aINS R 0.571 (0.232) 0.541 0.416 (0.136) 0.413 0.238 0.30 IFG R–vPMC R 0.264 (0.212) 0.188 0.084 (0.142) 0.096 0.125 0.39 aINS R–vPMC R 0.321 (0.191) 0.354 0.061 (0.151) 0.053 0.026* 0.58 Interhemispheric heterotopic IFG L–aINS R 0.164 (0.279) 0.007 0.060 (0.182) 0.075 0.409 0. 21 IFG L–vPMC R 0.023 (0.204) 0.046 0.134 (0.146) 0.179 0.288 0.27 IFG R–aINS L 0.150 (0.204) 0.144 0.309 (0.174) 0.306 0.125 0.40 IFG R–vPMC L 0.072 (0.152) 0.135 0.071 (0.148) 0.085 0.906 0.03 aINS L–vPMC R 0.050 (0.192) 0.060 0.001 (0.158) 0.036 0.814 0.06 aINS R–vPMC L 0.077 (0.194) 0.034 −0.059 (0.093) −0.057 0.077 0.46 aMann-Whitney U test, *Significant at p-level < 0.05, **Significant at p-level < 0.01. r, Mann-Whitney U test Effect Size. TABLE 6 Functional connectivity at A1, comparison between groups. Functional connectivity strength (Fisher’s Z) hypoperfusion and/or infarct affecting Broca’s area in the left hemisphere. Our result is also in keeping with meta-analyses by Turkeltaub et al. Intra–and interhemispheric IFG functional connectivity and AOS recovery Thus, even if areas in the vPMC are considered as the key region on which speech planning and programming TABLE 7 Relation between FC and behavioral results at the first assessment (A1). TABLE 7 Relation between FC and behavioral results at the first assessment (A1). FC at Assessment 1 ASRS A1 A-ning A1 BNT A1 NVOA A1 IFG L–IFG R −0.61 0.79 0.63 0.21 IFG L–aINS L −0.59 0.59 0.39 −0.11 IFG L–aINS R −0.29 0.49 0.32 0.13 IFG L–vPMC L 0.25 −0.31 −0.32 −0.21 IFG L–vPMC R −0.46 0.49 0.39 −0.11 IFG R –aINS L −0.06 −0.13 −0.12 0.07 IFG R –aINS R 0.14 −0.43 −0.10 0.33 IFG R–vPMC L −0.19 0.14 0.01 −0.43 IFG R–vPMC R 0.21 −0.23 −0.23 0.26 aINS L–aINS R −0.59 0.31 0.43 0.24 aINS L–vPMC L −0.08 −0.14 −0.01 0.13 aINS L–vPMC R −0.21 0.09 0.01 −0.06 aINS R–vPMC L −0.11 0.36 0.05 −0.08 aINS R–vPMC R −0.23 0.01 0.12 0.26 vPMC L–vPMC R −0.49 0.34 0.39 −0.14 Spearman’s rho, FDR-corrected significance level q < 0.003. TABLE 8 Relation between FC and behavioral results at the second assessment (A2). FC at Assessment 2 ASRS A2 A-ning A2 BNT A2 NVOA A2 IFG L–IFG R −0.29 0.22 −0.02 −0.02 IFG L–aINS L −0.40 −0.27 −0.47 −0.50 IFG L–aINS R −0.80 0.67 0.43 0.39 IFG L–vPMC L 0.07 −0.18 −0.38 −0.44 IFG L–vPMC R 0.06 0.05 −0.02 −0.08 IFG R –aINS L −0.23 −0.22 −0.45 −0.46 IFG R –aINS R 0.01 0.13 0.17 0.20 IFG R–vPMC L −0.13 0.47 0.30 0.23 IFG R–vPMC R 0.23 0.12 0.32 0.34 aINS L–aINS R −0.88* 0.43 0.27 0.22 aINS L–vPMC L −0.49 0.43 0.20 0.10 aINS L–vPMC R 0.25 −0.05 0.15 0.14 aINS R–vPMC L −0.26 0.53 0.40 0.33 aINS R–vPMC R −0.10 0.47 0.57 0.55 vPMC L–vPMC R −0.79 0.76 0.67 0.64 Spearman’s rho, *significant at FDR level q < 0.003. Spearman’s rho, *significant at FDR level q < 0.003. speech production when the homologous left side is lesioned (62, 63). In a longitudinal study of individuals with primary progressive aphasia and progressive AOS, worsening of AOS was significantly correlated with greater progressive cortical thinning in the right IFG (29). Our finding that interhemispheric IFG connectivity in the early phase after stroke may be an important predictor of AOS recovery fits with the Eickhoff model emphasizing the role of IFG as a starting point in speech production (19). Intra–and interhemispheric IFG functional connectivity and AOS recovery (59) who found a pattern of over activation both in left perilesional regions and in RH regions homotopic to the LH hemisphere speech-language network. Specifically, a stronger activation in the right IFG tended to be more often present in individuals with lesions in the left IFG than in those without a lesion in that area. Several studies investigating aphasia after stroke have also proposed that RH areas can contribute to the recovery (60, 61) and that the right IFG can support Frontiers in Neurology 10 10 frontiersin.org frontiersin.org Hybbinette et al. 10.3389/fneur.2022.1013652 TABLE 7 Relation between FC and behavioral results at the first assessment (A1). FC at Assessment 1 ASRS A1 A-ning A1 BNT A1 NVOA A1 IFG L–IFG R −0.61 0.79 0.63 0.21 IFG L–aINS L −0.59 0.59 0.39 −0.11 IFG L–aINS R −0.29 0.49 0.32 0.13 IFG L–vPMC L 0.25 −0.31 −0.32 −0.21 IFG L–vPMC R −0.46 0.49 0.39 −0.11 IFG R –aINS L −0.06 −0.13 −0.12 0.07 IFG R –aINS R 0.14 −0.43 −0.10 0.33 IFG R–vPMC L −0.19 0.14 0.01 −0.43 IFG R–vPMC R 0.21 −0.23 −0.23 0.26 aINS L–aINS R −0.59 0.31 0.43 0.24 aINS L–vPMC L −0.08 −0.14 −0.01 0.13 aINS L–vPMC R −0.21 0.09 0.01 −0.06 aINS R–vPMC L −0.11 0.36 0.05 −0.08 aINS R–vPMC R −0.23 0.01 0.12 0.26 vPMC L–vPMC R −0.49 0.34 0.39 −0.14 Spearman’s rho, FDR-corrected significance level q < 0.003. TABLE 8 Relation between FC and behavioral results at the second assessment (A2). FC at Assessment 2 ASRS A2 A-ning A2 BNT A2 NVOA A2 IFG L–IFG R −0.29 0.22 −0.02 −0.02 IFG L–aINS L −0.40 −0.27 −0.47 −0.50 IFG L–aINS R −0.80 0.67 0.43 0.39 IFG L–vPMC L 0.07 −0.18 −0.38 −0.44 IFG L–vPMC R 0.06 0.05 −0.02 −0.08 IFG R –aINS L −0.23 −0.22 −0.45 −0.46 IFG R –aINS R 0.01 0.13 0.17 0.20 IFG R–vPMC L −0.13 0.47 0.30 0.23 IFG R–vPMC R 0.23 0.12 0.32 0.34 aINS L–aINS R −0.88* 0.43 0.27 0.22 aINS L–vPMC L −0.49 0.43 0.20 0.10 aINS L–vPMC R 0.25 −0.05 0.15 0.14 aINS R–vPMC L −0.26 0.53 0.40 0.33 aINS R–vPMC R −0.10 0.47 0.57 0.55 vPMC L–vPMC R −0.79 0.76 0.67 0.64 Spearman’s rho, *significant at FDR level q < 0.003. speech production when the homologous left side is lesioned (62, 63). In a longitudinal study of individuals with primary production (19). Intra–and interhemispheric IFG functional connectivity and AOS recovery Thus, even if areas in the vPMC are considered as the key region on which speech planning and programming depends, the present results underscore the central role of the left IFG and its intra- and interhemispheric connections to the rest of the speech-language network in determining recovery from AOS. The reorganization of the speech-language network is however known to be more complex than just a direct Frontiers in Neurology 11 frontiersin.org frontiersin.org Hybbinette et al. 10.3389/fneur.2022.1013652 FIGURE 3 Functional connectivity strength between ROIs, within group diferences. Participants with AOS, aphasia and NVOA. IFG, inferior frontal gyrus; aINS, anterior insula; vPMC, ventral premotor cortex. Line thickness illustrates degree of FC strength. FIGURE 3 Functional connectivity strength between ROIs, within group diferences. Participants with AOS, aphasia and NVOA. IFG, inferior frontal gyrus; aINS, anterior insula; vPMC, ventral premotor cortex. Line thickness illustrates degree of FC strength. TABLE 9 Functional connectivity at first and second assessment, participants with AOS (n = 9). Functional connectivity strength (Fisher’s Z) Assessment 1 Assessment 2 Mean (SD) Median Mean (SD) Median aWithin group diff. Effect size (r) Interhemispheric homotopic IFG L–IFG R 0.130 (0.174) 0.132 0.134 (0.160) 0.127 0.594 0.14 aINS L–aINS R 0.187 (0.280) 0.170 0.178 (0.316) 0.094 0.859 0.05 vPMC L–vPMC R 0.079 (0.154) 0.039 0.123 (0.257) 0.178 0.515 0.17 Intrahemispheric LEFT IFG L–aINS L 0.211 (0.200) 0.202 0.394 (0.174) 0.376 0.110 0.41 IFG L–vPMC L 0.109 (0.180) 0.183 0.179 (0.154) 0.159 0.515 0.17 aINS L–vPMC L −0.007 (0.223) 0.025 0.024 (0.164) 0.105 0.767 0.08 Intrahemispheric RIGHT IFG R–aINS R 0.571 (0.232) 0.541 0.496 (0.205) 0.498 0.260 0.29 IFG R–vPMC R 0.264 (0.212) 0.188 0.296 (0.167) 0.331 0.515 0.16 aINS R–vPMC R 0.321 (0.191) 0.354 0.216 (0.254) 0.237 0.214 0.32 Interhemispheric heterotopic IFG L–aINS R 0.164 (0.279) 0.007 0.213 (0.230) 0.281 0.859 0.05 IFG L–vPMC R 0.023 (0.204) 0.046 0.097 (0.141) 0.082 0.314 0.26 IFG R–aINS L 0.150 (0.204) 0.144 0.096 (0.226) 0.029 0.594 0.14 IFG R–vPMC L 0.072 (0.152) 0.135 0.044 (0.125) 0.071 0.953 0.02 aINS L–vPMC R 0.050 (0.192) 0.060 −0.037 (0.219) 0.000 0.441 0.20 aINS R–vPMC L 0.077 (0.194) 0.034 0.054 (0.211) 0.116 0.374 0.23 aWilcoxon Signed Rank test, p = 0.05. r = Wilcoxon Signed Rank test Effect Size. TABLE 9 Functional connectivity at first and second assessment, participants with AOS (n = 9). node taking over synapses from its left homotopic hemisphere counterpart. Intra–and interhemispheric IFG functional connectivity and AOS recovery Nodes in the entire right network that are connected to this one node may then be activated and involved node taking over synapses from its left homotopic hemisphere counterpart. Nodes in the entire right network that are connected to this one node may then be activated and involved reengagement of homologous regions (26). Skipper-Kallal et al. (64) proposed that the increased activity of the RH may stem from a cascading effect from one right hemisphere reengagement of homologous regions (26). Skipper-Kallal et al. (64) proposed that the increased activity of the RH may stem from a cascading effect from one right hemisphere Frontiers in Neurology 1 Frontiers in Neurology 12 frontiersin.org frontiersin.org 10.3389/fneur.2022.1013652 Hybbinette et al. FIGURE 4 Correlation between ASRS recovery and FC between left and right IFG at A1 (rho = 0.92, p = 0.001/q = <0.003) in nine participants with AOS (with participant ID shown). that investigated mainly chronic AOS patients, we found that AOS severity also was not strongly associated to the bilateral vPMC connectivity strength at the early stage (rho = −0.49, p = 0.177). At the 6 months follow-up, however, a comparable pattern of strengthened connection between bilateral vPMC connectivity and AOS severity was found that almost reached an FDR corrected significance level (rho = −0.79, p = 0.012). At this time point, a significant correlation between bilateral aINS connectivity strength and AOS severity was also found (rho = −0.88, p = 0.002). In addition, a strengthened association was noticed between ASRS total score and FC strength between left IFG and right aINS (rho = −0.80, p = 0.009). These results accord with the current view of the importance of premotor regions for speech motor programming and the severity level of AOS, and further suggest that also the bilateral aINS connectivity may have an impact on AOS impairment level. We found no significant correlations regarding FC between any of the selected ROIs in relation to behavioral results in the language domain or for NVOA. Given the recognized association between areas in IFG and expressive language, it could have been expected that the aphasia recovery would be closely connected to intra- and/or interhemispheric IFG connectivity strength and to the increased activation of the right IFG. Intra–and interhemispheric IFG functional connectivity and AOS recovery However, the FC strength between the selected ROIs in our study displayed a closer connection to the measurements of AOS than those from the broad aphasia test (A-ning) and the naming test (BNT) (Table 10). As one possible explanatory factor, our selected ROI in the IFG area was in the posterior part, corresponding to the pars opercularis (Brodmann area (BA) 44) in Broca’s area. This area has been proposed to play an important role in motor speech programming (66) whereas the pars triangularis, BA 45, in the anterior part has been suggested to play a greater role at a linguistic level of speech production (15, 67). As another important factor, our results were based on a small study sample (further adressed in the limitation section) and needs to be confirmed in larger samples. in compensation, resulting in broad patterns of increased activity. In accordance with this suggestion, participants with AOS in our study displayed a significantly stronger right sided intrahemispheric connectivity between the right aINS and right vPMC in comparison to LH lesioned without a speech-language impairment (Figure 2). New et al. (36) observed the same pattern in a comparison with healthy controls and suggested that reallocating functions from the lesioned hemisphere to homologous areas in the RH could be used in compensation for the interrupted LH networks. They also speculated that for some patients with AOS, an upregulation of the right vPMC region may not be sufficient. To compensate, an increased activation of the right aINS may also occur (36). Ramage et al. (65) used the same data set as New et al. to study a network of brain regions considered as key regions for semantic and phonological processing in post stroke aphasia. Their results indicated that ipsilateral connections between temporal and frontal regions in both the left and the right hemisphere could predict access to semantic and phonological representations that serve as a basis for speech production. The findings from our study are in line with these interpretations and suggest that functional connectivity changes in both intra- and interhemispheric IFG connections can be involved in early AOS recovery. Functional connectivity in relation to severity of AOS, aphasia and NVOA As a comparison group in this study, a small sample of LH stroke patient without a speech-language impairment recruited from the ProHand study cohort was used. This option allowed us to further investigate if the FC pattern observed by New et al. (36) in healthy subjects also is visible in early LH stroke patients without AOS and aphasia. The comparison group had significantly smaller and mainly subcortical lesions, Despite the strong correlation between recovery of AOS and early interhemispheric IFG connectivity, the IFG connectivity strength did not correlate significantly with AOS severity early or late post-stroke. Unlike in the study by New et al. (36) Frontiers in Neurology 13 frontiersin.org Hybbinette et al. 10.3389/fneur.2022.1013652 TABLE 10 Correlation between recovery in behavioral measurements and functional connectivity between ROIs, results presented in descending order (based on the ASRS result). TABLE 10 Correlation between recovery in behavioral measurements and functional connectivity between ROIs, results presented in descending order (based on the ASRS result). FC at Assessment 1 ASRS recovery A-ning recovery BNT recovery NVOA recovery IFG L–IFG R 0.92* 0.56 0.26 0.55 IFG L–aINS L 0.81 0.45 0.29 0.54 IFG L–vPMC R 0.66 0.25 0.02 0.17 IFG L–aINS R 0.55 0.45 0.16 0.47 vPMC L–vPMC R 0.53 0.18 −0.26 0.18 aINS R–vPMC L 0.43 0.01 0.39 −0.05 aINS L–aINS R 0.36 0.08 0.23 0.35 IFG R–vPMC L 0.21 −0.18 0.04 −0.35 aINS L–vPMC R 0.20 −0.28 0.18 −0.08 aINS R–vPMC R 0.02 −0.15 0.24 0.13 IFG R–aINS L −0.04 −0.10 0.24 0.22 IFG R–vPMC R −0.21 −0.21 0.21 0.10 aINS L–vPMC L −0.24 −0.32 0.21 −0.22 IFG L–vPMC L −0.40 −0.38 −0.11 −0.42 IFG R–aINS R −0.46 −0.25 −0.26 0.07 Spearman’s rho, *significant at FDR level q < 0.003. cortical nodes at study. However, the vPMC was the region with the lowest percentage lesion overlap among our selected ROIs. The significantly reduced FC between bilateral vPMC can therefore not be interpreted as merely a direct consequence of a lesion overlap. As also can be seen in Table 4, seven out of the nine included in the AOS group had over 25% damage to the left IFG, and two of them (ID 6 and 9 in Figure 4) had an almost total overlap to all three selected ROIs. Functional connectivity in relation to severity of AOS, aphasia and NVOA However, Figure 4 shows that the relation between AOS recovery and interhemispheric IFG FC was maintained even in the patients with more than 25% of lesion-overlap to the left IFG (i.e., the relation was visible even in patients with the lowest FC values). To be added, we cannot rule out that other cortical and subcortical lesion profiles could affect the connectivity in speech and networks and the selected ROIs. The limited number of subjects included in this study prevented further testing of lesion-behavior relationships, as for example by voxel-based lesion symptom mapping analysis (VLSM). not affecting any of the selected cortical ROIs. In accordance with the previous findings, the AOS participants in our study had a significantly reduced interhemispheric homotopic vPMC seed connection in comparison with LH lesioned without a speech-language impairment. This reduced connectivity stayed over time and was only slightly strengthened at the 6 months follow-up. Considering that premotor areas, both in BA6 (corresponding to our ROI in vPMC) and in the posterior part of the IFG (corresponding to our IFG ROI) have been suggested to be involved in the hand motor movement (68, 69) signs of a disturbed network connectivity between the selected ROIs might have been present also in the No SLI group. However, in our No SLI group, we found a FC pattern similar to that observed by New et al. (36) in healthy individuals. Spearman’s rho, *significant at FDR level q < 0.003. Methodological considerations and study limitations four to five sessions a week during inpatient care and two to three times a week after discharge provided by the regional rehabilitation team. The exact dose, frequency and focus of the therapy sessions were however not possible to control for. In addition, the therapeutic effects and brain plasticity mechanisms in relation to improved speech-language functions and changes in FC cannot be disentangled. Finally, we only included three bilateral cortical seed regions in our analyses out of a much larger network related to speech production. A whole- brain analysis of FC might have revealed other key functional connectivity patterns important for AOS recovery. Investigation of subcortical areas and disruption of structural connectivity in white matter bundles should also be of highest interest. Future studies should consider adding other regions to the analyses that are regarded as key nodes in the speech production network. pattern emerged with the bilateral IFG at the subacute phase being the strongest predictor of the ASRS result at A2 (rho = −0.803, p = 0.009). All these results are presented in the Supplementary Table S2. Since all participants with AOS in this study also had aphasia and NVOA at the early assessment, it was not possible to make a direct group comparison to patients with isolated aphasia. In clinical settings, this co-occurrence is very common. According to Duffy (3), patients with Broca’s or non-fluent aphasia often also have AOS. We therefore chose to compare and report how clinical results in the different domains, both severity/score results and recovery of AOS, aphasia and NVOA, related to the FC results. Considering earlier observed limitations in assessing individuals with severe AOS with the ASRS (48), the choice to use it in this study may seem irrational. However, because of the increasing support for the ASRS, becoming a standard instrument in the AOS literature (45), the ASRS was still our choice. To adjust the ratings in line with the severity level in the study sample, a number of modifications presented in Table 1 were applied. High attention was paid to the influence of comorbidity and risk for perceptual overlap for every item rating, and all ratings with the ASRS were carried out in consensus by at least two raters. An additional point concerns the use of the ASRS 2.0, and not the current version ASRS 3.0. Methodological considerations and study limitations The ASRS 2.0 consists of the same 13 items as the ASRS 3.0 and uses the same 5-point grading scale with operationalized descriptors for each rating level. The only difference between the two versions is that ASRS 2.0 is organized in three different sections according to whether the features are considered as (a) primary distinguishing features (rare overlap with dysarthria or aphasia), (b) distinguishing features unless dysarthria present and (c) distinguishing features unless aphasia and/or dysarthria are present, while the items in ASRS 3.0 are organized in three sections according to whether they are (a) articulatory features, (b) prosodic features or (c) other features. In the present study, the ASRS 2.0 total score was used as an index of AOS severity. We thereby believe that the same result would have been found by use of the ASRS 3.0 and that the obtained result is a valid measure of the severity of AOS in this study cohort. Ethics statement Several limitations to this study need to be acknowledged. This report is based on the longitudinal follow-up of nine stroke patients. This is a rather small participant sample which inevitably limits the statistical strength in the performed analyses. Our results should thus be replicated in larger samples. However, the use of the same ROIs as New et al. (36) made it possible for us to add new data to previously published results and allowed a direct comparison with their findings. Hopefully, the results and tendencies observed in this small study sample may also be of value for future studies. Secondly, the cohort in this study was relatively young compared to the overall stroke population. There was also an imbalance in gender, with more men than women included. Thirdly, all participants with AOS received speech-language therapy with The studies involving human participants were reviewed and approved by the Regional Ethical Review Board in Stockholm. The patients/participants provided their written informed consent to participate in this study. Conclusions To our knowledge, this study provides the first longitudinal FC data in AOS, spanning from the early to the chronic phase post stroke. Despite the limited sample size, the results clearly indicated that early interhemispheric IFG connectivity may be a strong predictor of AOS recovery, thus adding to our knowledge about the neural mechanisms underlying AOS and to the recovery process. The results confirmed earlier findings regarding the role of the ventral premotor region in speech motor programming and severity level of AOS, and suggest that the involvement of the bilateral inferior frontal gyrus and an increased activation of homologous areas in the right hemisphere speech production network may contribute to the recovery of AOS. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Methodological considerations and study limitations Recovery in this study was primarily investigated focusing on the amount of change, defined as the percentage that a participant improves over time on a test in relation to the possible maximum improvement on that specific measure. This method has been applied in several studies as an alternative to investigate and describe the inter-individual variability in stroke recovery, as for example by Lazar et al. (72) and Marchi et al. (73). However, it has been recognized that the sensitivity to change may differ among different behavioral assessment instruments. To further investigate accuracy and predictive value of the recovery analysis and the role of bilateral IFG in AOS recovery, we therefore compared the behavioral test results at A2 with the initial FC at A1. Also in this analysis, the same The application of rs-fMRI in stroke research has increased during the past decade. Stroke lesions do however present some challenges for the FC analyses. There is no total consensus on how the BOLD signal should be preprocessed and how to analyze data from stroke patients with large lesions affecting selected ROIs (70, 71). For transparency, we therefore chose to report all lesion overlap to our selected ROIs. As can be seen in Table 4, most participants with AOS in the present study had over 25% lesion overlap to any of the selected ROIs, while the comparison group without speech-language impairment mainly had isolated subcortical lesions with no engagement of the Frontiers in Neurology 14 frontiersin.org Hybbinette et al. 10.3389/fneur.2022.1013652 10.3389/fneur.2022.1013652 four to five sessions a week during inpatient care and two to three times a week after discharge provided by the regional rehabilitation team. The exact dose, frequency and focus of the therapy sessions were however not possible to control for. In addition, the therapeutic effects and brain plasticity mechanisms in relation to improved speech-language functions and changes in FC cannot be disentangled. Finally, we only included three bilateral cortical seed regions in our analyses out of a much larger network related to speech production. A whole- brain analysis of FC might have revealed other key functional connectivity patterns important for AOS recovery. Investigation of subcortical areas and disruption of structural connectivity in white matter bundles should also be of highest interest. Future studies should consider adding other regions to the analyses that are regarded as key nodes in the speech production network. Frontiers in Neurology Hybbinette et al. Supplementary material 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. 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Detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in China
Virology journal
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* Correspondence: zlshi@wh.iov.cn 1CAS Key Laboratory of Special Pathogens and Biosafety and Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Full list of author information is available at the end of the article Abstract Background: In recent years, novel hepadnaviruses, hepeviruses, hepatoviruses, and hepaciviruses have been discovered in various species of bat around the world, indicating that bats may act as natural reservoirs for these hepatitis viruses. In order to further assess the distribution of hepatitis viruses in bat populations in China, we tested the presence of these hepatitis viruses in our archived bat liver samples that originated from several bat species and various geographical regions in China. Methods: A total of 78 bat liver samples (involving two families, five genera, and 17 species of bat) were examined using nested or heminested reverse transcription PCR (RT-PCR) with degenerate primers. Full-length genomic sequences of two virus strains were sequenced followed by phylogenetic analyses. Results: Four samples were positive for hepadnavirus, only one was positive for hepevirus, and none of the samples were positive for hepatovirus or hepacivirus. The hepadnaviruses were discovered in the horseshoe bats, Rhinolophus sinicus and Rhinolophus affinis, and the hepevirus was found in the whiskered bat Myotis davidii. The full-length genomic sequences were determined for one of the two hepadnaviruses identified in R. sinicus (designated BtHBVRs3364) and the hepevirus (designated BtHEVMd2350). A sequence identity analysis indicated that BtHBVRs3364 had the highest degree of identity with a previously reported hepadnavirus from the roundleaf bat, Hipposideros pomona, from China, and BtHEVMd2350 had the highest degree of identity with a hepevirus found in the serotine bat, Eptesicus serotinus, from Germany, but it exhibited high levels of divergence at both the nucleotide and the amino acid levels. Conclusions: This is the first study to report that the Chinese horseshoe bat and the Chinese whiskered bat have been found to carry novel hepadnaviruses and a novel hepevirus, respectively. The discovery of BtHBVRs3364 further supports the significance of host switches evolution while opposing the co-evolutionary theory associated with hepadnaviruses. According to the latest criterion of the International Committee on Taxonomy of Viruses (ICTV), we hypothesize that BtHEVMd2350 represents an independent genotype within the species Orthohepevirus D of the family Hepeviridae. Keywords: Bat, Natural reservoir, Hepadnavirus, Hepevirus, Genome characterization © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in China Bo Wang1,4, Xing-Lou Yang1, Wen Li1, Yan Zhu1, Xing-Yi Ge1, Li-Biao Zhang2, Yun-Zhi Zhang3, Claus-Thomas Bock4 and Zheng-Li Shi1* Wang et al. Virology Journal (2017) 14:40 DOI 10.1186/s12985-017-0706-8 Wang et al. Virology Journal (2017) 14:40 DOI 10.1186/s12985-017-0706-8 Open Access Open Access Genomic sequencing The complete genomic sequences of one hepadnavirus strain and one hepevirus strain were amplified using PCR with degenerate primers (the primers are available upon request). The genome ends were amplified using a 5′-Full RACE Kit (TaKaRa, Japan). The PCR products underwent gel purification with MinElute Gel Extraction Kit (Qiagen, Germany) and they were sequenced with both forward and reverse primers using the 3100 Se- quencer. The sequencing chromatograms were inspected for overlapping multicolor peaks, which are an indicator of sequence heterogeneity in the amplicons. The PCR products were cloned using the pGEM-T Easy Vector System (Promega, Germany) and at least three clones for each PCR fragment were sequenced to obtain a con- sensus sequence. There are around 120 species of bat in China; however, only limited information has been reported regarding the hepatitis viruses, a novel Orthohepadnavirus in pomona roundleaf bats from Yunnan province was identified in 2015 [9]. In this study, we report the discovery of four novel hepadnaviruses and a hepevirus in our archived bat liver samples that had been collected from several bat spe- cies and various geographical regions in China. Abstract 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. Wang et al. Virology Journal (2017) 14:40 Page 2 of 10 RNA extraction and PCR RNA was extracted from tissue using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions, and cDNA was synthesized using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Promega, Madison, WI, USA). The ex- tracted RNA from liver was tested by nested or heminested reverse transcription PCR (RT-PCR) using degenerate primers based on the conserved domain of the RNA- dependent RNA polymerase (RdRp) gene of viruses in the genus Hepatovirus, the polymerase gene of viruses in the family Hepadnaviridae, the RdRp gene of viruses in the genus Hepacivirus [11], and the RdRp gene of viruses in the family Hepeviridae [7] (Table 3). Standard precautions were taken to avoid contamination of the PCR procedure, and no false-positives were observed in the negative con- trols. The PCR products underwent gel purification with MinElute Gel Extraction Kit (Qiagen, Germany) and they were sequenced with both forward and reverse primers using the 3100 Sequencer (ABI, Waltham, MA, USA). Previous studies mainly focused on bat-borne viruses that are transmitted via respiratory droplets [3]. However, in recent years, several hepatitis virus-related sequences, including those associated with hepadnaviruses, hepe- viruses, hepatoviruses, and hepaciviruses, have been found in bats across the globe, indicating the importance of bats as the natural reservoirs of these viruses [5–9]. Hepatitis viruses include hepatitis viruses A, B, C, D, and E, which cause human hepatitis diseases. Hepatitis A virus (HAV) is classified as belonging to the genus Hepatovirus in the family Picornaviridae. Hepatitis B virus (HBV) is classified as belonging to the genus Orthohepadnavirus in the family Hepadnaviridae. Hepatitis C virus (HCV) is classified as belonging to the genus Hepacivirus in the fam- ily Flaviriridae. Hepatitis D virus (HDV) is considered to be a subviral satellite because it can only propagate in the presence of HBV. Hepatitis E virus (HEV) is classified as belonging to the genus Orthohepevirus in the family Hepe- viridae. Hepatovirus-related sequences have been identified in 13 species of bat collected in North America, Europe, and Africa [5]. Hepadnavirus-related sequences have been discovered in five species of bat collected in Panama, Gabon, Myanmar, and China [6, 8–10]. Highly diverse hepacivirus-related sequences have been detected in 20 species of bat across the world [11]. Hepevirus-related se- quences have been discovered in bats in Ghana, Panama, and Germany [7]. These results indicate that bats may be important reservoirs of these hepatitis viruses (Table 1). Sequence analysis The preliminary sequence management and analysis were carried out using Geneious version 9.1.3 (Biomatters Ltd., Auckland, New Zealand) and the sequence alignment and editing were performed using MAFFT [13]. The phylogen- etic analysis of hepadnavirus used the neighbor-joining Background comprised two families, five genera, and 17 species, and used for virus screening (Table 2). Different tissues (heart, liver, spleen, lung, kidney, brain and intestine) were collected separately and used for analyzing virus tissue tropism. The animals were firstly identified based on their morphology and then the species that they belonged to were further confirmed using DNA sequencing of the mitochondrial cytochrome b (CytB) gene following pre- viously described methods [12]. Nearly 60% of emerging infectious diseases in humans are zoonotic, with up to 70% of them being found to originate from wildlife [1]. Bats have been identified as natural reser- voirs of many viruses. Some of these viruses cause out- breaks of severe disease in humans [2], including the Ebola virus, the lyssavirus, the severe acute respiratory syndrome coronavirus, and henipaviruses [3]. Interestingly, these vi- ruses rarely cause apparent clinical signs in bats [2]. Bats possess unique characteristics that may contribute to their ability to act as a major natural reservoir for viruses, includ- ing a high level of species diversity, a long lifespan, a high population density, and high levels of spatial mobility [4]. Samples A total of 78 liver tissue samples were collected from dead bats caused by accident during sampling, which Wang et al. Virology Journal (2017) 14:40 Page 3 of 10 Table 1 Hepatitis virus-like sequences detected in bats Virus family Order: Chiroptera Sampling site (s) (year) Isolation source Genomic sequence Reference Family Genera Species Hepatoviridae Emballonuridae Coleura C. afra Ghana (2011), Gabon (2009) Feces, blood Full [5] Pteropodidae Eidolon E. helvum Ghana (2010/2011) Blood Full Natalidae Natalus N. lanatus Costa Rica (2010) Feces Full Rhinolophidae Rhinolophus R. landeri Ghana (2011) Feces Full R. ferrumequinum Romania (2008/2009), Bulgaria (2008/2009), Luxemburg (2011) Feces Full Rhinolophus R. hipposideros Bulgaria (2008/2009), Spain (2010) Feces Full Vespertilionidae Glauconycteris G. spec. Côte d’Ivoire (2013) Intestines Full Miniopterus M. cf. manavi Madagascar (2014) Liver Full M. schreibersii Romania (2008) Feces Full Myotis M. dasycneme Germany (2008) Feces Full M. myotis Romania (2008), Germany (2008/2010) Feces Full Nyctalus N. noctula Romania (2008/2009), Germany (2009) Feces Full Pipistrellus P. kuhlii Ukraine (2010/2011) Feces Full Hepadnaviridae Vespertilionidae Miniopterus Miniopterus spp. Myanmar (2010) Liver Full [8] Hipposideridae Hipposideros H. pomona China (2011) Liver Full [9] H. cf. ruber Gabon (2009) Blood Full [6] Phyllostomidae Uroderma U. bilobatum Panama (2010/2011) Blood Full Rhinolophidae Rhinolophus R. alcyone Gabon (2009) Blood Full R. sinicus China (2012) Liver Full This study R. affinis China (2013) Liver Partial Hepaciviridae Emballonuridae Taphozous Not identified Cameroon (2010) Blood Partial [11] Hipposideridae Hipposideros H. vittatus Kenya (2010) Blood Full H. gigas Nigeria (2008/2010) Blood Partial Molossidae Chaerephon Not identified Cameroon (2010), Kenya (2010) Blood Partial Mops M. condylurus DRC (2011) Blood Full Otomops O. martiensseni Kenya (2010) Blood Full Nyctinomops N. macrotis Mexico (2011) Blood Partial Phyllostomidae Artibeus A. watsoni Mexico (2011) Blood Partial Carollia C. perspicillata Guatemala (2010), Mexico (2011) Blood Full Desmodus D. rotundus Guatemala (2010), Blood Partial Glossophaga G. commissarisi Mexico (2011) Blood Partial Sturnira S. lilium Guatemala (2010) Blood Full S. ludovici Mexico (2011) Blood Partial Trachops T. cirrhosus Mexico (2011) Blood Partial Pteropodidae Eidolon E. helvum Cameroon (2010), DRC (2011) Blood Full Epomophorus E. labiatus Kenya (2010) Blood Partial Megaloglossus M. woermanni DRC (2011) Blood Full Rousettus R. aegyptiacus Kenya (2010), Nigeria (2008/2010) Blood Full Vespertilionidae Scotoecus Not identified Kenya (2010) Blood Partial Scotophilus S. dingani Kenya (2010) Blood Full S. Samples nigrita Nigeria (2008/2010) Blood Partial Unknown Nigeria (2008/2010), Mexico (2010/2011) Blood Partial Table 1 Hepatitis virus-like sequences detected in bats Page 4 of 10 Wang et al. Virology Journal (2017) 14:40 Table 1 Hepatitis virus-like sequences detected in bats (Continued) Hepeviridae Hipposideridae Hipposideros H. abae Ghana (2009) Feces Partial [7] Phyllostomidae Vampyrodes V. caraccioli Panama (2011) Blood Partial Vespertilionidae Eptesicus E. serotinus Germany (2008) Liver Full Myotis Myotis M. bechsteinii Germany (2008) Feces Partial M. daubentonii Germany (2008) Feces Partial M. davidii China (2011) Liver Full This study DRC Democratic Republic of the Congo Table 1 Hepatitis virus-like sequences detected in bats (Continued) DRC Democratic Republic of the Congo for 10 s, and 40 cycles of 95 °C 5 s and 60 °C 20 s with measurement of fluorescence. (NJ) method with Hasegawa-Kishino-Yano substitution model and complete deletion option and hepevirus used the maximum-likelihood (ML) method with the nucleotide percentage distance substitution matrix and the complete deletion option in MEGA version 7 [14]. The sequences and GenBank accession numbers of the representative vi- ruses in the families Hepadnaviridae and Hepeviridae used in the phylogenetic analyses are presented in Figs. 1 and 2. Detection of four hepadnaviruses and a hepevirus in bat liver samples Among the 78 bat liver samples, four were positive for hepadnavirus from Jinning city, Yunnan province and only one was positive for hepevirus from Xianning city, Hubei province (Fig. 3). However, none were positive for hepatovirus or hepacivirus. The nucleotide sequences of the four novel hepadnaviruses and the hepevirus de- scribed in this study are available from GenBank under the accession numbers KX513949–KX513953. Quantification real-time PCR Virus load of bat hepevirus and hepadnaviruese of dif- ferent tissues was measured by using photometrically quantified in vitro RNA transcripts and specific real- time RT-PCR primers (Table 3). Quantification was done by using 5 μL of RNA extract, 300 nM each primer, using the One Step SYBR PrimeScript™PLUS RT-PCR Kit (TaKaRa, Japan). Cycling in a Biorad CFX Connect instru- ment involved the following steps: 42 °C for 5 min, 95 °C Sequence analysis of the bat hepadnavirus All four of the hepadnavirus-positive samples were from horseshoe bats, two each from R. sinicus (designated Table 2 Detection of hepadnavirus and hepevirus in bats in China between 2008 and 2013 Family Genus Species No. of samples No. of hepadnavirus positive samples No. of hepevirus positive samples Sampling site (s) (year) Vespertilionidae Myotis M. adversus 4 Yunnan (2008) M. pilosus 7 Yunnan (2008) M. chinensis 1 Hubei (2008) M. davidii 12 1 Hubei (2011), Yunnan (2011) M. ikonnikovi 3 Yunan (2009), Hubei (2011) M. formosus 1 Yunnan (2011) Miniopterus M. fuliginosus 1 Henan (2010) Aselliscus A. stoliczkanus 1 Yunnan (2012) Rhinolophidae Rhinolophus R. sinicus 19 2 Hubei (2008/2011), Sichuan (2011), Yunnan (2009/2012/2013) R. monoceros 3 Hubei (2011), Chongqing (2011) R. affinis 7 2 Henan (2010), Hubei (2011) Yunnan (2012/2013), R. pusillus 4 Yunnan (2012/2013) R. pearsonii 1 Chongqing (2011) Hipposideros H. pratti 2 Hubei (2010) H. armiger 4 Sichuan (2011) H. pomona 7 Yunnan (2013) H. larvatus 1 Yunnan (2011) Total 5 genera 17 species 78 4 1 ble 2 Detection of hepadnavirus and hepevirus in bats in China between 2008 and 2013 Wang et al. Virology Journal (2017) 14:40 Page 5 of 10 Table 3 Primers used for virus RT-PCR screening and virus quantification Primer Sequence (5′-3′)a Polarity Targeted virus Reference HAV-3D-F1 CYTATHTRAARGATGAGCTKAGA + Hepatovirus This study HAV-3D-F2 ACRTCATCICCRTARCAIAGRA + HAV-3D-R1 RTCIAARACWAGRGCNATYG - HAV-3D-R2 TACCWAATCATRAATGGACT - HBV-pol-F1 TAGACTSGTGGTGGACTTCTC + Hepadnavirus This study HBV-pol-F2 AGTRAAYTGAGCCAGGAGAAAC + HBV-pol-R1 TGCCATCTTCTTGTTGGTTC - HBV-pol-R1 CATATAASTRAAAGCCAYACAG - BHV-1-F1 GTAGCGGAGAAGATGTATCTGGG + Hepacivirus [11] BHV-1-R1 GCCTTAGCCTTGAGAAAGCAGGTGAT + BHV-1-F2 GAGAAGATGTATCTGGGGGACGT + BHV-1-R2 AGAAAGCAGGTGATGGTATTGCC + BHV-2-F1 CCAAARGTWGTBAAGGCTGTGCT - BHV-2-R1 ACTTTGAKCCASGCAGTKARACAGTT - BHV-2-F2 GCTGTGCTSAAGGAMGAGTACGGCT + BHV-2-R2 CCASGCAGTKARACAGTTACTRGAG - DE-F4228 ACYTTYTGTGCYYTITTTGGTCCITGGTT + Hepevirus [7] DE-R4598 GCCATGTTCCAGAYGGTGTTCCA - DE-R4565 CCGGGTTCRCCIGAGTGTTTCTTCCA - BtHEV-qF ATGTCCGTGTTCAGGTTCC + Bat hepevirus This study BtHEV-qR GCCAACCCTCATTTGCAAC - BtHBV-qF TGTTGGTTCTCCTGGATTGGAG + Bat hepadnaviruses This study BtHBV-qR TGAAGGAATGGGCCAGCAGGTG - R: G/A; Y: C/T; S: G/C; W: A/T; M: A/C; K: G/T; H: A/C/T; N: A/T/C/G; I: inosine R: G/A; Y: C/T; S: G/C; W: A/T; M: A/C; K: G/T; H: A/C/T; N: A/T/C/G; I: inosine amino acid levels [9]. Notably, we found large differ- ences between BtHBVRs3364 and other hepadnaviruses from the African horseshoe and roundleaf bats, the long-fingered bat from Myanmar, and the tent-making bat from Panama. BtHBVRs3364 and BtHBVRs3366) and R. affinis (desig- nated BtHBVRa4325 and BtHBVRa4328) (Table 2). Phylogenetic analysis of the bat hepadnavirus A phylogenetic tree was constructed based on the align- ment of the full-length genomic sequence of BtHBVRs3364 with those of representative hepadnavirus strains available in GenBank. As shown in Fig. 1, the previously reported bat hepadnaviruses formed three clusters, with clear specificities for particular hosts. Although BtHBVRs3364 clustered with the bat hepadnaviruses, it formed an inde- pendent branch. Interestingly, the BtHBVRs3364 detected in the horseshoe bat is phylogenetically closer to viruses from the Asian roundleaf bat compared to viruses from the African horseshoe bat, despite the fact that it was found in an Asian horseshoe bat. The full-length genomic sequence of a sample from R. sinicus (designated bat HBV Rs3364, or BtHBVRs3364) was determined and it was found to have a length of 3,272 nucleotides. The virus has an identical genomic organization to other hepadnaviruses, with four open reading frames (ORFs) encoding the surface (S), polymer- ase (P), core (C), and X proteins. In addition, the typical direct repeat (DR) sequences for viral genome replication and the secondary structure Ɛ–loops for viral reverse transcription were present in the BtHBVRs3364 genome. A detailed comparison of the full-length genomic se- quence and the ORFs of the virus with other known hepadnaviruses is shown in Table 4. The results showed that the four genes of BtHBVRs3364 have the highest degree of identity with the roundleaf bat hepadnavirus from Yunnan province, at both the nucleotide and the Sequence analysis of the bat hepadnavirus The four partial polymerase gene sequences had 92.1–97.5% nucleotide sequence identity and they were found to be closely related to the roundleaf bat hepadnavirus from Yunnan province, China, with nucleotide identities of 88.8–95.5% [9]. Phylogenetic analysis of the bat hepevirus A phylogenetic tree was constructed based on the alignment of the full-length genomic sequence of BtHEVMd2350 with those of representative full-length hepevirus genomic sequences (Fig. 2). The results showed that bat hepeviruses (BtHEVMd2350 and BS7) cluster into a separate monophyletic clade within the family Hepeviridae. Sequence analysis of the bat hepevirus One sample found in the whiskered bat, M. davidii, from Hubei province was positive for hepevirus (designated bat Page 6 of 10 Wang et al. Virology Journal (2017) 14:40 Fig. 1 Phylogenetic analysis of bat hepadnavirus based on the full-length genomic sequences. Maximum likelihood phylogenetic tree was constructed based on the complete genomes of BtHBVRs3364 (in bold) and representative members of the family Hepadnaviridae using the Hasegawa-Kishino-Yano substitution model and complete deletion option in MEGA version 7. The values at the nodes indicate the bootstrap values (using 1,000 replications). The branches are labeled with the strain designation, the host species, and the GenBank accession number. The classification of the family Hepadnaviridae is indicated on the right Fig. 1 Phylogenetic analysis of bat hepadnavirus based on the full-length genomic sequences. Maximum likelihood phylogenetic tree was constructed based on the complete genomes of BtHBVRs3364 (in bold) and representative members of the family Hepadnaviridae using the Hasegawa-Kishino-Yano substitution model and complete deletion option in MEGA version 7. The values at the nodes indicate the bootstrap values (using 1,000 replications). The branches are labeled with the strain designation, the host species, and the GenBank accession number. The classification of the family Hepadnaviridae is indicated on the right HEV Md2350, or BtHEVMd2350). The genomic se- quence of BtHEVMd2350 was found to have a length of 6,607 nucleotides (excluding the poly(A) tail at the 3′ end). This is slightly shorter than BS7 (which has a genomic sequence length of 6,671 nucleotides), the only reported bat hepevirus with a fully sequenced genome, which was identified from the serotine bat, Eptesicus serotinus, in Germany [7]. BtHEVMd2350 was found to have a 5′ untranslated region (UTR) of 33 nucleotides and a 3′ UTR of 76 nucleotides. The three unique ORFs that are found in other members of the family Hepeviridae were also found in BtHEVMd2350: ORF1 encodes a nonstructural polyprotein that includes the RdRp, ORF2 encodes the capsid protein, and ORF3 encodes a multifunc- tional protein. Notably, most of the elements and do- mains characterized in BS7 could be found in BtHEVMd2350, but with a high level of divergence (Table 5). Quantification of novel viruses Viral load detected by qPCR in different tissues were presented in the Fig. 4. The highest viral load of the BtHEVMd2350 was found in the liver (1.9 × 1010 RNA copies per gram of tissue) and followed by spleen (7.3 × 108 RNA copies per gram of tissue), intestine and kidney, but not detectable in the brain. For bat hepadnavirus, the highest viral load was found in the liver of BtHBVRs3364 (2.0 × 1010 RNA copies per gram of tissue), the virus load of tissues of BtHBVRs3366, BtHBVRa4325, and Page 7 of 10 Wang et al. Virology Journal (2017) 14:40 Fig. 2 Phylogenetic analysis of bat hepevirus based on the full-length genomic sequences. Neighbor joining phylogenetic tree was constructed based on the alignment of the complete genomes of BtHEVMd2350 (in bold) and representative members of the family Hepeviridae using nucleotide percentage distance substitution matrix and complete deletion option implemented in MEGA version 7. The values at nodes indicate the bootstrap values (using 1,000 replications). The branches are labeled with the strain designation, the host species, and the GenBank accession number. The classification of the family Hepeviridae is indicated on the right Fig. 2 Phylogenetic analysis of bat hepevirus based on the full-length genomic sequences. Neighbor joining phylogenetic tree was constructed based on the alignment of the complete genomes of BtHEVMd2350 (in bold) and representative members of the family Hepeviridae using nucleotide percentage distance substitution matrix and complete deletion option implemented in MEGA version 7. The values at nodes indicate the bootstrap values (using 1,000 replications). The branches are labeled with the strain designation, the host species, and the GenBank accession number. The classification of the family Hepeviridae is indicated on the right Fig. 2 Phylogenetic analysis of bat hepevirus based on the full-length genomic sequences. Neighbor joining phylogenetic tree was constructed based on the alignment of the complete genomes of BtHEVMd2350 (in bold) and representative members of the family Hepeviridae using nucleotide percentage distance substitution matrix and complete deletion option implemented in MEGA version 7. The values at nodes indicate the bootstrap values (using 1,000 replications). The branches are labeled with the strain designation, the host species, and the GenBank accession number. Quantification of novel viruses The classification of the family Hepeviridae is indicated on the right BtHBVRa4328 were relatively similar (medien, 6.2 × 106 RNA copies per gram of tissue; range, 4.9 × 105 to 2.7 × 1010 RNA copies per gram of tissue). genomic sequences of one of the two hepadnaviruses from R. sinicus and the hepevirus from M. davidii were determined. The phylogenetic analysis indicates that the bat hepadnavirus found in this study is closely related to roundleaf bat hepadnaviruses, which were discovered in Pu’er city, Yunnan province in 2011 [9], but shows remarkable divergence when compared to the African horseshoe bat, despite the fact that it was found in an Asian horseshoe bat. A similar phylogenetic rela- tionship was found between hepadnaviruses from the African roundleaf bat and the African horseshoe bat [6], indicating the separate evolution of these viruses and their hosts. Conclusions and discussion Since the discovery of genetically diverse hepatitis virus-related sequences in bats, bats have been con- sidered to be important natural reservoirs for hepa- titis viruses, and potential sources of human diseases [10]. However, these hypotheses need to be proved by screening more bat samples from across the globe for hepatitis viruses. In this study, we screened for hepa- titis viruses in bats from China and discovered four novel hepadnaviruses circulating in two species of horseshoe bat in Jinning city, Yunnan province and one hepevirus in the whiskered bat M. davidii in Xianning city, Hubei province. The full-length Regarding the bat hepevirus, the phylogenetic analysis indicates that the known bat hepeviruses are highly di- vergent from other mammalian hepeviruses and that they form an independent branch in the family Page 8 of 10 Wang et al. Virology Journal (2017) 14:40 Fig. 3 Representation map of China and Yunnan province. Circle indicates the sampling site where the hepevirus (BtHEVMd2350) was detected. Triangles indicates the sampling sites where the hepadnaviruses were detected Fig. 3 Representation map of China and Yunnan province. Circle indicates the sampling site where the hepevirus (BtHEVMd2350) was detected. Triangles indicates the sampling sites where the hepadnaviruses were detected Hepeviridae. According to the latest proposal of the ICTV in 2016, amino acid distances of concatenated ORF1 and ORF2 (lacking hypervariable regions) greater than 0.088 could then act as threshold to demarcate intra- and inter- genotype distances [15]. The hepe- virus detected in the whiskered bat, M. davidii, and that found in the German serotine bat, E. serotinus (the only reported bat hepevirus with a full-length genome) shared significant diversity from both nucleotide and amino acid levels, we propose that they can be grouped into the species Orthohepevirus D which is divided into two genotypes: D1 and D2. Table 4 Nucleotide and amino acid sequence identity between BtHBVRs3364 and representative orthohepadnavirus strainsa Hepadnavirus (no. Conclusions and discussion of strains compared) Degree of identity (%) Full genome S gene (1–672) X gene (1217–1645) C gene (1657–2301) P gene (2147–1475) Nucleotides Nucleotides Amino acids Nucleotides Amino acids Nucleotides Amino acids Nucleotides Amino acids Asian roundleaf bat hepadnavirus (3) 81.2–81.9 90.5–93.9 83.5–90.2 86.7 75.4 85.2–85.3 91.2–91.7 80.1–80.9 77.4–77.9 African horseshoe bat hepadnavirus (1) 72.2 89.0 84.8 78.8 65.5 75.2 77.9 71.8 65.9 African roundleaf bat hepadnavirus (4) 72.0–72.1 89.0–89.2 86.2 81.8–82.1 70.4–71.1 76.9–77.1 77.9 72.3–72.4 65.6–66.0 Long-fingered bat hepadnavirus (3) 69.0–69.2 79.7–80.1 67.4 69.2–70.2 49.3–51.4 74.2–74.5 75.6–76.0 68.1–68.3 61.5–61.8 Tent-making bat hepadnavirus (4) 56.7–56.8 73.5–74.2 61.0–61.9 58.9–59.3 43.4–44.1 51.6 49.5 56.8–57.1 49.1–49.2 Primate HBV (15) 59.5–61.1 76.5–77.8 65.0–66.8 62.1–64.9 46.5–52.1 61.0–67.9 63.0–66.5 57.0–59.7 50.2–52.0 Woolly monkey hepadnavirus (1) 61.5 75.3 67.1 63.2 50.0 64.2 61.4 60.3 51.9 Ground squirrel hepadnavirus (2) 64.0–65.1 78.7–78.8 67.9–68.3 60.6–63.9 45.1–46.5 71.1–71.4 70.9–74.0 58.9–59.1 49.3–50.2 Woodchuck hepadnavirus (1) 59.8 77.9 67.4 63.9 47.2 69.8 72.5 58.8 50.9 Duck hepadnavirus (1) 31.1 44.1 28.7 NA NA 31.3 20.9 35.1 23.2 aThe sequences were aligned using MAFFT. The evolutionary analyses were conducted using MEGA version 7. The GenBank accession numbers are as follows: KF939648, KF939649, and KF939650 for the Asian roundleaf bat hepadnaviruses; KC790377 for the African horseshoe bat hepadnavirus; KC790374, KC790375, KC790373, and KC790376 for the African roundleaf bat hepadnaviruses; KC790378, KC790379, KC790380, and KC790381 for the Tent-making bat hepadnaviruses; AF160501, AY090454, X69798, AF193863, U46935, AB486012, AF305327, AB117758, D23678, AM282986, AF241409, AB205192, FJ349213, AB126581, and JN040752 for the Primate HBVs; AF046996 for the woolly monkey hepadnavirus; NC 001484 and U29144 for the ground squirrel hepadnaviruses; NC_004107 for the woodchuck hepadnavirus; and EU429324 for the duck hepadnaviruses. NA not available Page 9 of 10 Wang et al. Virology Journal (2017) 14:40 Table 5 Nucleotide and amino acid sequence identity between BtHEVMd2350 and representative hepevirus strainsa Hepevirus (no. Conclusions and discussion of strains compared) Degree of identity (%) Full genome ORF1 (genome positions 56–4699) ORF2 (genome positions 4700–6607) ORF3 (genome positions 4779–5192) Nucleotides Nucleotides Amino acids Nucleotides Amino acids Nucleotides Amino acids Bat hepevirus (1) 67.8 65.5 72.0 70.9 79.2 73.2 44.3 Avian hepevirus (4) 50.9–51.7 48.2–49.0 44.3–44.5 46.0–47.3 44.4–44.9 19.9–21.1 5.3–6.1 HEV genotype 3 (22) 44.6–46.0 43.4–45.5 39.1–40.9 47.4–49.9 44.6–47.4 30.7–31.8 14.8–18.9 HEV genotype 4 (5) 45.2–46.0 44.9–45.9 40.4–41.0 47.5–48.2 45.8–47.1 30.0–31.6 14.6–16.3 HEV genotype 1 (3) 45.6–45.9 45.0–45.5 40.5–41.1 48.7–49.3 46.6–46.9 31.9–32.6 17.1 HEV genotype 2 (1) 45.6 45.3 40.8 49.7 47.1 31.6 17.1 Rodent hepevirus (3) 44.0–44.4 45.9–46.1 41.6–42.0 47.9–48.4 45.4–46.7 25.2–27.4 9.0 Ferret hepevirus (1) 44.0 46.4 42.6 48.3 46.0 25.5 8.0–12.8 Trout hepevirus (1) 33.7 34.9 24.5 30.2 15.5 21.1 9.8 aThe sequences were aligned using MAFFT. The evolutionary analyses were conducted using MEGA version 7. The GenBank accession numbers are as follows: JQ001749 for the bat hepevirus; AM943646, AM943647, KF511797, and AY535004 for the avian hepeviruses; AB189070, AB189075, AP003430, AB091394, AB222183, AB073912, AY115488, AF060669, AF082843, HQ389544, JN564006, AB290312, FJ998008, FJ705359, KC618402, AF455784, AB248521, EU360977, EU495148, FJ956757, FJ906895, and JQ013793 for the HEVs genotype 3; AB856243, AB602440, AB161717, AJ272108, and EU366959 for the HEVs genotype 4; AY230202, AF076239, and M80581 for the HEVs genotype 1; M74506 for HEV genotype 2; GU345042, AB847306, and JX120573 for the rodent hepeviruses; JN998607 for the ferret hepevirus; and NC_015521 for the trout hepevirus aThe sequences were aligned using MAFFT. The evolutionary analyses were conducted using MEGA version 7. The GenBank accession numbers are as follows: JQ001749 for the bat hepevirus; AM943646, AM943647, KF511797, and AY535004 for the avian hepeviruses; AB189070, AB189075, AP003430, AB091394, AB222183, AB073912, AY115488, AF060669, AF082843, HQ389544, JN564006, AB290312, FJ998008, FJ705359, KC618402, AF455784, AB248521, EU360977, EU495148, FJ956757, FJ906895, and JQ013793 for the HEVs genotype 3; AB856243, AB602440, AB161717, AJ272108, and EU366959 for the HEVs genotype 4; AY230202, AF076239, and M80581 for the HEVs genotype 1; M74506 for HEV genotype 2; GU345042, AB847306, and JX120573 for the rodent hepeviruses; JN998607 for the ferret hepevirus; and NC_015521 for the trout hepevirus Our results provide further evidence to support the theory regarding the long-term co-evolution of hepadna- viruses and hepeviruses with their hosts, and the theory that bats act as major natural reservoirs for these hepa- titis viruses. Abbreviations 6. Drexler JF, Geipel A, Konig A, Corman VM, van Riel D, Leijten LM, Bremer CM, Rasche A, Cottontail VM, Maganga GD, et al. Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes. Proc Natl Acad Sci U S A. 2013;110:16151–6. C: Core; HAV: Hepatitis A virus; HBV: Hepatitis B virus; HCV: Hepatitis C virus; HDV: Hepatitis D virus; HEV: Hepatitis E virus; ORFs: Open reading frames; P: Polymerase; RT-PCR: Reverse transcription polymerase chain reaction; S: Surface Rasche A, Cottontail VM, Maganga GD, et al. Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes. Proc Natl Acad Sci U S A. 2013;110:16151–6. infecting human hepatocytes. Proc Natl Acad Sci U S A. 2013;110:1615 7. Drexler JF, Seelen A, Corman VM, Tateno AF, Cottontail V, Zerbinati RM, Gloza-Rausch F, Klose SM, Adu-Sarkodie Y, Oppong SK, et al. Bats worldwide carry hepatitis E virus-related viruses that form a putative novel genus within the family Hepeviridae. J Virol. 2012;86:9134–47. Availability of data and materials Not applicable. 13. Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30:3059–66. Acknowledgements We appreciate Wei Zhang, Bei Li and Yu-Tao Gao (all Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China) for the excellent technical assistance. 8. He B, Fan Q, Yang F, Hu T, Qiu W, Feng Y, Li Z, Li Y, Zhang F, Guo H, et al. Hepatitis virus in long-fingered bats, Myanmar. Emerg Infect Dis. 2013;19:638–40. 9. He B, Zhang F, Xia L, Hu T, Chen G, Qiu W, Fan Q, Feng Y, Guo H, Tu C. Identification of a novel Orthohepadnavirus in pomona roundleaf bats in China. Arch Virol. 2015;160:335–7. Consent for publication Not applicable. Consent for publication Not applicable. Not applicable. Ethics approval and consent to participate The study protocol was reviewed and approved by the Ethics Committee of the Yunnan Institute of Endemic Disease Control and Prevention. All the animals were treated in strict accordance with the Guidelines for the Use and Care of Laboratory Animals from the Chinese CDC and the Rules for the Implementation of Laboratory Animal Medicine (1998) from the Ministry of Health, China. The protocols followed for the use of the animals were approved by the National Institute for Communicable Disease Control and Prevention, China. All surgery was performed under ether anesthesia, and all efforts were made to minimize suffering. Conclusions and discussion Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature. 2013;503:535–8. 13. Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30:3059–66. 14. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870–4. 15. Smith DB, Simmonds P, members of the International Committee on the Taxonomy of Viruses Hepeviridae Study G, Jameel S, Emerson SU, Harrison TJ, Meng XJ, Okamoto H, Van der Poel WH, Purdy MA. Consensus proposals for classification of the family Hepeviridae. J Gen Virol. 2015;96:1191–2. Authors’ contributions BW conducted the experiments and drafted the manuscript. X-LY, WL and YZ conducted molecular studies. BW, X-LY, X-YG, L-BZ, and Y-ZZ performed the sampling. L-ZS devised the study design and provided scientific oversight. The manuscript was revised by X-LY, C-TB, and L-ZS with input from all the contributing authors. All authors read and approved the final manuscript. 14. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870–4. 15. Smith DB, Simmonds P, members of the International Committee on the Taxonomy of Viruses Hepeviridae Study G, Jameel S, Emerson SU, Harrison TJ, Meng XJ, Okamoto H, Van der Poel WH, Purdy MA. Consensus proposals for classification of the family Hepeviridae. J Gen Virol. 2015;96:1191–2. Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: Funding Thi k This work was jointly funded by the National Natural Science Foundation of China (81290341), China Mega-Project for Infectious Disease (2014ZX10004001), the Scientific and Technological Basis Special Project (2013FY113500) and Funds for Environment Construction & Capacity Building of GDAS’ Research Platform (2016GDASPT-0215). BW was supported by the China Scholarship Council (CSC), Beijing, China. 10. Rasche A, Souza BF, Drexler JF. Bat hepadnaviruses and the origins of primate hepatitis B viruses. Curr Opin Virol. 2016;16:86–94. 11. Quan PL, Firth C, Conte JM, Williams SH, Zambrana-Torrelio CM, Anthony SJ, Ellison JA, Gilbert AT, Kuzmin IV, Niezgoda M, et al. Bats are a major natural reservoir for hepaciviruses and pegiviruses. Proc Natl Acad Sci U S A. 2013;110:8194–9. 12. Ge XY, Li JL, Yang XL, Chmura AA, Zhu G, Epstein JH, Mazet JK, Hu B, Zhang W, Peng C, et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature. 2013;503:535–8. 1. Jones KE, Patel NG, Levy MA, Storeygard A, Balk D, Gittleman JL, Daszak P. Global trends in emerging infectious diseases. Nature. 2008;451:990–3. 2. Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T. Bats: important reservoir hosts of emerging viruses. Clin Microbiol Rev. 2006;19:531–45. 3. Shi ZL. Emerging infectious diseases associated with bat viruses. Sci China-Life Sci. 2013;56:678–82. 4. Wibbelt G, Moore MS, Schountz T, Voigt CC. Emerging diseases in Chiroptera: why bats? Biol Lett. 2010;6:438–40. 5. Drexler JF, Corman VM, Lukashev AN, van den Brand JM, Gmyl AP, Brunink S, Rasche A, Seggewibeta N, Feng H, Leijten LM, et al. Evolutionary origins of hepatitis A virus in small mammals. Proc Natl Acad Sci U S A. 2015;112:15190–5. Author details 1C S b 1CAS Key Laboratory of Special Pathogens and Biosafety and Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China. 2Guangdong Institute of Applied Biological Resource, Guangzhou 510260, China. 3Laboratory for Zoonosis Control and Prevention, Yunnan Institute of Endemic Diseases Control and Prevention, 1CAS Key Laboratory of Special Pathogens and Biosafety and Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China. 2Guangdong Institute of Applied Biological Resource, Guangzhou 510260, China. 3Laboratory for Zoonosis Control and Prevention, Yunnan Institute of Endemic Diseases Control and Prevention, Dali 671000, China. 4Department of Infectious Diseases, Robert Koch Institute, Berlin, Germany. Dali 671000, China. 4Department of Infectious Diseases, Robert Koch Institute, Berlin, Germany. Competing interests g The authors declare that they have no competing interests. Received: 17 August 2016 Accepted: 4 February 2017 Received: 17 August 2016 Accepted: 4 February 2017 10. Rasche A, Souza BF, Drexler JF. Bat hepadnaviruses and the origins of primate hepatitis B viruses. Curr Opin Virol. 2016;16:86–94. 4. Wibbelt G, Moore MS, Schountz T, Voigt CC. Emerging diseases in Chiroptera: why bats? Biol Lett. 2010;6:438–40. Conclusions and discussion Our results have limitations due to the small sample size used, which was a result of the protec- tion of bat populations in China, as bats play important roles in the pollination of plants and in pest control, as they feed on insects. However, based on our discovery of hepatitis viruses in bats, it is expected that there are many more hepatitis viruses circulating in numerous bat species and in various geographic regions. In order to obtain larger sample sizes, non-invasive methods of virus detection should be considered for future studies. Fig. 4 Tissue distribution of bat hepevirus and hepadnaviruses. Virus concentrations assessed by virus-specific one-step real-time RT-PCR using quantified in vitro-transcribed RNA controls Fig. 4 Tissue distribution of bat hepevirus and hepadnaviruses. Virus concentrations assessed by virus-specific one-step real-time RT-PCR using quantified in vitro-transcribed RNA controls Page 10 of 10 Page 10 of 10 Page 10 of 10 Wang et al. Virology Journal (2017) 14:40 6. Drexler JF, Geipel A, Konig A, Corman VM, van Riel D, Leijten LM, Bremer CM, Rasche A, Cottontail VM, Maganga GD, et al. Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes. Proc Natl Acad Sci U S A. 2013;110:16151–6. 7. Drexler JF, Seelen A, Corman VM, Tateno AF, Cottontail V, Zerbinati RM, Gloza-Rausch F, Klose SM, Adu-Sarkodie Y, Oppong SK, et al. Bats worldwide carry hepatitis E virus-related viruses that form a putative novel genus within the family Hepeviridae. J Virol. 2012;86:9134–47. 8. He B, Fan Q, Yang F, Hu T, Qiu W, Feng Y, Li Z, Li Y, Zhang F, Guo H, et al. Hepatitis virus in long-fingered bats, Myanmar. Emerg Infect Dis. 2013;19:638–40. 9. He B, Zhang F, Xia L, Hu T, Chen G, Qiu W, Fan Q, Feng Y, Guo H, Tu C. Identification of a novel Orthohepadnavirus in pomona roundleaf bats in China. Arch Virol. 2015;160:335–7. 10. Rasche A, Souza BF, Drexler JF. Bat hepadnaviruses and the origins of primate hepatitis B viruses. Curr Opin Virol. 2016;16:86–94. 11. Quan PL, Firth C, Conte JM, Williams SH, Zambrana-Torrelio CM, Anthony SJ, Ellison JA, Gilbert AT, Kuzmin IV, Niezgoda M, et al. Bats are a major natural reservoir for hepaciviruses and pegiviruses. Proc Natl Acad Sci U S A. 2013;110:8194–9. 12. Ge XY, Li JL, Yang XL, Chmura AA, Zhu G, Epstein JH, Mazet JK, Hu B, Zhang W, Peng C, et al. 15. Smith DB, Simmonds P, members of the International Committee on the Taxonomy of Viruses Hepeviridae Study G, Jameel S, Emerson SU, Harrison TJ, Meng XJ, Okamoto H, Van der Poel WH, Purdy MA. Consensus proposals for classification of the family Hepeviridae. J Gen Virol. 2015;96:1191–2. References • We accept pre-submission inquiries 1. Jones KE, Patel NG, Levy MA, Storeygard A, Balk D, Gittleman JL, Daszak P. Global trends in emerging infectious diseases. Nature. 2008;451:990–3. 2. Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T. Bats: important reservoir hosts of emerging viruses. Clin Microbiol Rev. 2006;19:531–45. 3. Shi ZL. Emerging infectious diseases associated with bat viruses. Sci China-Life Sci. 2013;56:678–82. 4. Wibbelt G, Moore MS, Schountz T, Voigt CC. Emerging diseases in Chiroptera: why bats? Biol Lett. 2010;6:438–40. 5. Drexler JF, Corman VM, Lukashev AN, van den Brand JM, Gmyl AP, Brunink S, Rasche A, Seggewibeta N, Feng H, Leijten LM, et al. Evolutionary origins of hepatitis A virus in small mammals. Proc Natl Acad Sci U S A. 2015;112:15190–5. 5. Drexler JF, Corman VM, Lukashev AN, van den Brand JM, Gmyl AP, Brunink S, Rasche A, Seggewibeta N, Feng H, Leijten LM, et al. Evolutionary origins of hepatitis A virus in small mammals. Proc Natl Acad Sci U S A. 2015;112:15190–5.
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Aortic Wave Dynamics and Its Influence on Left Ventricular Workload
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Introduction Congestive heart failure (CHF) has reached an epidemic proportion in the US and worldwide with serious consequences in terms of human suffering and economic impact. In the US alone, there are 60,000 patients dying each year with CHF as the underlying cause. Approximately 5,800,000 Americans have been diagnosed with this condition and this number is increasing every year [1]. In the absence of myocardial infarction, hypertension is a primary risk factor of CHF [2] mainly due to the chronic elevation of the left ventricular (LV) workload and the development of left ventricular hypertrophy (LVH) [3,4,5]. The wave dynamics in a compliant tube is controlled by the fundamental frequency of the propagating waves, the material properties of the tube, and wave reflections [12,13]. Similarly, aortic wave dynamics depend on heart rate, aortic compliance, and the locations of reflection sites. We hypothesized that there exists a specific heart rate at which LV pulsatile load becomes a minimum for a given physiological condition. Pulsatile flow generated by the heart enters the compliant aorta as pressure and flow waves. These waves propagate and reflect throughout arterial vasculature, thus playing a dominant role in the hemodynamics of the arterial system. The hemodynamic load on the heart has two parts: steady and pulsatile. The steady load is the result of the resistance from the arterial network to the mean part of the flow. The pulsatile load depends on the interaction between the heart’s pumping characteristics (stroke volume, heart rate, and ejection fraction) and arterial wave dynamics. Significant efforts have been made in the past to elucidate the role of wave reflections in heart failure [6,7,8]. Clinical studies have confirmed that abnormal pulsatile loads play an important role in the pathogenesis of left ventricular hypertrophy (LVH) and CHF [4,9,10]. O’Rourke [11] suggested four important factors that control the level of the pulsatile load applied to the heart: 1-rigidity of the aorta and other large vessels, 2-interaction between the left ventricular and the terminal of the vasculature in the upper and lower parts of the body, 3-wave reflection, and 4-balance between the heart rate and the body length. He showed in an animal (dog) To test our hypothesis, we implemented a computational approach in order to be able to examine a large spectrum of wave states. Abstract The pumping mechanism of the heart is pulsatile, so the heart generates pulsatile flow that enters into the compliant aorta in the form of pressure and flow waves. We hypothesized that there exists a specific heart rate at which the external left ventricular (LV) power is minimized. To test this hypothesis, we used a computational model to explore the effects of heart rate (HR) and aortic rigidity on left ventricular (LV) power requirement. While both mean and pulsatile parts of the pressure play an important role in LV power requirement elevation, at higher rigidities the effect of pulsatility becomes more dominant. For any given aortic rigidity, there exists an optimum HR that minimizes the LV power requirement at a given cardiac output. The optimum HR shifts to higher values as the aorta becomes more rigid. To conclude, there is an optimum condition for aortic waves that minimizes the LV pulsatile load and consequently the total LV workload. Citation: Pahlevan NM, Gharib M (2011) Aortic Wave Dynamics and Its Influence on Left Ventricular Workload. PLoS ONE 6(8): e23106. doi:10.1371/ journal.pone.0023106 Editor: Aravinthan Samuel, Harvard University, United States of America Citation: Pahlevan NM, Gharib M (2011) Aortic Wave Dynamics and Its Influence on Left Ventricular Workload. PLoS ONE 6(8): e23106. doi:10.1371/ journal.pone.0023106 Editor: Aravinthan Samuel, Harvard University, United States of America Received March 30, 2011; Accepted July 6, 2011; Published August 11, 2011 Received March 30, 2011; Accepted July 6, 2011; Published August 11, 2011 Copyright:  2011 Pahlevan, Gharib. 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. Funding: This research effort has been funded by unrestricted (research topic or publication approval) gift funds from Fletcher Jones foundation, Medtronic Corp, and Charyk Family foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. mpeting Interests: Medtronic Corp is one of the funders. This does not alter the authors’ adherence to all the PLoS ONE policie terials. Medtronic Corp is one of the funders. Niema M. Pahlevan1, Morteza Gharib2* 1 Option of Bioengineering, Division of Engineering & Applied Sciences, California Institute of Technology, Pasadena, California, United States of America, 2 Graduate Aerospace Laboratories, Division of Engineering & Applied Sciences, California Institute of Technology, Pasadena, California, United States of America Abstract This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and * E-mail: mgharib@caltech.edu study that the ratio of the pulsatile load over the total load decreased as the heart rate increased, and that the ratio increased as the aortic distensibility decreased. However, previous studies did not investigate the interplay between aortic rigidity and heart rate (HR) [11]. Introduction This approach enabled us to study aortic rigidity and HR while having a better control on parameters such as the aortic input flow wave (ventricular ejection wave), terminal compliance, peripheral resistance (PR), cardiac output (CO), and the locations of reflection sites. The goal of this study is to investigate the effects of different states of aortic wave dynamics on the LV power requirement (LVPR). Various states of aortic wave dynamics are produced by changing the heart rate and aortic wall rigidity (aortic compliance) while fixing other determinant factors of wave dynamics and power requirements. Aortic Wave Dynamics and Its Influence on Left Ventricular Workload Niema M. Pahlevan1, Morteza Gharib2* Physical Model A three-dimensional axisymmetric model of the aorta was used (Figure 1). The geometrical data of the aortic model, such as aortic PLoS ONE | www.plosone.o August 2011 | Volume 6 | Issue 8 | e23106 August 2011 | Volume 6 | Issue 8 | e23106 PLoS ONE | www.plosone.org 1 Aortic Waves and Left Ventricular Workload Figure 1. Computational model of the aorta. doi:10.1371/journal.pone.0023106.g001 Figure 1. Computational model of the aorta. doi:10.1371/journal.pone.0023106.g001 length, inner diameter, and wall thickness, were within the average physiological range [14]. The aortic wall was assumed to be elastic and isotropic. These wall material assumptions are applicable when modeling large central arteries, especially the aorta, but it may not be suitable for radial arteries or arterioles since these vessels are more anisotropic and viscoelastic [15]. A further concern about our wall assumption might be the nonlinear dependency of the vessel’s wall elasticity on the mean arterial pressure; however, for the normal mean pressure (less than 125 mmHg) the relation is linear [15]. The material properties of the wall were taken from Nichols et al. [15] The tapering and the change of wall stiffness along the aorta were considered, though the aortic arc and bifurcation were excluded since the model is 3D axisymmetric. The blood was assumed to be an incompressible Newtonian fluid, and the different levels of aortic rigidity considered were multiplicative factors of a minimum rigidity level E1(x) (x is the distance from the heart) of a healthy 30-year-old man taken from Nichols et al. [15]. The physical parameters of the aortic model are summarized in Table 1. ~+:~V~0, ð3Þ ð3Þ rf L~V Lt z~V:~+~V ! z~+p~m+2~Vz~Fb, ð4Þ ð4Þ where ~V~ vy,vz   represents the flow velocity vector, rf is the fluid density, p is the static pressure, m is the dynamic viscosity, and ~Fb is the body force. Since our fluid domain has moving boundaries, an arbitrary Lagrangian-Eulerian (ALE) formulation is used for the analysis of the fluid flow [19,20,21,22]. This formulation can be directly coupled with the Lagrangian formulation of the solid domain. Physical Model In an ALE formulation, the total time derivative (d/dt) for all the solution variables is given by [23] d :ð Þ dt ~ d :ð Þ dt z ~V{ ~ W   :+ :ð Þ, ð5Þ At the inlet, we imposed a physiological flow wave (Figure 2), from Matthys [16], with a flat velocity profile and scaled to give a cardiac output (CO) of 4.6 L/min for any given heart rate. ð5Þ where d(.)/dt is the transient term at the mesh position, ~ W is the mesh velocity, and ~V is the actual fluid particle velocity. Applying equation 5 in equation 4 gives [23] Mathematical method Solid Model. Large deformation with small strain theory was assumed for the formulation of elastic wall motion [17]. We assume the walls are composed of elastic isotropic material. Dynamic motion of the aortic wall was formulated by a constitutive relation for linear elastic isotropic materials and balance of momentum equations in Lagrangian form as [17,18] rf L~V Lt z ~V{ ~ W   :~+~V ! z~+p~m+2~Vz~Fb: ð6Þ ð6Þ Coupling conditions. With no-slip boundary conditions at the wall, the coupling equations at the solid-fluid interface are sij,jzFi~rs:€ui ð1Þ ð1Þ ~Vf ~~_u_us Velocity condition ð Þ ð7Þ ~n: s ~f ~~n: s ~s : Traction equilibrium ð Þ, ð8Þ 2Þ ~Vf ~~_u_us Velocity condition ð Þ ð7Þ ð7Þ sij~lekkdijz2mleij: ð2Þ ð2Þ ~n: s ~f ~~n: s ~s : Traction equilibrium ð Þ, ð8Þ ð8Þ Here, sij is the wall stress tensor, F is the external force, u is the displacement vector, rs is the wall density, and l, ml are Lame´ constants. where ~Vf and ~_u_us are the respective velocities of the fluid and solid at the interface, s ~f is fluid stress tensor, s ~s is solid stress tensor, d ~ i h i i h l di i Fluid Model. To solve for pressure and flow fields in the fluid domain, we use the full Navier-Stokes (N-S) equations. In Eulerian form, they are and ~n is the unit vector in the normal direction. PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e23106 August 2011 | Volume 6 | Issue 8 | e23106 August 2011 | Volume 6 | Issue 8 | e23106 2 Aortic Waves and Left Ventricular Workload Table 1. Physical parameters*. Mathematical method This boundary model extends the computational domain with a straight elastic tube connected to a contracted rigid tube [24]. Parameters such as the ratio of the radii of the rigid tube together with the length and elasticity of the elastic boundary tube are selected to represent the effects of a truncated vascular network (resistance, compliance, and wave reflection) [24]. Mathematical method Physical parameters Symbol Value Blood density rf 1050 kg/m3 Blood viscosity m 0.0051 kg/m.sec Aortic wall density rs 1050 kg/m3 Young’s modulus of the sinus of Valsalva Esin 3:5|105 Pa Young’s modulus of the ascending aorta Easc 4:2|105 Pa Young’s modulus of the descending aorta Edes 4:7|105 Pa Young’s modulus of the upper part of the abdominal aorta Eupab 5:3|105 Pa Young’s modulus of the lower part of the abdominal aorta Elowab 8:6|105 Pa Aortic wall Poisson’s ratio u 0.45 Wall thickness of the sinus of Valsalva hsin 1.6 mm Wall thickness of the ascending aorta hasc 1.4 mm Wall thickness of the descending aorta hdes 1 mm Wall thickness of the upper part of the abdominal aorta hupab 0.9 mm Wall thickness of the lower part of the abdominal aorta hlowab 0.8 mm *Since the physical parameters vary in each aortic section, the average values within the sections have been given. The Young’s modulus values in this Table are for the case of minimum rigidity (E1). doi:10.1371/journal.pone.0023106.t001 *Since the physical parameters vary in each aortic section, the average values within the sections have been given. The Young’s modulus values in this Table are for the case of minimum rigidity (E1). doi:10.1371/journal.pone.0023106.t001 *Since the physical parameters vary in each aortic section, the average values within the sections have been given. The Young’s modulus values in this Table are for the case of minimum rigidity (E1). doi:10.1371/journal.pone.0023106.t001 original radius before the contraction. The parameters of outflow boundary condition were kept the same for all simulations. Boundary Conditions. A novel extension tube boundary model was used for the outflow boundary condition at the terminal of the abdominal aorta. This boundary model extends the computational domain with a straight elastic tube connected to a contracted rigid tube [24]. Parameters such as the ratio of the radii of the rigid tube together with the length and elasticity of the elastic boundary tube are selected to represent the effects of a truncated vascular network (resistance, compliance, and wave reflection) [24]. The parameters of the outflow boundary condition model are given in Table 2, where the contraction ratio is the ratio of the radius of the rigid boundary tube beyond the contraction to the Boundary Conditions. A novel extension tube boundary model was used for the outflow boundary condition at the terminal of the abdominal aorta. Results The simulations were run for seven different levels of aortic rigidities, ranging from a 30-year old healthy individual (E1(x)) to a 70-year old sick individual suffering from aortic stiffening (E7(x)) (values taken from Nichols et al) [15]. Here, Ei(x) indicates that Young’s modulus changes along the aorta, where x is the axial distance from aortic input. The different levels used were mul- tiplicative factors of E1(x) (Table 1) as E2=1.25E1, E3=1.5E1, E4= 1.75 E1, E5 =2E1, E6 =2.5E1, and E7 =3E1. Each case of the aortic rigidity was run for eight heart rates (70.5, 75, 89.5, 100, 120, 136.4, 150, and 187.5 beats per minute (bpm)). In all simulations, CO, PR, the terminal compliance, and the shape of the inflow wave were kept constant. An implicit Euler backward method with Newton-Raphson iteration was used for the time integration with a time step of 0.00125 s. A total of 2420 nine-node axisymmetric elements were used to mesh the solid domain, and a total of 17,416 three-node axisymmetric elements were used for the fluid domain. Further simulations with different time steps and mesh sizes confirmed that these results were independent of spatial and temporal discretiza- tions. All simulations started from rest until the mean of the aortic input pressure reached a steady state as shown in Figure 3. Aortic Waves and Left Ventricular Workload Aortic Waves and Left Ventricular Workload Table 2. Outflow boundary parameters. Description value The inner radius of the elastic boundary tube 6.31 mm Total length of the elastic boundary tube 105 mm Wall thickness of the elastic boundary tube 0.65 mm Contraction ratio of the rigid boundary tube 0.76 Total volume compliance of the boundary model 9:96|10{11 m3 Pa doi:10.1371/journal.pone.0023106.t002 Table 2. Outflow boundary parameters. The steady power (Ps) was the product of mean pressure (pmean) and mean flow (qmean), and the pulsatile power (Ppulse) is the difference of the total power and steady power. Ptotal~ 1 T ðT 0 p tð Þq tð Þdt ð12Þ ð12Þ Ps~pmeanqmean ð13Þ Ps~pmeanqmean ð13Þ Ppulse~Ptotal{Ps ð14Þ ð13Þ Ppulse~Ptotal{Ps ð14Þ ð14Þ In summary, the general computational steps in the employed direct coupling method were (i) to assemble the solid and fluid equations separately into a single fluid and single solid model; (ii) to assemble the solid matrix, fluid matrix, and the coupling matrices into one coupled matrix system; (iii) to solve the linearized equation of the coupled system and to update the solution; and (iv) to compute and check the residuals against the specified tolerance. If the solution did not converge, the process was restarted from step (i) [19,24]. Numerical method A finite-element scheme was applied to solve the equations of the solid and fluid models incrementally in time using the commercial package ADINA 8.6 (ADINA R&D, Inc., MA). A direct two-way coupling fluid-structure interaction (FSI) method (simultaneous solution method) was used to couple the fluid and solid domains at the interface. In this method, the discretized fluid, solid, and coupling equations are all combined in one matrix [19]. The parameters of the outflow boundary condition model are given in Table 2, where the contraction ratio is the ratio of the radius of the rigid boundary tube beyond the contraction to the Figure 2. Inflow wave. doi:10.1371/journal.pone.0023106.g002 PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23106 Figure 2. Inflow wave. doi:10.1371/journal.pone.0023106.g002 PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23106 Figure 2. Inflow wave. Figure 2. Inflow wave. doi:10.1371/journal.pone.0023106.g002 August 2011 | Volume 6 | Issue 8 | e23106 August 2011 | Volume 6 | Issue 8 | e23106 Power Calculation To verify the model, we compared results with well-known features of aortic pressure waves [15,25]: (i) the pulse pressure The total power (Ptotal) was calculated as the average of the product of the pressure (p(t)) and flow (q(t)) over a cardiac cycle. Figure 3. The mean pressure at the aortic input reaches a steady state for HR = 70.5 bpm. doi:10.1371/journal.pone.0023106.g003 Figure 3. The mean pressure at the aortic input reaches a steady state for HR = 70.5 bpm. doi:10.1371/journal.pone.0023106.g003 August 2011 | Volume 6 | Issue 8 | e23106 August 2011 | Volume 6 | Issue 8 | e23106 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org 4 Aortic Waves and Left Ventricular Workload Figure 4. Simulated results from our aortic model showing pulse pressure amplification, pressure narrowing, shifting of the second-highest peak, and the existence of the dicrotic notch. The average per cycle decreases due to viscous losses, consistent with realistic results. Data is for a 100 bpm heart rate and aortic rigidity of E1 taken during one cycle after reaching steady state. doi:10.1371/journal.pone.0023106.g004 Figure 4. Simulated results from our aortic model showing pulse pressure amplification, pressure narrowing, shifting of the second-highest peak, and the existence of the dicrotic notch. The average per cycle decreases due to viscous losses, consistent with realistic results. Data is for a 100 bpm heart rate and aortic rigidity of E1 taken during one cycle after reaching steady state. doi:10.1371/journal.pone.0023106.g004 Effects of Aortic Compliance on Left Ventricular Power Requirement amplification; (ii) the narrowing of the pressure waves as they travel down the aorta; (iii) the existence of a dicrotic notch; and (iv) the shifting of the second-highest pressure peak to the end of the cycle. Figure 4 shows that our computational model captured all four characteristics of the pressure wave. The model additionally captured another important physical feature of the blood flow—the mean pressure decreased down the aorta (see Figure 4). Figure 5 shows average values per cycle for the total LV power requirement—external LV power (Ptotal) —and the steady power (Ps) versus aortic rigidity for an HR of 75 bpm. It shows that both Ptotal and Ps, as well as the pulsatile power (Ppulse = Ptotal2Ps), increased at higher rigidities. This is in agreement with clinical findings [26]. Figure 5. The blue line is average per cycle of the total external left ventricular power (Ptotal) and the red line is the external steady power (Ps) at different levels of aortic rigidities. The value of E = E1 corresponds to the level of aortic rigidity given in Table 1. The difference between the two curves shown by the double arrow is pulsatile power (Ppulse). The results are for HR = 75 bpm. doi:10.1371/journal.pone.0023106.g005 Figure 5. The blue line is average per cycle of the total external left ventricular power (Ptotal) and the red line is the external steady power (Ps) at different levels of aortic rigidities. The value of E = E1 corresponds to the level of aortic rigidity given in Table 1. The difference between the two curves shown by the double arrow is pulsatile power (Ppulse). The results are for HR = 75 bpm. doi:10.1371/journal.pone.0023106.g005 August 2011 | Volume 6 | Issue 8 | e23106 PLoS ONE | www.plosone.org 5 Aortic Waves and Left Ventricular Workload Figure 6. Average value per cycle of pulsatile power Ppulse versus HR for three different levels of aortic rigidities. E1 (black) corresponds to the values given in Table 1, E2 = 1.25E1 (blue), and E3 = 1.5E1 (red). Green circles are the heart rates corresponding to the minimum Ppulse value at each level of aortic rigidity. doi:10.1371/journal.pone.0023106.g006 Figure 6. Average value per cycle of pulsatile power Ppulse versus HR for three different levels of aortic rigidities. E1 (black) corresponds to the values given in Table 1, E2 = 1.25E1 (blue), and E3 = 1.5E1 (red). Effects of Aortic Compliance on Left Ventricular Power Requirement Green circles are the heart rates corresponding to the minimum Ppulse value at each level of aortic rigidity. doi:10.1371/journal.pone.0023106.g006 Discussion The main findings of this study are as follows: (i) an increase in aortic rigidity leads to an increase in both the steady and pulsatile loads on LV; (ii) at higher aortic rigidities, the effect of pulsatile load becomes more dominant; (iii) at a given heart pumping condition (cardiac output and inlet flow wave), there is an optimum heart rate that minimizes the pulsatile LV power; and (iv) the optimum HR shifts to higher values as aortic rigidity increases. Pulsatile load on the LV is the result of complex wave dynamics in the arterial network. As the largest and most compliant vessel extending from the heart, the aorta dominates the wave dynamics that the LV experiences. Although pulsatile load on the LV only accounts for a small portion of the total energy of the heart [27,28], its adverse affect on the LV has been well accepted. Indeed, clinical studies have confirmed that abnormal pulsatile load plays an important role in the development of LVH and progression of LVH to CHF [4,8,9,10]. Hence, aortic wave dynamics—as the determinant of LV pulsatile load—plays an important role in pathogenesis of LVH and CHF. First, we investigated the effect of aortic wave dynamics on LV power requirements by changing aortic rigidity at a fixed HR( = 75 bpm) while keeping constant all the other aortic wave dynamic determinants such as CO, shape of inflow wave, peripheral resistance, terminal compliance, and the locations of the reflection sites. Both steady power and pulsatile power increase at higher rigidities. The increase in pulsatile load is due to both reduced compliance and wave dynamics. Second, for a fixed cardiac output (CO = 4.6 L/min), we studied the effect of aortic wave dynamics on the pulsatile LV power requirement across a physiological range of heart rates. Our results reveal that there is an optimum HR (within physiological range) at witch pulsatile LV power requirement becomes a minimum, thereby confirming a prediction by O’Rourke [29]. Figure 8. (a) Percentage of Ppulse Ptotal versus HR for the cases given in Figure 6; E1 (black) corresponds to the values given in Table 1, E2 = 1.25E1 (blue), and E3 = 1.5E1 (red). (b) Percentage of Ppulse Ptotal versus HR for the cases given in Figure 7; E5 = 2E1 (black), E6 = 2.5E1 (blue), and E7 = 3E1 (red). Discussion doi:10.1371/journal.pone.0023106.g008 The pulsatile power continues to decrease with increasing HR until it reaches its minimum point. Beyond the minimum point, the aortic waves start acting destructively, and as a result, the pulsatile power starts elevating with the HR (see Figures 6 and 7). The same pattern has been shown in an animal study performed by O’Rourke [11]. He studied the affect of HR on the ratio of the pulsatile to total LV power. In Figure 1 of his paper, he showed the pulsatile/total percentage versus HR for 3 dogs (dog 7, 9, and 32) where dog 32 showed the same pattern as in Figure 5 and it had an optimum HR of around 120 bpm; however, he did not explain the existence of this minimum point in the paper. Notice that we sketch ‘‘pulsatile’’ load versus HR in figure 6 and 7. However, as Figure 8 shows, the shape of the graphs in Figures 6 and 7 will be preserved if one sketches the percentage of pulsatile load over total power versus HR. or long-term smoking—the optimum wave condition shifts to a higher HR and therefore the heart increases the HR to reach the new optimum. Some may argue that increasing HR as a compensatory mechanism for decreasing LV pulsatile load can have a metabolic disadvantage for the heart in terms of increased myocardial oxygen consumption and impaired ventricular-arterial coupling. However, it has been shown in previous studies that under nor- mal conditions and in the absence of heart diseases (e,g. dilated cardiomyopathy), increased HR may even results in enhanced LV systolic and diastolic performance [36,37]. Furthermore, under normal conditions, the LV-arterial coupling remains optimal after a moderate increase in HR [38]. Clinical example: Smoking, Aortic Stiffness, and Heart rate Effect of Heart Rate on Left Ventricular Power Requirement minimized (and as a result Ptotal is minimized). The Ppulse increases with HR beyond this optimum point. This phenomenon is present for all three cases. Interestingly, the optimum point shifted towards higher HR as aortic rigidity increased. In fact, as Figure 7 demonstrates, these phenomena still exist at very high rigidities, two- or three-fold greater than those of Figure 6. Figure 6 shows Ppulse as a function of HR for three levels of aortic rigidity. As mentioned before, the cardiac output (average flow per cardiac cycle) is kept the same for all cases. As the HR increases, the Ppulse decreases until the HR reaches an optimum point where Ppulse is Figure 7. Ppulse versus HR for extreme values of aortic rigidity. E5 = 2E1 (black), E6 = 2.5E1 (blue), and E7 = 3E1 (red), where E1 corresponds to the values given in Table 1. Green circles are the heart rates corresponding to the minimum Ppulse value at each level of aortic rigidity. doi:10.1371/journal.pone.0023106.g007 Figure 7. Ppulse versus HR for extreme values of aortic rigidity. E5 = 2E1 (black), E6 = 2.5E1 (blue), and E7 = 3E1 (red), where E1 corresponds to the values given in Table 1. Green circles are the heart rates corresponding to the minimum Ppulse value at each level of aortic rigidity. doi:10.1371/journal.pone.0023106.g007 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e23106 6 Aortic Waves and Left Ventricular Workload Figure 8. (a) Percentage of Ppulse Ptotal versus HR for the cases given in Figure 6; E1 (black) corresponds to the values given in Table 1, E2 = 1.25E1 (blue), and E3 = 1.5E1 (red). (b) Percentage of Ppulse Ptotal versus HR for the cases given in Figure 7; E5 = 2E1 (black), E6 = 2.5E1 (blue), and E7 = 3E1 (red). doi:10.1371/journal.pone.0023106.g008 PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e23106 References Laskey W, Kussmaul W (1987) Arterial wave reflection in heart failure. Circulation 75: 711–722. 8. Mitchell GF, Tardif J-C, Arnold JMO, Marchiori G, O’Brien TX, et al. (2001) Pulsatile Hemodynamics in Congestive Heart Failure. Hypertension 38: 1433–1439. 30. Levenson J, Simon A, Cambien F, Beretti C (1987) Cigarette smoking and hypertension. Factors independently associated with blood hyperviscosity and arterial rigidity. Arterioscler Thromb Vasc Biol 7: 572–577. 9. Ooi H, Chung W, Biolo A (2008) Arterial Stiffness and Vascular Load in Heart Failure. Congestive Heart Failure 14: 31–36. 31. Levent E, Ozyu¨rek AR, U¨ lger Z (2004) Evaluation of aortic stiffness in tobacco- smoking adolescents. Journal of Adolescent Health 34: 339–343. Safar M, O’Rourke MF (2006) Arterial Stiffness in Hypertension Else 32. Stefanadis C, Tsiamis E, Vlachopoulos C, Stratos C, Toutouzas K, et al. (1997) Unfavorable Effect of Smoking on the Elastic Properties of the Human Aorta. Circulation 95: 31–38. 11. O’Rourke MF (1967) Steady and Pulsatile Energy Losses in the Systemic Circulation under Normal Conditions and in Simulated Arterial Disease. Cardiovascular Research 1: 313–326. 33. Giannattasio C, Mangoni AA, Stella ML, Carugo S, Grassi G, et al. (1994) Acute effects of smoking on radial artery compliance in humans. Journal of Hypertension 12: 691–696. 12. Avrahami I, Gharib M (2008) Computational studies of resonance wave pumping in compliant tubes. Journal of Fluid Mechanics 608: 139–160. 13. Hickerson A, Rinderknecht D, Gharib M (2005) Experimental study of the behavior of a valveless impedance pump. Experiments in Fluids 38: 534–540. 34. Kool MJF, Hoeks APG, Struijker Boudier HAJ, Reneman RS, Van Bortel LMAB (1993) Short and long-term effects of smoking on arterial wall properties in habitual smokers. Journal of the American College of Cardiology 22: 1881–1886. p p p p 14. Caro CG, Pedley JG, Schroter RC, Seed WA (1978) The Mechanics of the Circulation. Oxford: Oxford University Press. y 15. Nichols WW, O’Rourke MF (1998) McDonald’s Blood Flow in Arteries: Theoretical, Experimental and Clinical Principles. London: Arnold 564 p. 35. Mahmud A, Feely J (2003) Effect of Smoking on Arterial Stiffness and Pulse Pressure Amplification. Hypertension 41: 183–187. 16. Matthys KS, Alastruey J, Peiro J, Khir AW, Segers P, et al. (2007) Pulse wave propagation in a model human arterial network: Assessment of 1-D numerical simulations against in vitro measurements. Journal of Biomechanics 40: 11. 36. Feldman MD, Alderman JD, Aroesty JM, Royal HD, Ferguson JJ, et al. Conclusion We are grateful to Dr. Fazle Hussain for productive discussion. Our results in this computational study show that at a given heart’s pumping condition, there is an optimum condition for aortic waves that minimizes the pulsatile load (and consequently total workload) on the heart. In addition, based on clinical observations, our results suggest that the heart may use this fact as a temporary compensatory mechanism to reduce myocardial workload. Therefore, controlling and modifying aortic wave Model limitation However, the influences of branches in terms of wave reflection August 2011 | Volume 6 | Issue 8 | e23106 PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e23106 7 Aortic Waves and Left Ventricular Workload dynamics—as the determinant of LV pulsatile load—can be a therapeutic approach for the reversal of LVH and the prevention of HF. were included in the outflow boundary condition of our model (by assuming that the branches produce summated reflected waves). Exclusion of the arch can be justified by considering the fact that the curve has an insignificant effect on pressure waves [15]. Model limitation It has been shown in clinical studies that both aortic rigidity and HR are higher in habitual smokers compared to nonsmokers [10,30,31,32]. It has also been shown that even smoking a single cigarette leads to a transient increase in pulse wave velocity (aortic rigidity), whether habitual or not [10,33]. Hence in all cases the HR—even for habitual smokers whose HR level is generally higher—will increase during and immediately after smoking even one cigarette [34,35]. We showed that the optimum wave condition leads to a minimum pulsatile load at a specific HR. Hence, our results suggest a possible explanation for HR elevation in smoking. As aortic rigidity increases—such as from short-term In this study, we assumed that the heart acts as a flow source and hence specified the flow wave at the inlet. Although in general the heart is neither a flow nor a pressure source, the behavior of a normal heart is closer to a flow source [39]. In fact, in the case of a hypertrophied left ventricle (LVH condition), the heart acts completely as a flow source [39]. For a given metabolic condition, body requires a certain flow rate and therefore we found it reasonable to consider the heart as a flow source in our study. Additionally, the aortic curve (arch) and the aortic branches were not included in our computational model (see figure 1). However, the influences of branches in terms of wave reflection In this study, we assumed that the heart acts as a flow source and hence specified the flow wave at the inlet. Although in general the heart is neither a flow nor a pressure source, the behavior of a normal heart is closer to a flow source [39]. In fact, in the case of a hypertrophied left ventricle (LVH condition), the heart acts completely as a flow source [39]. For a given metabolic condition, body requires a certain flow rate and therefore we found it reasonable to consider the heart as a flow source in our study. Additionally, the aortic curve (arch) and the aortic branches were not included in our computational model (see figure 1). References 1. WRITING GROUP MEMBERS, Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, et al. (2010) Heart Disease and Stroke Statistics–2010 Update: A Report From the American Heart Association. Circulation 121: e46–215. 1. WRITING GROUP MEMBERS, Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, et al. (2010) Heart Disease and Stroke Statistics–2010 Update: A Report From the American Heart Association. Circulation 121: e46–215. 22. Bathe M, Kamm RD (1999) A Fluid-Structure Interaction Finite Element Analysis of Pulsatile Blood Flow Through a Compliant Stenotic Artery. Journal of Biomechanical Engineering 121: 361–369. 2. Lloyd-Jones DM, Larson MG, Leip EP, Beiser A, D’Agostino RB, et al. (2002) Lifetime Risk for Developing Congestive Heart Failure: The Framingham Heart Study. Circulation 106: 3068–3072. g g 23. Bathe K-J, Hou Z, Ji S (1999) Finite element analysis of fluid flows fully coupled with structural interactions. Computers & Structures 72: 1–16. 24. Pahlevan NM, Amlani F, Gorji H, Hussain F, Gharib M (2011) A Physiologically Relevant, Simple Outflow Boundary Model for Truncated Vasculature. Annals of Biomedical Engineering 39: 1470–1481. 3. Kannel WB, Ho K, Thom T (1994) Changing epidemiological features of cardiac failure. British heart journal 72: S3–S9. j 4. Mitchell GF, Hwang S-J, Vasan RS, Larson MG, Pencina MJ, et al. (2010) Arterial Stiffness and Cardiovascular Events: The Framingham Heart Study. Circulation 121: 505–511. 25. Fung YC (1997) Biomechanics of the Circulation. New York, NY: Springer- Verlag. 26. Kelly R, Tunin R, Kass D (1992) Effect of reduced aortic compliance on cardiac efficiency and contractile function of in situ canine left ventricle. Circ Res 71: 490–502. 5. Moser M, Hebert P (1996) Prevention of disease progression, left ventricular hypertrophy and congestive heart failure in hypertension treatment trials. J Am Coll Cardiol 27: 1214–1218. 27. Elzinga G, Westerhof N (1991) Matching between ventricle and arterial load. An evolutionary process. Circ Res 68: 1495–1500. 6. Curtis SL, Zambanini A, Mayet J, McG Thom SA, Foale R, et al. (2007) Reduced systolic wave generation and increased peripheral wave reflection in chronic heart failure. Am J Physiol Heart Circ Physiol 293: H557–562. 28. Milnor WR (1989) Haemodynamics. Baltimore, Md: Williams & Wilkins Co. y g p p chronic heart failure. Am J Physiol Heart Circ Physiol 293: H557– 29. O’Rourke MF (1996) Toward Optimization of Wave Reflection: Therapeutic Goal for Tomorrow? Clinical and Experimental Pharmacology and Physiology 23: s11–s15. y y 7. Author Contributions Conceived and designed the experiments: NMP MG. Performed the experiments: NMP MG. Analyzed the data: NMP MG. Contributed reagents/materials/analysis tools: NMP MG. Wrote the paper: NMP MG. Conceived and designed the experiments: NMP MG. Performed the experiments: NMP MG. Analyzed the data: NMP MG. Contributed reagents/materials/analysis tools: NMP MG. Wrote the paper: NMP MG. References (1988) Depression of systolic and diastolic myocardial reserve during atrial pacing tachycardia in patients with dilated cardiomyopathy. The Journal of Clinical Investigation 82. g J 17. Bathe KJ (2006) Finite Element Procedures Prentice-Hall. 17. Bathe KJ (2006) Finite Element Procedures Prentice-Hall. 18. ADINA R&D I (2009) Theory and modeling guide, Volume I: ADINA Solids & Structures. Watertown MA . 37. Laniado S, Yellin E, Yoran C, Strom J, Hori M, et al. (1982) Physiologic mechanisms in aortic insufficiency. I. The effect of changing heart rate on flow dynamics. II. Determinants of Austin Flint murmur. Circulation 66: 226–235. 19. ADINA R&D I (2009) Theory and modeling guide, Volume III: ADINA CFD & FSI. Watertown MA . hte N, Cheng C-P, Little WC (2003) Tachycardia exacerbates abnor 38. Ohte N, Cheng C-P, Little WC (2003) Tachycardia exacerbates abnormal left ventricular–arterial coupling in heart failure. Heart and Vessels 18: 136–141. 20. Bathe KJ, Zhang H, Wang MH (1995) Finite element analysis of incompressible and compressible fluid flows with free surfaces and structural interactions. Computers & Structures 56: 193–213. 39. Westerhof N, O’Rourke MF (1995) Haemodynamic basis for the development of left ventricular failure in systolic hypertension and for its logical therapy. Journal of Hypertension 13: 943–952. 21. Bathe KJ, Zhang H, Zhang X (1997) Some advances in the analysis of fluid flows. Computers & Structures 64: 909–930. PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e23106 8
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Ältere Freiwillige im Bundesfreiwilligendienst
Blätter der Wohlfahrtspflege
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Eine Befragung zu Motivation und Zufriedenheit des ersten Jahrgangs Der Bundesfreiwilligendienst steht allen Altersgruppen offen. Das Wohlfahrtswerk für Baden-Württemberg hat die ersten älteren Absolventen des Bundesfreiwilligendienstes nach ihren Erfahrungen befragt. Danach zeichnet sich bei den über 27-jährigen Teilnehmenden am Bundesfreiwilligendienst ein klares Bild ab: Es handelt sich um Personen, die überwiegend ohne feste Anstellung waren und sich neben der Möglichkeit zum sozialen Engagement auch eine berufliche Perspektive erhoffen. Für die Bildungszentren stellt sich damit die Aufgabe, die vom alten Zivildienst geprägten Bildungsinhalte weiterzuentwickeln. Gisela Gölz Gisela Gölz, Diplom-Pädagogin, ist seit 1984 beim Wohlfahrtswerk für Baden-Württemberg tätig; zu- nächst als pädologische Mitarbeite- rin, ab 1990 als Leiterin des Freiwil- ligen Sozialen Jahres und ab 2011 als Gesamtleiterin Freiwilliges Soziales Jahr und Bundesfreiwilligendienst. www.wohlfahrtswerk.de Das Wohlfahrtswerk für Baden-Würt- temberg hat als Träger des Freiwilligen Sozialen Jahres seit über 35 Jahren Er- fahrung mit Gewinnung und Betreuung von jungen Freiwilligen und befragt die- se regelmäßig nach ihrer Zufriedenheit. So konnte man bei der Übernahme der Trägerschaft für den Bundesfreiwil- ligendienst ab Sommer 2011 bei den unter 27-jährigen Teilnehmenden am Bundesfreiwilligendienst auf breite Er- fahrung zurückgreifen. ersten Jahrgang beim Wohlfahrtswerk für Baden-Württemberg waren 32 Teil- nehmende (19%) älter als 27 Jahre. Sie engagierten sich in der Altenhilfe, Be- hindertenhilfe, einer Einrichtung für Kinder und einer Schule. Abbrecher gab es in dieser Altersklasse keine, während von den unter 27-Jährigen Teilnehmen- den am Bundesfreiwilligendienst 10% das Freiwilligenjahr vorzeitig beende- ten. Der Schwerpunkt der Tätigkeit der Freiwilligen über 27 Jahre liegt in der Betreuung. Grundpflege, Fahrdienste, Hausmeistertätigkeiten etc. werden nur ergänzend übernommen. Offen war dagegen die Motivation und Zufriedenheit von Älteren mit dem neuen Freiwilligendienst: Aus welchen Gründen entscheiden sich Menschen über 27 Jahre für einen Bundesfrei- willigendienst? Wie zufrieden sind sie mit dem Freiwilligendienst? Welche Erwartungen haben sie an den Dienst und werden diese erfüllt? Ist der Bun- desfreiwilligendienst nach ihrer Mei- nung gesellschaftlich angesehen? Diese Fragen stellte das Wohlfahrtswerk als Bundesfreiwilligendienst-Träger den Teilnehmenden gegen Ende ihres meist zwölfmonatigen Dienstes. Gisela Gölz Gisela Gölz, Diplom-Pädagogin, ist seit 1984 beim Wohlfahrtswerk für Baden-Württemberg tätig; zu- nächst als pädologische Mitarbeite- rin, ab 1990 als Leiterin des Freiwil- ligen Sozialen Jahres und ab 2011 als Gesamtleiterin Freiwilliges Soziales Jahr und Bundesfreiwilligendienst. www.wohlfahrtswerk.de THEMA THEMA Ergebnisse der Befragung Nachfolgend werden die wichtigsten Re- sultate der Studie des Wohlfahrtswerkes für Baden-Württemberg vorgestellt. Die detaillierten Ergebnisse können auf der Website der Stiftung eingesehen werden (www.wohlfahrtswerk.de). ■ ■Allgemeine Daten: 44% der Befrag- ten sind männlich, 56% weiblich. Besonders stark vertreten ist die Al- tersgruppe der 27- bis 35-Jährigen ■ ■Allgemeine Daten: 44% der Befrag- ten sind männlich, 56% weiblich. Besonders stark vertreten ist die Al- tersgruppe der 27- bis 35-Jährigen Von den insgesamt 161 Teilnehmen- den am Bundesfreiwilligendienst im 6 | 2012 Blätter der Wohlfahrtspflege 2-6-213 24, 06:03:27. Fs ist nicht zulässig 213 THEMA mit 32% und der 56- bis 65-Jähri- gen mit 28%. Die Bildungsvoraus- setzungen sind heterogen: 44% der Teilnehmenden verfügen über einen Hauptschulabschluss, 44% über (Fach-) Hochschulreife. 44% haben eine Berufsausbildung und 32% ein abgeschlossenes Studium. 20% ha- ben keinen Berufsabschluss. Diese finden sich ausschließlich unter den Personen ohne Schulabschluss oder mit Hauptschulabschluss. 48% wa- ren vor dem Bundesfreiwilligendienst arbeitslos oder arbeitsuchend, 20% hatten einen Ein-Euro-Job, 12% wa- ren angestellt tätig, 4% in Rente und 4% gaben Sonstiges an (Sprachkurse, Elternzeit etc.). mit 32% und der 56- bis 65-Jähri- gen mit 28%. Die Bildungsvoraus- setzungen sind heterogen: 44% der Teilnehmenden verfügen über einen Hauptschulabschluss, 44% über (Fach-) Hochschulreife. 44% haben eine Berufsausbildung und 32% ein abgeschlossenes Studium. 20% ha- ben keinen Berufsabschluss. Diese finden sich ausschließlich unter den Personen ohne Schulabschluss oder mit Hauptschulabschluss. 48% wa- ren vor dem Bundesfreiwilligendienst arbeitslos oder arbeitsuchend, 20% hatten einen Ein-Euro-Job, 12% wa- ren angestellt tätig, 4% in Rente und 4% gaben Sonstiges an (Sprachkurse, Elternzeit etc.). Das Wohlfahrtswerk für Baden-Württemberg Mit den meisten Älteren wird ein Semi­ nartag pro Dienstmonat vertraglich fest- gelegt; von den ersten 32 Personen haben alle diese Option gewählt. Das Bildungs- angebot findet als Einzeltag in der Grup- pe statt. Dabei sind die Teilnehmenden im hohen Maße an der Gestaltung und Festlegung der Inhalte beteiligt. Darüber hinaus besuchen hauptamtliche päda- gogische Mitarbeitende die Freiwilligen mindestens einmal während ihres Diens- tes in der Einsatzstelle. ist Träger des Freiwilligen Sozialen Jahres und des Bundesfreiwilligendienst. Die Zahl der Teilnehmenden am Freiwilligen Sozialen Jahr im Jahrgang 2011/2012 be- trug 1.048 in 339 Einsatzstellen in ganz Ba- den-Württemberg. Am Stichtag 15. Januar 2012 gab es 161 Teilnehmende im Bundes- freiwilligendienst in 63 Einsatzstellen in den Einsatzfeldern Behindertenhilfe, Al- tenhilfe, Gesundheitspflege, Kinder- und Jugendarbeit. ist Träger des Freiwilligen Sozialen Jahres und des Bundesfreiwilligendienst. Die Zahl der Teilnehmenden am Freiwilligen Sozialen Jahr im Jahrgang 2011/2012 be- trug 1.048 in 339 Einsatzstellen in ganz Ba- den-Württemberg. Am Stichtag 15. Januar 2012 gab es 161 Teilnehmende im Bundes- freiwilligendienst in 63 Einsatzstellen in den Einsatzfeldern Behindertenhilfe, Al- tenhilfe, Gesundheitspflege, Kinder- und Jugendarbeit. Das Wohlfahrtswerk für Baden-Württem- berg geht auf die Initiative von Königin Katharina von Württemberg im Jahre 1817 zurück. Die russische Zarentochter und Gemahlin König Wilhelms I. woll- te die Armut im Lande bekämpfen und legte damit den Grundstein für die Sozi- alpolitik in Württemberg. Heute ist das Wohlfahrtswerk für Baden-Württemberg als Stiftung des bürgerlichen Rechts ei- ner der größten Träger der Altenhilfe in Baden-Württemberg und betreut mit 1.400 Mitarbeitern rund 2.000 Menschen. Obwohl Name, Rechtsform und Aufgaben der Stiftung mehrfach gewechselt haben, sind die beiden wesentlichen Aufgaben doch über die Jahrhunderte hinweg gleich geblieben: Soziale Dienste und Einrichtun- gen zu unterhalten und Innovationen auf sozialem Gebiet anzuregen und durchzu- setzen. Der Bewerbungsprozess beinhaltet stets ein persönliches Gespräch sowie einen Hospitationstag in der Einsatzstelle. Bei Freiwilligen im Freiwilligen Sozialen Jahr, die ein bevorzugtes Einsatzfeld nennen, wird angestrebt, dem nachzukommen, was überwiegend gelingt. Bei den über 27-jährigen Interessierten am Bundesfrei- willigendienst konnte im Sommer 2011 in allen Fällen das gewünschte Einsatzfeld geboten werden. ■ ■Informationsquellen zum Bundes- freiwilligendienst: Die Bedeutung der Medien für die Gewinnung von älteren Freiwilligen ist hoch: 52% der Befragten erhielten ihre Infor- mationen über die Medien. Eine In- terviewpartnerin sagt: »Habe durch die Presse davon erfahren und es hat mich einfach interessiert: Was pas- siert da? 6 | 2012 Blätter der Wohlfahrtspflege https://doi.org/10.5771/0340-8574-2012-6-213 Generiert durch IP '195.242.1.92', am 24.10.2024, 06:03:27. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. Das Wohlfahrtswerk für Baden-Württemberg Welche Möglichkeiten gibt es für mich?« Etwa ein Drittel (32%) hat Informationen über die Einsatz- stelle genutzt. An dritter Stelle folgen Bekannte und Verwandte (12%). Ar- beitsagentur (8%) und Messen (4%) hatten nur geringe Bedeutung als Informationsquellen. ■ ■Informationsquellen zum Bundes- freiwilligendienst: Die Bedeutung der Medien für die Gewinnung von älteren Freiwilligen ist hoch: 52% der Befragten erhielten ihre Infor- mationen über die Medien. Eine In- terviewpartnerin sagt: »Habe durch die Presse davon erfahren und es hat mich einfach interessiert: Was pas- siert da? Welche Möglichkeiten gibt es für mich?« Etwa ein Drittel (32%) hat Informationen über die Einsatz- stelle genutzt. An dritter Stelle folgen Bekannte und Verwandte (12%). Ar- beitsagentur (8%) und Messen (4%) hatten nur geringe Bedeutung als Informationsquellen. Das Wohlfahrtswerk für Baden-Württem- berg hat getrennte Bildungskonzepte für unter und für über 27-Jährige konzipiert. Für die Jüngeren finden die Bildungsange- bote gemeinsam mit den Teilnehmenden im Freiwilligen Sozialen Jahr in gemisch- ten Gruppen statt. www.wohlfahrtswerk.de ■ ■Motive für den Bundesfreiwilligen- dienst: Als Entscheidungsgründe für den Bundesfreiwilligendienst kristal- lisieren sich zwei Schwerpunkte he- raus: 50% hegten die Hoffnung auf eine anschließende Festanstellung oder ein Fußfassen im Arbeitsmarkt. Hierzu die beispielhafte Aussage ei- ■ ■Motive für den Bundesfreiwilligen- dienst: Als Entscheidungsgründe für den Bundesfreiwilligendienst kristal- lisieren sich zwei Schwerpunkte he- raus: 50% hegten die Hoffnung auf eine anschließende Festanstellung oder ein Fußfassen im Arbeitsmarkt. Hierzu die beispielhafte Aussage ei- haben, die sie bei Problemen um Rat fragen können. Schwierigkeiten am Arbeitsplatz hatten 24% der Teilneh- menden, 76% verneinen diese Fra- ge. In sehr hohem oder hohem Maße empfinden 83% ihre Tätigkeit in der Einsatzstelle als abwechslungsreich und 66% sagen, dass sie ihre eigenen Ideen in die Arbeit einbringen kön- nen. Die Möglichkeit, persönlichen Fähigkeiten und Wissen einzubrin- gen, haben 78% in hohem oder sehr hohem Maße. 66% bestätigen einen hohen oder sehr hohen Einfluss da- rauf, welche Arbeit ihnen zugeteilt wird. Dasselbe gilt für 74% bei der Frage danach, ob ihre Bedürfnisse bei der Planung der Arbeitszeit berück- sichtigt werden. 86% der Befragten geben an, sich in persönlicher Weise durch ihre Tätigkeit im Bundesfrei- willigendienst weiterentwickelt zu haben. Bezogen auf die berufliche Perspektive in ihrer Einsatzstelle ge- ben sein«, in einem anderen: »Wollte die Erfahrung machen im direkten Umgang mit Menschen.« Die andere Hälfte der Freiwilligen (50%) suchte die Möglichkeit, einmal in den so- zialen Bereich hineinzuschauen und etwas Gutes und Soziales zu tun. Teilnehmenden haben sich neue berufliche Perspektiven eröffnet« nes Teilnehmers der schriftlichen Befragung: »War die einzige Mög- lichkeit wieder in den Beruf einzu- steigen, da ich lange in Elternzeit war und keine Berufserfahrung hatte.« Damit hängen die Erwartungen an den Bundesfreiwilligendienst zusam- men. In einem Fragebogen heißt es: »Es sollte ein Neustart ins Berufsle- Eine Teilnehmerin drückt dies so aus: »Das Soziale hat bei mir immer eine geringe Rolle gespielt, in meinem Berufsleben und ich da irgendwie ein Bedürfnis gehabt habe, so etwas zu machen.« ■ ■Zufriedenheit in der Einsatzstelle: Alle Befragten geben an, dass sie in ihrer Einsatzstelle Ansprechpartner 6 | 2012 Blätter der Wohlfahrtspflege https://do Generiert durch Das Erstellen und Weit Blätter der Wohlfahrtspflege Das E 214 THEMA dienst hat meine Psyche und mein Selbstwertgefühl geformt und allein dafür hat es sich gelohnt, das Jahr. Es hat schon enorm viel bewegt.« ■ ■Begleitung durch den Träger des Bundesfreiwilligendienstes: Die Be- gleitung durch das Wohlfahrtswerk für Baden-Württemberg wird insge- samt positiv bewertet. 92% der Be- fragten stimmen in sehr hohem oder hohem Maße zu, dass sie mit der Begleitung durch die pädagogischen Mitarbeitenden insgesamt zufrieden sind. Deren Besuch in den Einsatz- stellen fanden 57% hilfreich. An den Seminaren des Wohlfahrtswerks ha- ben 80% gerne teilgenommen. Insbe- sondere der Austausch mit anderen Teilnehmenden am Bundesfreiwilli- gendienst wurde als sehr bereichernd empfunden. für sie nur in geringem Maß gelte. für sie nur in geringem Maß gelte. ben dies immerhin 43% an. Darun- ter sind auch vier Teilnehmende am Bundesfreiwilligendienst, die nur in den Sozialbereich »hineinschnuppern wollten«. g g g ■ ■Persönlicher Nutzen: Persönlich aus dem Bundesfreiwilligendienst mitge- nommen haben die Teilnehmenden beispielsweise einen «guten Umgang mit Stärken, Schwächen und Gren- zen«. Manche fanden ihr Talent im Umgang mit behinderten Menschen. »Toleranz, Situationen durchstehen und Kontaktaufnahme« sind weitere Aspekte, welche die Teilnehmenden benennen. Besonders häufig geben sie an, durch den Bundesfreiwilli- gendienst wertvolle Erfahrungen mitzunehmen und dass eine Weiter- entwicklung der Lebenseinstellung stattfindet. Auch das Gefühl »etwas Gutes getan zu haben«, ist für viele Freiwillige wichtig. Bezeichnend für alle Antworten soll folgende Aussa- ge stehen: »Der Bundesfreiwilligen- p ■ ■Perspektiven nach Beendigung des Bundesfreiwilligendienst und beruf- licher Nutzen: 36% werden weiter in ihrer Einsatzstelle arbeiten und fünf Personen (20%) werden eine Ausbildung beginnen (Altenpflege, Gesundheits- und Krankenpflege, Heilerziehungspflege, Arbeitserzie- her). Für drei von ihnen wird es die Erstausbildung sein. 12% werden sich arbeitsuchend melden und 8% werden in einer angestellten Beschäf- tigung arbeiten. Teilnehmenden haben sich neue berufliche Perspektiven eröffnet« Die Antworten zum beruflichen Nutzen des Bundesfrei- willigendienst zeigen: Für einige hat sich ein neues Berufsfeld eröffnet, ebenso konnten sich einige Befragte neu orientieren oder berufliche Kon- ■ ■ ■ ■Identifikation und Anerkennung: Auf die Frage, ob sie die Ergebnisse ihrer Arbeit sehen, antworten 90% mit ja und 87% identifizieren sich mit ihrer Arbeit. Sogar 96% stimmen der Aus- sage, dass ihre Arbeit gesellschaftlich nützlich und sinnvoll sei, in hohem oder sehr hohem Maße zu. Gefragt danach, ob der Bundesfreiwilligen- dienst einen hohen gesellschaftlichen Bekanntheitsgrad habe, geben 44% an, dass dies zuträfe. Ebenfalls 44% geben an, dass sie sich als Teilneh- mende am Bundesfreiwilligendienst gesellschaftlich anerkannt fühlen, je- doch antworten auch 44%, dass dies ■ ■Identifikation und Anerkennung: Auf die Frage, ob sie die Ergebnisse ihrer Arbeit sehen, antworten 90% mit ja und 87% identifizieren sich mit ihrer Arbeit. Sogar 96% stimmen der Aus- sage, dass ihre Arbeit gesellschaftlich nützlich und sinnvoll sei, in hohem oder sehr hohem Maße zu. Gefragt danach, ob der Bundesfreiwilligen- dienst einen hohen gesellschaftlichen Bekanntheitsgrad habe, geben 44% an, dass dies zuträfe. Ebenfalls 44% geben an, dass sie sich als Teilneh- mende am Bundesfreiwilligendienst gesellschaftlich anerkannt fühlen, je- doch antworten auch 44%, dass dies Sowohl offen als auch reglementiert – der neue Bundesfreiwilligendienst Der Bundesfreiwilli- gendienst als Chance zum Wieder- einstieg in den Arbeitsmarkt kann für diese ersten älteren Freiwilligen damit überwiegend als gelungen be- zeichnet werden (vgl. Abb.). Die Erwartungen von Freiwilligen nach beruflichen Perspektiven gilt es ernst zu nehmen. Hier sehen wir den Bedarf, gemeinsam mit den Einsatzstel- len geeignete Konzepte zu entwickeln, die sowohl die Förderung der Freiwil- ligen hinsichtlich ihrer beruflichen Kompetenzen als auch die Schaffung von Ausbildungs- und Beschäftigungs- möglichkeiten nach dem Freiwilligen- dienst beinhaltet. Eine kritische Ausei- nandersetzung mit der Gratwanderung zwischen Beschäftigungsförderung und Freiwilligendienst muss diesen Prozess jedoch begleiten. n n Die Erhebung des Wohlfahrtswerkes für Baden-Württemberg kann erste Ten- denzen zu Motivation und Zufrieden- heit aufzeigen, jedoch keinen Anspruch auf Allgemeingültigkeit erheben. Doch zeichnet sich bei den über 27-jährigen Teilnehmenden am Bundesfreiwilligen- dienst ein klares Bild ab: Es handelt sich um Personen, die überwiegend vor ih- rem Bundesfreiwilligendienst ohne feste Anstellung waren und sich neben der Möglichkeit zum sozialen Engagement im Bundesfreiwilligendienst auch eine berufliche Perspektive erhoffen. Im An- schluss an den Bundesfreiwilligendienst haben sich für immerhin 56% berufli- che Perspektiven eröffnet. Wirtschafts- und Sozialpolitik Nomos Der demografische Wandel als europäische Herausforderung Lösungen durch Sozialen Dialog Demographic change as a European challenge Solutions by Social Dialogue Harald Kohler | Josef Schmid [Hrsg.] Wirtschafts- und Sozialpolitik | 8 Der demografi sche Wandel als europäische Herausforderung. Demographic change as a European challenge Lösungen durch Sozialen Dialog. Solutions by Social Dialogue Herausgegeben von Dr. Harald Kohler und Prof. Dr. Josef Schmid 2012, Band 8, 232 S., brosch., 22,– € ISBN 978-3-8329-6578-5 Schlussfolgerungen Das Engagement im Bundesfreiwilligen- dienst kann auch für ältere Erwachsene in sehr hohem Maße mit persönlichem Nutzen verbunden sein. Die Zufrie- denheit dieser Teilnehmergruppe gegen Ende des Jahres ist hoch bis sehr hoch. Sinkende Geburtenraten und eine steigende Lebensdauer führen zu Veränderungen in den Altersstrukturen der Erwerbsbevölkerung in den EU-Staaten. Gegen diese sich abzeichnende Problematik wird politisch gegengesteuert. Eine Form der politischen Intervention ist die Verlängerung der Lebensarbeitszeit, die auf die unmittelbaren Auswirkungen des demografi schen Wandels die Struktur der Erwerb- spersonen in den Unternehmen zielt und damit die betrieblichen wie auch die kollektiven Arbeitsbeziehungen durch die Verschiebung der Grenzlinien zwischen Berufstätigkeit und dem Ausscheiden aus dem Berufsleben tangiert. Der persönliche Gewinn nimmt einen hohen Stellenwert ein, in den Einsatzstellen können sich die meisten Freiwilligen in hohem Maße einbringen und fühlen sich dort sehr wertgeschätzt. Ebenso wie bei jungen Menschen kön- nen bei älteren Freiwilligen die Erfah- rungen zur Stabilisierung und Weiter- entwicklung der Persönlichkeit dienen. g Als Träger des Bundesfreiwilligen- dienstes richtet sich das primäre Ziel des Wohlfahrtswerkes für Baden- Württemberg auf die Sicherung und Weiterentwicklung einer hohen Qua- lität beim Freiwilligendienst für Ältere. Die Interviews der Teilnehmenden über 27 Jahre zeigen klar, dass die vom Zi- vildienst geprägten Bildungsinhalte der Bildungszentren geringe Attraktivität für die Erwachsenen haben, während sie noch mehr Angebote zur persönli- chen Weiterentwicklung, zum Umgang mit den erlebten Alltagssituationen und fachliche Hintergrundinfos begrüßen würden. Hierbei wäre eine fachliche Umstellung der Bildungszentren wün- schenswert. Im Band wird der demografi sche Wandel in drei Rubriken betrachtet: das Europäische Sozialmodell und die Situation von Älteren; der de- mografi sche Wandel, Rentensysteme und Reformen in EU-Staaten sowie Arbeitsmarktpolitik und Beschäftigungsfähigkeit (älterer Arbeit- nehmer). Die Beiträge sind sowohl wissenschaftlich als auch praxis- orientiert und weisen zum Teil eine komparative Perspektive auf, indem die Länder Deutschland, Frankreich, Großbritannien, Italien, Kroatien, Slowakei und Spanien in die Betrachtung einbezogen werden. Weitere Informationen: www.nomos-shop.de/13584 Nomos Sowohl offen als auch reglementiert – der neue Bundesfreiwilligendienst Sowohl offen als auch reglementiert – der neue Bundesfreiwilligendienst ch reglementiert – der neue Bundesfreiwilligendienst Nach der Aussetzung der allgemeinen Wehrpflicht und damit des Zivildienstes wurde zum 1.Juli 2012 der Bundesfreiwil- ligendienst etabliert. In seiner Zielsetzung soll er Menschen jeden Alters soziales En- gagement für das Allgemeinwohl ermög- lichen und lebenslanges Lernen fördern, so das Bundesfreiwilligendienstegesetz (BFDG) in Paragraf 1. Sozialversicherungspflicht vom Ehrenamt. Anders als bei den zivilgesellschaftlich ausgestalteten Diensten Freiwilliges So- ziales Jahr und Freiwilliges Ökologisches Jahr liegen Vertragsgestaltung sowie ar- beitsrechtlichen Befugnisse nicht bei den Trägern und Einsatzstellen, sondern beim Bundesministerium für Familie, Senioren, Frauen und Jugend. Dies wird bei Trägern, Wohlfahrtsverbänden und in politischen Gremien kritisch diskutiert. nehmende unter 27 Jahre im wesentlichen denen in den Jugendfreiwilligendiensten. Die Rahmenbedingungen für Menschen über 27 Jahre sind im Bundesfreiwilligen- dienstegesetz flexibler gestaltet. So kann der Bundesfreiwilligendienst in Teilzeit (mindestens jedoch 20,5 Stunden) absol- viert werden. nehmende unter 27 Jahre im wesentlichen denen in den Jugendfreiwilligendiensten. Die Rahmenbedingungen für Menschen über 27 Jahre sind im Bundesfreiwilligen- dienstegesetz flexibler gestaltet. So kann der Bundesfreiwilligendienst in Teilzeit (mindestens jedoch 20,5 Stunden) absol- viert werden. Sozialversicherungspflicht vom Ehrenamt. Anders als bei den zivilgesellschaftlich ausgestalteten Diensten Freiwilliges So- ziales Jahr und Freiwilliges Ökologisches Jahr liegen Vertragsgestaltung sowie ar- beitsrechtlichen Befugnisse nicht bei den Trägern und Einsatzstellen, sondern beim Bundesministerium für Familie, Senioren, Frauen und Jugend. Dies wird bei Trägern, Wohlfahrtsverbänden und in politischen Gremien kritisch diskutiert. Die Festlegung der Seminartage obliegt dem Träger des Freiwilligendienstes. Durch diese Regelung soll den vielfältigen Moti- ven und Lebenslagen älterer Menschen entsprochen werden. Analog zu den Ju- gendfreiwilligendiensten unterscheidet sich der Bundesfreiwilligendienst durch vertragliche Regelungen wie Dauer, Tä- tigkeitsumfang, Urlaub, Vergütung sowie Die Öffnung des neuen Freiwilligendiens- tes für alle Generationen war ein wichti- ger Schritt in der Engagementpolitik in Deutschland. Als Vorbild für den Bundes- freiwilligendienst dienten die erfolgreichen Jugendfreiwilligendienste Freiwilliges Sozi- ales Jahr und Freiwilliges Ökologisches Jahr. Daher entsprechen die Eckpunkte für Teil- Weitere Informationen zum Bundesfreiwil- ligendienst vermitteln zahlreichen Websei- ten im Internet, beispielsweise: www.wohl- fahrtswerk.de, www.derparitaetische.de, www.paritaet-bw.de und www.bafza.de. 6 | 2012 Blätter der Wohlfahrtspflege -6-213 24, 06:03:27. Fs ist nicht zulässig. 6 Blätter der Wohlfahrtspflege -6-213 24, 06:03:27. Fs ist nicht zulässig 215 THEMA takte knüpfen. Der Bundesfreiwilli- gendienst als Chance zum Wieder- einstieg in den Arbeitsmarkt kann für diese ersten älteren Freiwilligen damit überwiegend als gelungen be- zeichnet werden (vgl. Abb.). takte knüpfen. Nomos 6 | 2012 https://doi.org/10.5771/0340-8574-2012-6-213 eneriert durch IP '195.242.1.92', am 24.10.2024, 06:03:27. ellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. 6 | 2012 ätter der Wohlfahrtspflege https://doi.org/10.5771/0340-8574-2012-6-213 Generiert durch IP '195.242.1.92', am 24.10.2024, 06:03:27. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. 6 | 2012 Blätter der Wohlfahrtspflege https://do Generiert durch Das Erstellen und Weit 216
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Predictors of School Students’ Leisure-Time Physical Activity: An Extended Trans-Contextual Model Using Bayesian Path Analysis
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Permalink https://escholarship.org/uc/item/2b21f050 Journal PLOS ONE, 16(11) ISSN 1932-6203 Authors Polet, Juho Schneider, Jekaterina Hassandra, Mary et al. Publication Date 2021 DOI 10.1371/journal.pone.0258829 Peer reviewed UC Merced UC Merced Previously Published Works Title Title Predictors of school students’ leisure-time physical activity: An extended trans-contextual model using Bayesian path analysis Permalink https://escholarship.org/uc/item/2b21f050 Powered by the California Digital Library University of California eScholarship.org PLOS ONE RESEARCH ARTICLE Juho Polet1, Jekaterina SchneiderID1, Mary Hassandra2, Taru Lintunen1, Arto Laukkanen1, Nelli Hankonen3, Mirja HirvensaloID1, Tuija H. Tammelin4, Kyra Hamilton5, Martin S. HaggerID1,6* Juho Polet1, Jekaterina SchneiderID1, Mary Hassandra2, Taru Lintunen1, Arto Laukkanen1, Nelli Hankonen3, Mirja HirvensaloID1, Tuija H. Tammelin4, Kyra Hamilton5, Martin S. HaggerID1,6* Juho Polet1, Jekaterina SchneiderID1, Mary Hassandra2, Taru Lintunen1, Arto Laukkanen1, Nelli Hankonen3, Mirja HirvensaloID1, Tuija H. Tammelin4, Kyra Hamilton5, Martin S. HaggerID1,6* 1 Faculty of Sport and Health Sciences, University of Jyva¨skyla¨, Jyva¨skyla¨, Finland, 2 Department of Physical Education and Sport Science, University of Thessaly, Trikala, Greece, 3 Faculty of Social Sciences, University of Helsinki, Helsinki, Finland, 4 LIKES Research Centre for Physical Activity and Health, Jyva¨skyla¨, Finland, 5 School of Applied Psychology, Griffith University, Brisbane, Queensland, Australia, 6 Psychological Sciences, University of California, Merced, Merced, CA, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * mhagger@ucmerced.edu * mhagger@ucmerced.edu Abstract The present study aimed to examine effects of motivational and social cognition constructs on children’s leisure-time physical activity participation alongside constructs representing implicit processes using an extended trans-contextual model. The study adopted a correla- tional prospective design. Secondary-school students (N = 502) completed self-report mea- sures of perceived autonomy support from physical education (PE) teachers, autonomous motivation in PE and leisure-time contexts, and social cognition constructs (attitudes, sub- jective norms, perceived behavioral control), intentions, trait self-control, habits, and past behavior in a leisure-time physical activity context. Five weeks later, students (N = 298) self- reported their leisure-time physical activity participation. Bayesian path analyses supported two key premises of the model: perceived autonomy support was related to autonomous motivation in PE, and autonomous motivation in PE was related to autonomous motivation in leisure time. Indirect effects indicated that both forms of autonomous motivation were related to social cognition constructs and intentions. However, intention was not related to leisure-time physical activity participation, so model variables reflecting motivational pro- cesses did not account for substantive variance in physical activity participation. Self-con- trol, attitudes, and past behavior were direct predictors of intentions and leisure-time physical activity participation. There were indirect effects of autonomous motivation in lei- sure time on intentions and physical activity participation mediated by self-control. Specify- ing informative priors for key model relations using Bayesian analysis yielded greater precision for some model effects. Findings raise some questions on the predictive validity of constructs from the original trans-contextual model in the current sample, but highlight the value of extending the model to incorporate additional constructs representing non-con- scious processes. OPEN ACCESS Citation: Polet J, Schneider J, Hassandra M, Lintunen T, Laukkanen A, Hankonen N, et al. (2021) Predictors of school students’ leisure-time physical activity: An extended trans-contextual model using Bayesian path analysis. PLoS ONE 16(11): e0258829. https://doi.org/10.1371/ journal.pone.0258829 Introduction Research indicates that low levels of physical activity have deleterious effects on the health of young people [1]. However, children and adolescents in many nations are not sufficiently active to confer health benefits and reduce disease risk [2]. As a consequence, national and international health organizations have developed guidelines and national strategies aimed at promoting physical activity in this population [3]. Given the imperative for promoting physi- cal activity among young people, public health organizations and educators have sought to identify optimally effective strategies to enhance physical activity in this population, and the contexts in which these strategies will have maximal reach. Funding: This project was supported by the Finnish Ministry of Education and Culture under grant number OKM/62/626/2016) awarded to MSH and TL; and a Finland Distinguished Professor (FiDiPro) award from Business Finland under grant number 1801/31/2105 awarded to MSH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Physical education (PE) has been suggested as a potentially useful existing network that can be utilized to deliver interventions promoting physical activity both inside school, and, impor- tantly, outside school, in children and adolescents [4]. Researchers have, therefore, aimed to explore the potentially efficacious strategies delivered in PE to promote increased physical activity outside of school. Such an endeavor necessitates an understanding of the determinants of children and adolescents’ physical activity participation in a PE context and, importantly, whether those determinants relate to physical activity participation outside of school in stu- dents’ leisure time [5]. Understanding how factors linked to engagement in physical activity in school relate to physical activity performed in another context, leisure time, is critical to informing potential strategies delivered in PE that promote physical activity participation in children and adolescents in their leisure time. Such an approach is also consistent with one of the key pedagogical aims of PE to provide young people with the necessary skills to lead an active lifestyle [6]. Competing interests: The authors have declared that no competing interests exist. materials are available on the Open Science Framework (https://osf.io/z8axj/). materials are available on the Open Science Framework (https://osf.io/z8axj/). PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 Editor: Lambros Lazuras, Sheffield Hallam University, UNITED KINGDOM Editor: Lambros Lazuras, Sheffield Hallam University, UNITED KINGDOM Received: January 27, 2021 Accepted: October 6, 2021 Published: November 12, 2021 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0258829 Copyright: © 2021 Polet 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: Data, analysis scripts and output, study materials, and supplemental 1 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 PLOS ONE Motivation and behavior in PE and leisure time materials are available on the Open Science Framework (https://osf.io/z8axj/). The trans-contextual model The trans-contextual model [5] was developed to provide a theoretical explanation of the con- structs and associated processes that link engagement in physical activity in school PE with physical activity participation in leisure time. Specifically, the model draws on multiple theo- ries to outline relations between school students’ motivation toward physical activity in PE and their motives and beliefs toward, and actual participation in, physical activity in their lei- sure time. The model integrates core constructs and processes from self-determination theory [7], the hierarchical model of intrinsic and extrinsic motivation [8], and the theory of planned behavior [9]. Next, we outline the key premises of the model relating to the determinants of children and adolescents’ leisure-time physical activity and the processes involved. y y Based on self-determination theory, the first premise of the trans-contextual model focuses on the origins of school students’ motivation toward activities in PE, and how their motivation relates to their behavior in PE. The model predicts that the social environment in educational settings fostered by social agents and leaders (e.g., PE teachers) will determine the type or form of motivation students experience when performing tasks (e.g., physical activities in PE) and, importantly, their persistence on tasks. Central to the theory is the distinction between autono- mous and controlled forms of motivation [10]. Autonomous motivation is a form of motivation reflecting self-endorsed reasons for acting and autonomously motivated individuals tend to per- sist on tasks and exhibit behavioral persistence. Fostering autonomous motivation through the display of autonomy-supportive behaviors by social agents such as PE teachers will promote autonomous motivation toward physical activities performed in PE. Students who perceive their PE teacher as displaying behaviors that support their autonomy are more likely to report autonomous motivation toward activities in PE [11]. This prediction forms the basis of the first premise of the trans-contextual model: students’ perceived autonomy support from their teach- ers in PE will relate to their autonomous motivation toward physical activity in a PE context. PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 2 / 25 PLOS ONE Motivation and behavior in PE and leisure time A central prediction of the trans-contextual model is that there will be a trans-contextual relationship between students’ autonomous motivation toward physical activities across PE and leisure-time contexts. This prediction is based on Vallerand’s [8] hierarchical model, which describes the process by which motivation is transferred across contexts. The trans-contextual model Vallerand pro- posed that individuals experiencing autonomous motivation toward activities in one context will also cite autonomous motives toward similar behaviors in other related contexts. This forms the second premise of the trans-contextual model: school students’ level of autonomous motivation toward physical activities in a PE context will be related to their autonomous moti- vation toward physical activities performed outside of school in their leisure time. A final prediction of the trans-contextual model is that autonomous motivation toward physical activities in a leisure-time context will be related to students’ beliefs and intentions toward, and future participation in, leisure-time physical activity. If an individual has experi- enced a behavior as autonomously motivated, it is likely to be internalized and integrated into the individual’s repertoire of behaviors that satisfy their psychological need for autonomy. They are therefore more likely to actively seek out opportunities to engage in the behavior in future. To do so, they need to align their system of beliefs and intentions involved in the decision to perform that behavior in future. In the trans-contextual model, this process is represented by associations between forms of motivation from self-determination theory and the sets of beliefs from the theory of planned behavior, a leading social cognition theory [9]. Autonomously-moti- vated individuals are likely to form intentions to perform the behavior in future, and report favorable attitudes, subjective norms, and perceived behavioral control, the immediate belief- based determinants of intentions [12–14]. This forms the third premise of the trans-contextual model: students’ autonomous motivation toward physical activity in leisure time will be related to their future participation in leisure-time physical activity mediated by the belief-based social cognition determinants (attitudes, subjective norms, and perceived behavioral control) and their intentions toward participating in leisure-time physical activity in future. The key premises of the trans-contextual model have received substantial empirical support [5, 15–17]. Furthermore, a meta-analysis of studies applying the model in PE and leisure-time physical activity contexts provides converging evidence supporting model predictions across multiple studies [18]. PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 Extending the model While the trans-contextual model has displayed utility in identifying the determinants of lei- sure-time physical activity participation, it is not without limitations. One limitation is the exclusive focus on motivational and social cognition determinants of leisure-time physical activity participation without regard for the influence of implicit beliefs and motives that affect individuals’ behavior beyond their awareness [19]. In contrast, dual-process theories of moti- vation and social cognition propose that individuals’ behavior is determined by constructs that reflect conscious, reasoned decision making (e.g., autonomous motivation, social cognition constructs) but also by constructs that reflect implicit decision-making that impact behavior with little reasoned deliberation. Such constructs include implicit attitudes, habits, and indi- vidual difference constructs [20–23]. Non-conscious processes are adaptive because they lead to effective, efficient decision-making when reasoned deliberation is unnecessary or particu- larly costly [24]. Constructs reflecting these non-conscious processes are proposed to impact behavior directly without mediation by intentions, independent of the reasoned processes. These constructs may, therefore, account for the additional variance in leisure-time physical activity participation in the trans-contextual model and serve to provide a more comprehen- sive prediction of physical activity motivation and behavior in PE and leisure time. PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 3 / 25 PLOS ONE Motivation and behavior in PE and leisure time Prominent behavioral determinants that reflect non-conscious processes are habit, trait self-control, and affective attitudes. Focusing on habit, although research has historically con- sidered effects of past behavior as a viable proxy for habitual effects [25], recent research has focused on habit as a psychological construct [21, 26]. Theories of habit suggest that habitual behaviors are a function of behavioral experiences in the presence of consistent environmental, situational, or internal cues, and are often experienced as automatic, effortless, and highly accessible [26]. These components have been captured by self-report measures of habit, which are meta-cognitive measures which tap individuals’ experience of target behaviors as ‘unthink- ing’ and ‘automatic’ [21]. Such measures have been shown to predict behavior independent of intention-mediated measures [27–30], and are also associated with action accessibility and behavioral performance in stable contexts [31, 32]. While habits are expected to predict intentions and behavior, they may also be related to motives that form part of the ‘motivational sequence’ proposed in the trans-contextual model. Extending the model Individuals that hold autonomous motives toward behaviors like leisure-time physical activity, are more likely to persist with those behaviors, because the behavior fulfils psychological needs and leads to adaptive outcomes in the absence of external contingencies. This has been sup- ported in the research literature on self-determination theory with consistent links between autonomous motivation and physical activity [33]. Related to this, autonomously motivated individuals are also more likely to form habits for those behaviors because they are likely to perform those behaviors in a consistent fashion with high frequency and in stable contexts–the key conditions under which habits form [34, 35]. This has been supported by previous research on the autonomous motivation-habit relationship [36]. Based on these premises, it follows that autonomous motivation toward physical activity in PE and leisure-time contexts is expected to be indirectly related to intentions and physical activity behavior mediated by habits. This pro- posed effect will be independent of the ‘motivational sequence’ proposed in the model, consis- tent with the dual-process approaches outlined previously. Trait self-control reflects individual differences in capacities and self-regulatory skills that enable individuals to resist impulses and temptations, and engage in sustained, effortful behav- ior to attain long-term goal-directed outcomes [37]. Trait self-control has been consistently related to adaptive behaviors, including physical activity, across multiple contexts and popula- tions [38]. Research has also demonstrated that behavioral effects of trait self-control may be direct, independent of intentions [39]. Such effects reflect generalized tendencies to engage in adaptive behaviors without the need for deliberation or consideration. However, a case has also been made for effects of trait self-control on behavior mediated by intentions [39]. Such effects reflect situations where individuals have to actively engage in effortful deliberation to overcome a maladaptive behavior, or engage in a new behavior, that requires deliberation. Effects of trait self-control in motivational and social cognition theories may, therefore, relate to behavior via two pathways, directly, and indirectly through intentions. Research incorporat- ing trait self-control in the model has supported these dual effects, with direct and intention- mediated effects on physical activity participation [39]. While self-control has been identified as an independent determinant of intentions and behavior, there is also research that has linked self-control, and self-regulatory processes in general, with the forms of motivation implicated in the motivational sequence of the trans- contextual model. PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 The present study In the present study, we aimed to extend the trans-contextual model by including self-control, habits, and attitudes as additional direct determinants of physical activity intentions and behavior participation. This extension is expected to provide additional information on the determinants of leisure-time physical activity behavior, particularly effects of constructs repre- senting non-conscious processes not accounted for in the original model. We also applied the Bayesian analytic approach to test model effects using informative prior values for key model effects derived from a meta-analysis of the model [18]. We also capitalized on previous research on self-reported habit [36, 46] and trait self-control [38] to specify informative priors for these parameters in our test of extensions to the model. We expected to see a reduced level of uncertainty in the distributions of the parameters of the model specified for the current data when informative priors for key model parameters derived from the meta-analysis are speci- fied, reflected in narrowed credibility intervals, compared to the distributions when non-infor- mative priors are specified. Specifically, the study adopted survey methods and a five-week prospective design with measures of motivational and social cognition constructs, habit, trait-self-control, and past physical activity participation taken at an initial occasion, and self-reported leisure-time physi- cal activity participation taken at follow-up, five weeks later. This time period was selected to provide reasonable medium-term prediction, which exceeds typical time frames in model tests [47]. In addition to testing effects of the motivational and social cognition determinants from the trans-contextual model on students’ intentions toward, and actual participation in, leisure- time physical activity, we also tested direct effects of the constructs reflecting non-conscious processes as direct determinants of leisure-time physical activity participation: self-reported habit, trait self-control, and attitudes. Further, effects of autonomous motivation in both con- texts were expected to be indirectly related to physical activity intentions and behavior in lei- sure time mediated by both habit and self-control. Such effects represent processes by which self-determined motivation promotes behavioral enactment by promoting greater perceived self-regulatory capacity [42] and experience of the behavior as automatic [48]. Finally, we also expected model effects to hold in the presence of past behavior, and that there would be an indirect effect of past physical activity behavior on leisure-time physical activity mediated by habit. Such a relationship would illustrate the extent to which past behavior is a function of habit formation, consistent with previous theory and research [27, 48]. Extending the model For example, research has highlighted that individuals reporting self-deter- mined motives are less likely to be vulnerable to self-control failure and ego-depletion [40–42], and more likely to report intentions toward, and participate in, future behaviors, including physical activity [36, 39, 43]. These findings are consistent with the self-determination theory hypothesis that autonomous motivation is ‘energizing’ and individuals with autonomous motives toward behaviors are likely to report greater capacity to perform the behavior, and PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 4 / 25 PLOS ONE Motivation and behavior in PE and leisure time hence greater self-control [36]. This hypothesis also aligns with the premise of the trans-con- textual model that autonomous motives lead individuals to mobilize their resources to perform need-satisfying behaviors in future. Consistent with these proposals, it is reasonable to expect that autonomous motivation will be indirectly related to leisure-time physical activity inten- tions and behavior through self-control. There is also research demonstrating that attitudes may predict behavior directly, and such direct effects may also reflect non-conscious decision making [44, 45]. The original conceptu- alization of the theory of planned behavior specifies that attitudes represent cognitive reflec- tions on future participation in a target behavior and should relate to behavior mediated by intentions. However, direct effects of attitudes have been identified [45], and have been attrib- uted to the affective or emotional component of attitude. Research separating the cognitive and affective attitude components has demonstrated independent effects. The affective compo- nent is proposed to encompass visceral approach or avoidance responses learned through behavioral experience [44]. Direct effects of attitude on behavior may reflect a further sponta- neous, automatic process, which affects behavior independent of intentions. PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 Participants Participants were lower secondary school students (N = 502, 43.82% female; M age = 14.52, SD = 0.71) recruited from selected schools across Jyva¨skyla¨, Finland with support from the City Education Department. The University institutional review board and Education Depart- ment approved the study protocol prior to data collection. Informed consent was sought from the head teacher of each school, and, subsequently, PE teachers and eligible students’ parents or legal guardians via the schools’ online administration and communication software or via email or post. Opt-in consent was sought from the head teachers and PE teachers, while opt- out consent was sought from students’ parents and legal guardians. Qualified full-time PE teachers teaching regular PE lessons in lower secondary schools were eligible to participate in the study and were asked to select one of their PE classes to take part. Students in grades 7 to 9 (typical ages 13 to 15 years) in lower secondary schools were eligible to participate. Students with existing physical or mental health conditions that prevented participation in PE lessons, regular leisure-time physical activity, or completing surveys were excluded. Design and procedure Data for this study was collected as part of a larger randomized controlled trial (trial registra- tion: ISRCTN39374060; PETALS). The trial adopted a cluster-randomized, waitlist control, sin- gle-group intervention design with randomization by school. The trial comprised a teacher training phase and an implementation phase; full details of the intervention design and content have been published previously [50]. Secondary school PE teachers (N = 29) from 11 secondary schools and their students (N = 502) were invited to participate in the study. The pool of poten- tially eligible students numbered approximately 5000 across the 11 schools. Baseline data was collected prior to the teacher training phase and participants completed self-report question- naires assessing demographic, psychological, and behavioral measures. The baseline data collec- tion period was followed by the teacher training phase (12 hours over two weeks) and the implementation phase (one month), after which post-intervention data was collected compris- ing the same self-report questionnaires as at baseline. Follow-up data was further collected one, three, and six months post-intervention. The present study used measures of motivation and social cognition constructs and leisure-time physical activity participation taken at baseline and post-intervention leisure-time physical activity participation controlling intervention effects at baseline. Data for the present study were collected between September and December 2018. The present study The specific predictions PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 5 / 25 PLOS ONE Motivation and behavior in PE and leisure time of the proposed model, including direct and indirect effects in the proposed model are sum- marized in Table 1 and Fig 1. of the proposed model, including direct and indirect effects in the proposed model are sum- marized in Table 1 and Fig 1. Measures Measures of study constructs were adapted from instruments used in previous applications of the trans-contextual model. Measures included in the surveys were: perceived autonomy support from PE teachers [51]; autonomous motivation derived from items measuring self-determined forms of motivation from the perceived locus of causality scales for the PE and leisure-time phys- ical activity contexts [52]; intentions, attitudes, subjective norms, and perceived behavioral con- trol from the theory of planned behavior [53]; self-reported habit [21] and trait self-control [54]; and self-reported leisure-time physical activity participation [55]. All self-report measures were previously translated from English to Finnish using a back-translation process by two bilingual researchers. All measures used in the current research exhibited acceptable construct validity in PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 6 / 25 PLOS ONE Motivation and behavior in PE and leisure time Table 1. (Continued) Hypothesis (H) Independent variable Dependent variable Mediator(s) Informative priors β σ2 H35 Aut. mot. (LT) Intention Self-control – – H36 Aut. mot. (LT) Phys. act. Attitude – – Intention H37 Aut. mot. (LT) Phys. act. Sub. norm. – – Intention H38 Aut. mot. (LT) Phys. act. PBC – – Intention H39 Aut. mot. (LT) Habit Phys. act – – H40 Aut. mot. (LT) Self-control Phys. act – – H41 Past beh. Habit Phys. act – – Note. aPrior values derived from Hagger and Chatzisarantis [18]; bPrior values derived from Gardner et al. [49]; cPrior values derived from de Ridder et al. [38]; dPrior value derived from Kaushal et al. [36]. PAS = Perceived autonomy support; Aut. mot. = Autonomous motivation; PE = Physical education context; LT = Leisure- time context; PBC = Perceived behavioral control; Sub. norm = Subjective norm; Phys. act = Self-reported leisure-time physical activity participation; Past. beh. = Past leisure-time physical activity behavior. https://doi.org/10.1371/journal.pone.0258829.t001 Prior values derived from de Ridder et al. [38]; dPrior value derived from Kaushal et al. [36]. PAS = Perceived autonomy support; Aut. mot. = Autonomous motivation; PE = Physical education context; LT = Leisure- time context; PBC = Perceived behavioral control; Sub. norm = Subjective norm; Phys. act = Self-reported leisure-time physical activity participation; Past. beh. = Past leisure-time physical activity behavior. dPrior value derived from Kaushal et al. [36]. PAS = Perceived autonomy support; Aut. mot. = Autonomous motivation; PE = Physical education context; LT = Leisure- time context; PBC = Perceived behavioral control; Sub. norm = Subjective norm; Phys. act = Self-reported leisure-time physical activity participation; Past. beh. = Past leisure-time physical activity behavior. previous research applying confirmatory factor analyses. Items tapping each construct exhibited satisfactory factor loadings, average variance extracted, and composite reliability estimates [5, 46, 56]. Furthermore, we also conducted a pilot study in which the validity of the translated trans- contextual measures was tested in the target population [57]. This study used single-indicator latent variable model using omega reliability estimates to control for measurement error. Simula- tion research using full structural equation models and single-indicator models has revealed little difference in the parameter estimates in models either approach [58]. PLOS ONE Motivation and behavior in PE and leisure time PLOS ONE Table 1. Summary of hypothesized direct and indirect effects in the extended trans-contextual model. Hypothesis (H) Independent variable Dependent variable Mediator(s) Informative priors β σ2 Direct effects H1 PAS Aut. mot. (PE)a – 0.42 0.10 H2 Aut. mot. (PE) Aut. mot. (LT)a – 0.56 0.17 H3 PAS Aut. mot. (LT)a – 0.29 0.18 H4 Aut. mot. (LT) Attitudea – 0.60 0.12 H5 Aut. mot. (LT) Subjective norma – 0.26 0.26 H6 Aut. mot. (LT) PBCa – 0.51 0.19 H7 Aut. mot. (LT) Intentiona – 0.31 0.13 H8 Attitude Intentiona – 0.68 0.09 H9 Subjective norm Intentiona – 0.42 0.25 H10 PBC Intentiona – 0.63 0.28 H11 Habit Intention – – – H12 Self-control Intention – – – H13 Intention Phys. act.a – 0.60 0.20 H14 Attitude Phys. act.a – 0.43 0.21 H15 PBC Phys. act.a – 0.43 0.21 H16 Habit Phys. act.b – 0.43 0.13 H17 Self-control Phys. act.c – 0.26 0.09 H18 Aut. mot. (LT) Phys. act. – – – H19 Aut. mot. (LT) Habitd – 0.20 0.43 H20 Aut. mot. (LT) Self-control – – – H21 Past behavior Habit – – – H22 Past behavior Phys. act. – – – Indirect effects H23 PAS Aut. mot. (LT) Aut. mot. (PE) – – H24 Aut. mot. (PE) Intention Aut. mot. (LT) – – Attitude H25 Aut. mot. (PE) Intention Aut. mot. (LT) – – Sub. norm. H26 Aut. mot. (PE) Intention Aut. mot. (LT) – – PBC H27 Aut. mot. (PE) Phys. act. Aut. mot. (LT) – – Attitude Intention H28 Aut. mot. (PE) Phys. act. Aut. mot. (LT) – – Sub. norm. Intention H29 Aut. mot. (PE) Phys. act. Aut. mot. (LT) – – PBC Intention H30 Aut. mot. (PE) Phys. act. Aut. mot. (LT) – – PBC H31 Aut. mot. (LT) Intention Attitude – – H32 Aut. mot. (LT) Intention Sub. norm. – – H33 Aut. mot. (LT) Intention PBC – – H34 Aut. mot. (LT) Intention Habit – – (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 7 / 25 https://doi.org/10.1371/journal.pone.0258829.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 PLOS ONE These data provided sup- port for the use of these measures in this population, particularly the construct and predictive validity of the measures in the context of the trans-contextual model. Full details of the measures used are available in Appendix A in S1 File. Fig 1. Hypothesized relations among constructs of the extended trans-contextual model. Effects of past behavior on all other model constructs have been omitted for clarity. https://doi.org/10.1371/journal.pone.0258829.g001 Fig 1. Hypothesized relations among constructs of the extended trans-contextual model. Effects of past behavior on all other model constructs have been omitted for clarity. Fig 1. Hypothesized relations among constructs of the extended trans-contextual model. Effects of past behavior on all other model constructs have been omitted for clarity. https://doi.org/10.1371/journal.pone.0258829.g001 https://doi.org/10.1371/journal.pone.0258829.g001 8 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 PLOS ONE Motivation and behavior in PE and leisure time Perceived autonomy support. Students’ perceived autonomy support from their PE teacher was measured using items from the Perceived Autonomy Support Scale for Exercise Settings [51]. The scale comprised 13 items (e.g., “I feel that my PE teacher provides me with choices and options to . . .”) with responses provided on seven-point scales (1 = strongly dis- agree and 7 = strongly agree). Autonomous motivation. Autonomous motivation toward in-school and out-of-school physical activities was measured using items from the Perceived Locus of Causality Question- naire [52]. Two items measured identified regulation (e.g., “I do PE/physical activity because it is important to me to do well in PE/physical activity”) and two items measured intrinsic moti- vation (e.g., “I do PE/physical activity because it is fun”). Responses were provided on seven- point scales (1 = not true for me and 7 = very true for me). For each of the PE and leisure-time contexts, a composite autonomous motivation score was computed by averaging scores on the identified regulation and intrinsic motivation items. Theory of planned behavior constructs. Measures of students’ attitudes, subjective norms, perceived behavioral control, and intentions with respect to their future participation in leisure-time physical activity were developed according to guidelines [53]. Attitudes were measured on three items in response to a common stem: “Participating in physical activity in the next five weeks will be. . .”, with responses provided on seven-point scales (e.g., 1 = unen- joyable and 7 = enjoyable). PLOS ONE Subjective norms (e.g., “Most people who are important to me think I should do active sports and/or vigorous physical activities during my leisure time in the next five weeks”), perceived behavioral control (e.g., “I am confident I could do active sports and/or vigorous physical activities during my leisure time in the next five weeks”), and intentions (e.g., “I intend to do active sports and/or vigorous physical activities during my lei- sure time in the next five weeks”) were measured using two items each with responses pro- vided on seven-point scales (e.g., 1 = strongly disagree and 7 = strongly agree). Habit. Habit was measured using automaticity items from the Self-Reported Habit Index [21] which focuses on personal experience of the behavior as ‘automatic’ and excludes items related to past behavior. The scale comprised four items (e.g., “Physical activity is something I do without thinking”) with responses provided on seven-point scales (1 = completely disagree and 7 = completely agree). Habit. Habit was measured using automaticity items from the Self-Reported Habit Index [21] which focuses on personal experience of the behavior as ‘automatic’ and excludes items related to past behavior. The scale comprised four items (e.g., “Physical activity is something I do without thinking”) with responses provided on seven-point scales (1 = completely disagree and 7 = completely agree). Trait self-control. Students’ trait self-control was measured using the 10-item Self-Disci- pline Scale (e.g., “I tend to carry out my plans”) from the IPIP-HEXACO scales [59] with responses provided on four-point scales (1 = not like me at all and 4 = very much like me). Trait self-control. Students’ trait self-control was measured using the 10-item Self-Disci- pline Scale (e.g., “I tend to carry out my plans”) from the IPIP-HEXACO scales [59] with responses provided on four-point scales (1 = not like me at all and 4 = very much like me). Behavior. Past leisure-time physical activity at baseline and leisure-time physical activity participation at follow-up was measured using the short form of the International Physical Activity Questionnaire (IPAQ; 55). The IPAQ comprises four items recording the frequency (number of days) and duration (minutes per day) of engagement in moderate and vigorous physical activity, walking, and sitting over the past seven days. IPAQ data were processed according to established guidelines [60]. PLOS ONE The procedure gives an estimate of physical activity in MET-minutes per week with higher MET-minute values indicating higher level of physical activity engagement. Full details of calculations used to produce physical activity estimates are presented in Appendix B in S1 File. Internal consistency values for the short-form IPAQ exceed guideline cut-off values and scores on the scale demonstrate reasonable agreement with the long form in previous research [55]. Data analysis The proposed hypotheses of the extended trans-contextual model (see Table 1 and Fig 1) were tested using Bayesian path analytic models estimated with the Mplus 7.31 statistical software. PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 9 / 25 PLOS ONE Motivation and behavior in PE and leisure time We computed composite scales study measures by computing an average of the items for each construct. We controlled for the effects of the intervention in the model by including effects of a binary variable representing intervention group membership (1 = allocated autonomy sup- port intervention group, 0 = allocated to control group) on follow-up leisure-time physical activity participation. We controlled for effects of gender as a binary variable (0 = female, 1 = male) and age as a continuous variable by estimating effects of these variables on all other constructs in the model. Missing data for the model components were imputed using full information maximum likelihood (FIML) in Mplus. A Markov Chain Monte Carlo (MCMC) simulation process using Gibbs’ algorithm [61] was applied to estimate our Bayesian path models in the present study. The first 50% of the iterations (N = 100,000) was used as a burn-in phase with the remainder used to test the speci- fied model parameters. We established convergence of the parameter estimates in the Bayesian models according to the Gelman-Rubin [62] criterion based on a potential scale reduction (PSR) value of 1.01. Our analysis required estimation of two models. We first estimated a model that adopted the non-informative default priors available in Mplus to estimate model parameters. The defaults assumed a normal distribution with a mean of zero and a variance value essentially equivalent to infinity (1010). In our second model, we applied informative prior values taken from previous research [18, 36, 38, 49] to estimate model parameters. Spe- cifically, priors for relations among the trans-contextual model constructs were taken from Hagger and Chatzisarantis’ [18] meta-analysis. The prior value for the effect of self-reported habit on leisure-time physical activity participation was derived from Gardner et al.’s [46] meta-analysis of self-reported habit in physical activity. The prior value for the effect of trait self-control on leisure-time physical activity participation was taken from de Ridder et al.’s [38] meta-analysis of trait self-control in health behaviors. The prior value for the effect of autonomous motivation in leisure time on habit was taken from Kaushal et al.’s [36] integrated model test. PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 Path analyses Bayesian path analytic models using non-informative (Bayesian posterior predictive χ2 95% CI = [-26.087, 57.542]; PPP = 0.225; BIC = 9202.749) and informative priors for key model rela- tionships (Bayesian posterior predictive χ2 95% CI = [-18.211, 67.825]; prior PPP = 0.126; BIC = 9081.715) exhibited adequate goodness-of-fit with the data. In addition, the BIC indi- cated that the model that included informative priors exhibited better fit than the model with non-informative priors. Parameter estimates and 95% credibility intervals for the analysis with non-informative priors (Model 1) and the analysis including informative priors for key model relationships (Model 2) are presented in Table 2 and Fig 2. It is important to note that we did not conduct an a priori statistical power analysis for the current study as the main purpose of the trial was to test the effects of the intervention [50]. Nevertheless, we conducted a posteriori power analysis to check whether we had adequate power to test the current model. Our analysis was based on MacCallum, Browne, and Suga- wara’s [64] statistical power determination based on the RMSEA. The final sample size (N = 298), a null hypothesis RMSEA of 0 and a study hypothesis RMSEA of 0.064, alpha set at 0.05, and 16 degrees of freedom were inputs for the analysis, which was conducted using the Webpower package in R [65]. The resulting power estimate of 0.805 indicated that we had suf- ficient statistical power to detect effects of the stipulated size. Assuming the selected priors derived from meta-analyses were indicative of the population point estimates and distributions of effects among model constructs, we expected that Model 2 would yield greater precision in model parameter estimates compared to Model 1 [63]. This was evaluated by examining the extent to which the credibility intervals about each parameter estimate differed across the models (Table 2). Results indicated that the width of the credibility intervals was narrowed for a few of the effects in Model 2 relative to Model 1, but not by a sub- stantial margin in most cases. The adequate fit of both models suggests that including informa- tive priors in the analyses for key model relationships did not have a substantial bearing on the pattern of effects in the model. Preliminary analyses Of the participants who completed the initial survey (N = 502), 370 provided complete data at baseline and 298 participants (50% female; M age = 14.51, SD = 0.70) provided data for analysis after the second survey (19.46% attrition rate). Attrition was due to school absences. Percent- age of missing data for the psychological constructs over time was low (M = 1.4%; range = 0.0% to 4.0%) and Little’s MCAR test (χ2 = 63.882, df = 70, p = .683) suggested the data were missing at random. In addition, there were no significant differences between those who completed study measures at both time points and those who did not on gender distribu- tion, (χ2(1) = 0.403, p = .526), age (t(368) = -0.463, p = .643), or baseline physical activity (t (368) = -1.103, p = .271). We also conducted a one-way MANOVA to examine differences in psychological variables between those who completed study measures at both time points and those who did not, which was not significant (F(9, 360) = 0.775, p = .639; Wilks’ Λ = .981; par- tial η2 = .019). All constructs exhibited adequate internal consistency. Means, standard devia- tions, omega internal consistency coefficients and zero-order intercorrelations among study constructs are presented in Appendix C in S1 File. Data analysis Finally, we used non-informative prior values for remaining parameters without user-specified priors. We used multiple published criteria to establish the goodness-of-fit of our proposed models with the data across the iterations of the Bayesian analysis [61]. These criteria included the 95% confidence interval of the difference in the goodness-of-fit chi-square value across the observed and replicated models, as well as the posterior predictive p-value (PPP). The good- ness-of-fit chi-square value should have a positive upper limit, a negative lower limit, and be centered about zero, and the PPP value should be greater than .05 and approach .50, for well- fitting models. In addition, we also report the Bayesian Information Criterion (BIC), a relative fit index which allows researchers to identify the model with the greatest parsimony relative to fit as it includes a term that ‘penalizes’ overfitting. Cut-off values of .95 or greater for the CFI and TLI, and .06 for the RMSEA, have been proposed as indicative of good model fit. Further- more, the 90% confidence intervals of each index should ideally exceed the cut-off values. With respect to model parameters, a point estimate and 95% posterior credibility interval was produced for each parameter in the models. A non-zero credibility interval for a parame- ter provides confirmatory support for the proposed effect in the model. Point estimates and credibility intervals were also produced for the proposed indirect effects in the models [63]. In addition, we also expected that specification of informative prior values in the second model would lead to increased precision in the point estimates. To demonstrate changes in precision, we followed Yuan and McKinnon’s method which evaluates the extent to which the posterior credibility intervals about each parameter estimate is narrowed with the introduction of infor- mative priors. If the width of the confidence intervals is reduced, we have sharp confirmation that inclusion of the prior values alongside the current data leads to increased precision in esti- mates. Data, syntax, and output files for our analyses are available online: https://osf.io/z8axj. PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 10 / 25 PLOS ONE Motivation and behavior in PE and leisure time PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 PLOS ONE PLOS ONE PLOS ONE Table 2. Parameter estimates (β) with 95% credibility intervals for hypothesized effects from the Bayesian path analyses of the extended trans-contextual model for leisure-time physical activity. H Independent variable Dependent variable Mediator(s) Model 1 Model 2 %diff β 95% CrI β 95% CrI LL UL LL UL Direct effects H1 PAS Aut. mot. (PE)† – 2.403 1.485 3.018 1.222 0.625 1.634 -34.18 H2 Aut. mot. (PE) Aut. mot. (LT)† – 0.449 0.364 0.536 0.452 0.366 0.537 -0.58 H3 PAS Aut. mot. (LT)† – 0.140 0.012 0.264 0.138 0.011 0.265 0.79 H4 Aut. mot. (LT) Attitude† – 0.433 0.343 0.523 0.437 0.348 0.526 -1.11 H5 Aut. mot. (LT) Sub. norm† – 0.164 0.007 0.321 0.166 0.012 0.322 -1.27 H6 Aut. mot. (LT) PBC† – 0.221 0.119 0.322 0.225 0.125 0.326 -0.99 H7 Aut. mot. (LT) Intention† – 0.410 0.304 0.516 0.403 0.298 0.507 -1.42 H8 Attitude Intention† – 0.166 0.057 0.275 0.182 0.076 0.289 -2.29 H9 Sub. norm Intention† – 0.118 0.058 0.178 0.118 0.059 0.178 -0.83 H10 PBC Intention† – 0.325 0.227 0.422 0.324 0.228 0.420 -1.54 H11 Habit Intention – 0.057 -0.029 0.144 0.058 -0.028 0.144 -0.58 H12 Self-control Intention – 0.217 0.032 0.400 0.214 0.030 0.399 0.27 H13 Intention Phys. act.† – -0.001 -0.062 0.060 0.000 -0.060 0.061 -0.82 H14 Attitude Phys. act.† – 0.092 0.032 0.151 0.093 0.034 0.152 -0.84 H15 PBC Phys. act.† – -0.029 -0.085 0.026 -0.029 -0.084 0.026 -0.90 H16 Habit Phys. act.† – -0.005 -0.051 0.040 -0.004 -0.049 0.042 0.00 H17 Self-control Phys. act.† – 0.104 0.003 0.206 0.107 0.007 0.207 -1.48 H18 Aut. mot. (LT) Phys. act. – -0.017 -0.079 0.045 -0.019 -0.080 0.043 -0.81 H19 Aut. mot. (LT) Habit† – 0.121 -1.592 2.637 0.532 0.392 0.671 -93.40 H20 Aut. mot. (LT) Self-control – 0.108 0.046 0.171 0.138 0.071 0.207 8.80 H21 Past behavior Habit – 1.521 -3.089 0.658 0.799 0.397 1.206 -78.41 H22 Past beh. (LT) Phys. act. – 0.675 0.519 0.829 0.672 0.518 0.827 -0.32 Indirect effects H23 PAS Aut. mot. (LT) Aut. mot. (PE) 1.059 0.643 1.449 0.547 0.276 0.777 -37.84 H24 Aut. mot. (PE) Intention Aut. mot. (LT) 0.032 0.011 0.057 0.035 0.014 0.061 2.17 Attitude H25 Aut. mot. (PE) Intention Aut. mot. (LT) 0.008 0.000 0.020 0.008 0.001 0.020 -5.00 Sub. norm. H26 Aut. mot. (PE) Intention Aut. mot. (LT) 0.032 0.015 0.053 0.032 0.016 0.054 0.00 PBC H27 Aut. mot. (PE) Phys. Path analyses Nevertheless, given that some parameters were more precise, particularly the direct effects of perceived autonomy support on autonomous motivation in PE and autonomous motivation in PE on autonomous motivation in leisure time, and the indirect effects of autonomous motivation in PE on autonomous motivation in leisure time and PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 11 / 25 Motivation and behavior in PE and leisure time PLOS ONE act Habit 0.000 -0.071 0.056 -0.002 -0.027 0.023 -60.63 H40 Aut. mot. (LT) Phys. act Self-control 0.011 0.000 0.026 0.014 0.001 0.033 23.08 H41 Past beh. Phys. act Habit -0.002 -0.127 0.108 -0.003 -0.042 0.035 -67.23 Note. †Parameters with informative priors. Model 1 = Bayesian path model with non-informative priors; Model 2 = Bayesian path model including informative priors; H = Hypothesis; β = Parameter estimate; 95% CrI = 95% credibility interval of path coefficient; %diff = Percent difference in 95% credibility interval of path coefficients of path analysis including informative priors for specified model relationships compared to analysis using non-informative priors (negative numbers indicate a narrowing of credibility intervals when using informative priors); PAS = Perceived autonomy support; Aut. mot. = Autonomous motivation; PE = Physical education contexts; LT = Leisure-time context; PBC = Perceived behavioral control; Sub. norm = Subjective norm; Phys. act = Self-reported leisure-time physical activity participation; Past. Beh. = Past leisure-time physical activity behavior. p < .05  https://doi.org/10.1371/journal.pone.0258829.t002 intention, we elected to evaluate our hypothesis tests based on the model using informative priors (Model 2). If the posterior distribution for each effect, represented by the credibility intervals about the coefficients, did not include zero, then the effect was considered supported and the posterior probability of a non-zero value for the coefficient exceeds 0.975. In terms of direct effects, we found a non-zero effect of perceived autonomy support in PE on autonomous motivation in PE (H1; β = 1.222, 95% CI [0.6425, 1.634], p < .001). There was also a non-zero trans-contextual effect of autonomous motivation in PE on autonomous moti- vation in leisure time (H2; β = 0.452, 95% CI [0.366, 0.537], p < .001), and perceived autonomy support in PE also had a non-zero effect on autonomous motivation in leisure time (H3; β = 0.138, 95% CI [0.011, 0.265], p < .001). We also found non-zero effects of autonomous motiva- tion in leisure time on attitudes (H4; β = 0.437, 95% CI [0.348, 0.526], p < .001), subjective norms (H5; β = 0.166, 95% CI [0.012, 0.322], p = .017), and perceived behavioral control (H6; β = 0.225, 95% CI [0.125, 0.326], p < .001). PLOS ONE Table 2. (Continued) H Independent variable Dependent variable Mediator(s) Model 1 Model 2 %diff β 95% CrI β 95% CrI LL UL LL UL H33 Aut. mot. (LT) Intention PBC 0.071 0.035 0.115 0.072 0.037 0.115 -2.50 H34 Aut. mot. (LT) Intention Habit 0.003 -0.117 0.240 0.030 -0.015 0.079 -73.67 H35 Aut. mot. (LT) Intention Self-control 0.022 0.003 0.052 0.028 0.004 0.064 22.45 H36 Aut. mot. (LT) Phys. act. Attitude 0.000 -0.005 0.005 0.000 -0.005 0.005 0.00 Intention H37 Aut. mot. (LT) Phys. act. Sub. norm. 0.000 -0.001 0.001 0.000 -0.001 0.001 0.00 Intention H38 Aut. mot. (LT) Phys. act. PBC 0.000 -0.005 0.005 0.000 -0.005 0.005 0.00 Intention H39 Aut. mot. (LT) Phys. act Habit 0.000 -0.071 0.056 -0.002 -0.027 0.023 -60.63 H40 Aut. mot. (LT) Phys. act Self-control 0.011 0.000 0.026 0.014 0.001 0.033 23.08 H41 Past beh. Phys. act Habit -0.002 -0.127 0.108 -0.003 -0.042 0.035 -67.23 Note. †Parameters with informative priors. Model 1 = Bayesian path model with non-informative priors; Model 2 = Bayesian path model including informative priors; H = Hypothesis; β = Parameter estimate; 95% CrI = 95% credibility interval of path coefficient; %diff = Percent difference in 95% credibility interval of path coefficients of path analysis including informative priors for specified model relationships compared to analysis using non-informative priors (negative numbers indicate a narrowing of credibility intervals when using informative priors); PAS = Perceived autonomy support; Aut. mot. = Autonomous motivation; PE = Physical education contexts; LT = Leisure-time context; PBC = Perceived behavioral control; Sub. norm = Subjective norm; Phys. act = Self-reported leisure-time physical activity participation; Past. Beh. = Past leisure-time physical activity behavior. p < .05 p < .01 Table 2. (Continued) H Independent variable Dependent variable Mediator(s) Model 1 Model 2 %diff β 95% CrI β 95% CrI LL UL LL UL H33 Aut. mot. (LT) Intention PBC 0.071 0.035 0.115 0.072 0.037 0.115 -2.50 H34 Aut. mot. (LT) Intention Habit 0.003 -0.117 0.240 0.030 -0.015 0.079 -73.67 H35 Aut. mot. (LT) Intention Self-control 0.022 0.003 0.052 0.028 0.004 0.064 22.45 H36 Aut. mot. (LT) Phys. act. Attitude 0.000 -0.005 0.005 0.000 -0.005 0.005 0.00 Intention H37 Aut. mot. (LT) Phys. act. Sub. norm. 0.000 -0.001 0.001 0.000 -0.001 0.001 0.00 Intention H38 Aut. mot. (LT) Phys. act. PBC 0.000 -0.005 0.005 0.000 -0.005 0.005 0.00 Intention H39 Aut. mot. (LT) Phys. PLOS ONE act. Aut. mot. (LT) 0.000 -0.002 0.002 0.000 -0.002 0.002 0.00 Attitude Intention H28 Aut. mot. (PE) Phys. act. Aut. mot. (LT) 0.000 -0.001 0.001 0.000 -0.001 0.001 0.00 Sub. norm. Intention H29 Aut. mot. (PE) Phys. act. Aut. mot. (LT) 0.000 -0.002 0.002 0.000 -0.002 0.002 0.00 PBC Intention H30 Aut. mot. (PE) Phys. act. Aut. mot. (LT) -0.003 -0.009 0.003 -0.003 -0.009 0.003 0.00 PBC H31 Aut. mot. (LT) Intention Attitude 0.071 0.024 0.123 0.079 0.032 0.131 0.00 H32 Aut. mot. (LT) Intention Sub. norm. 0.018 0.001 0.043 0.019 0.001 0.044 2.38 (Continued) credibility intervals for hypothesized effects from the Bayesian path analyses of the extended trans-contextual model for PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 12 / 25 Motivation and behavior in PE and leisure time PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 Note. †Parameters with informative priors. Model 1 = Bayesian path model with non-informative priors; Model 2 = Bayesian path model including informative priors; H = Hypothesis; β = Parameter estimate; 95% CrI = 95% credibility interval of path coefficient; %diff = Percent difference in 95% credibility interval of path coefficients of path analysis including informative priors for specified model relationships compared to analysis using non-informative priors (negative numbers indicate a narrowing of credibility intervals when using informative priors); PAS = Perceived autonomy support; Aut. mot. = Autonomous motivation; PE = Physical education contexts; LT = Leisure-time context; PBC = Perceived behavioral control; Sub. norm = Subjective norm; Phys. act = Self-reported leisure-time physical activity participation; Past. Beh. = Past leisure-time physical activity behavior.  PLOS ONE PE = Physical education context; LT = Leisure time context. Model parameters omitted for clarity: past physical activity behavior!perceived autonomy support, β = 0.390, 95% CI [0.116, 0.664], p = .003; past physical activity behavior!autonomous motivation (PE), β = 0.368, 95% CI [-0.076, 0.799], p = .051; past physical activity behavior!autonomous motivation (LT), β = 1.382, 95% CI [1.109, 1.654], p < .001; past physical activity behavior!attitude, β = 0.194, 95% CI [-0.109, 0.495], p = .106; past physical activity behavior!subjective norms, β = 0.337, 95% CI [-0.193, 0.867], p = .103; past physical activity behavior!perceived behavioral control, β = 0.593, 95% CI [0.249, 0.938], p < .001; past physical activity behavior!intention, β = 0.293, 95% CI [0.014, 0.574], p = .019; past physical activity behavior!habit, β = 0.799, 95% CI [0.397, 1.206], p < .001; past physical activity behavior!self-control, β = 0.153, 95% CI [-0.041, 0.348], p = .061; past physical activity behavior!physical activity behavior, β = 0.672, 95% CI [0.518, 0.827], p = .001. https://doi.org/10.1371/journal.pone.0258829.g002 https://doi.org/10.1371/journal.pone.0258829.g002 control (H15), habit (H16), and autonomous motivation in leisure time (H18) were no different from zero. In addition, there were non-zero effects of autonomous motivation in leisure time on habit (H19; β = 0.532, 95% CI [0.392, 0.671], p < .001) and trait self-control (H20; β = 0.138, 95% CI [0.071, 0.207], p < .001). Finally, we found a non-zero effect of past behavior on habit (H21; β = 0.799, 95% CI [0.397, 1.206], p < .001). All effects were small-to-medium in size. Focusing on the indirect effects, we found non-zero indirect effects of perceived autonomy support on autonomous motivation in leisure time mediated by autonomous motivation in PE (H23; β = 0.547, 95% CI [0.276, 0.777], p < .001). There were also non-zero effects of autono- mous motivation in PE on intentions mediated by autonomous motivation in leisure time and attitude (H24; β = 0.035, 95% CI [0.014, 0.061], p < .001), subjective norms (H25; β = 0.008, 95% CI [0.001, 0.020], p = .017), and perceived behavioral control (H26; β = 0.032, 95% CI [0.016, 0.054], p < .001). PLOS ONE There were also non-zero effects of attitudes (H8; β = 0.182, 95% CI [0.076, 0.289], p < .001), subjective norms (H9; β = 0.118, 95% CI [0.059, 0.178], p < .001), and perceived behavioral control (H10; β = 0.324, 95% CI [0.228, 0.420], p < .001) on intentions. Moreover, there were non-zero effects of autonomous motivation in leisure time (H7; β = 0.403, 95% CI [0.298, 0.507], p < .001) and trait self-control (H12; β = 0.214, 95% CI [0.030, 0.399], p = .012) on intentions, but the effect of habit on intention (H11) was no dif- ferent from zero. We found non-zero effects of attitude (H14; β = 0.093, 95% CI [0.034, 0.152], p = .001), trait self-control (H17; β = 0.107, 95% CI [0.007, 0.207], p = .018), and past physical activity participation (H22; β = 0.672, 95% CI [0.518, 0.827], p < .001) on leisure-time physical activity participation at follow-up, while effects of intention (H13), perceived behavioral PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 13 / 25 PLOS ONE Motivation and behavior in PE and leisure time Fig 2. Parameter estimates from the Bayesian path analysis of the extended trans-contextual model for leisure- time physical activity including informative priors. PE = Physical education context; LT = Leisure time context. Model parameters omitted for clarity: past physical activity behavior!perceived autonomy support, β = 0.390, 95% CI [0.116, 0.664], p = .003; past physical activity behavior!autonomous motivation (PE), β = 0.368, 95% CI [-0.076, 0.799], p = .051; past physical activity behavior!autonomous motivation (LT), β = 1.382, 95% CI [1.109, 1.654], p < .001; past physical activity behavior!attitude, β = 0.194, 95% CI [-0.109, 0.495], p = .106; past physical activity behavior!subjective norms, β = 0.337, 95% CI [-0.193, 0.867], p = .103; past physical activity behavior!perceived behavioral control, β = 0.593, 95% CI [0.249, 0.938], p < .001; past physical activity behavior!intention, β = 0.293, 95% CI [0.014, 0.574], p = .019; past physical activity behavior!habit, β = 0.799, 95% CI [0.397, 1.206], p < .001; past physical activity behavior!self-control, β = 0.153, 95% CI [-0.041, 0.348], p = .061; past physical activity behavior!physical activity behavior, β = 0.672, 95% CI [0.518, 0.827], p = .001. https://doi org/10 1371/journal pone 0258829 g002 Fig 2. Parameter estimates from the Bayesian path analysis of the extended trans-contextual model for leisure- time physical activity including informative priors. PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 Discussion The purpose of the current research was to examine the determinants of lower secondary school students’ leisure-time physical activity participation using an extended version of the trans-contextual model [5]. Specifically, the model was augmented to include two constructs that reflect non-conscious processes as predictors of leisure-time physical activity participa- tion: self-reported habit [21] and trait self-control [37]. In addition, attitude was also set as a direct predictor of leisure-time physical activity participation, representing a further non-con- scious process [44, 45]. Hypothesized relations among the extended trans-contextual model constructs were tested using a two-wave prospective survey design in a convenience sample of lower secondary school students. Data were analyzed using two Bayesian path analytic models: one specifying non-informative priors and one in which informed priors for key relations in the model derived from previous research were specified. Results indicated adequate fit of both models with the data. Perceived autonomy support predicted autonomous motivation in PE and leisure-time contexts, autonomous motivation in PE predicted autonomous motiva- tion in a leisure-time context, and autonomous motivation in a leisure-time context predicted social cognition constructs (attitudes, perceived behavioral control) and intentions toward lei- sure-time physical activity participation. There were also indirect effects of perceived auton- omy support on autonomous motivation in leisure time mediated by autonomous motivation in PE, and of autonomous motivation in PE and leisure time on intentions through the social cognition constructs. In contrast, the hypothesized indirect effects of autonomous motivation in both contexts on leisure-time physical activity participation were not supported, primarily due to effects of intention and perceived behavioral control on behavior that were no different from zero. However, attitudes and trait self-control predicted both intentions and behavior. Furthermore, there were indirect effects of autonomous motivation in leisure-time on inten- tions and physical activity participation mediated by self-control, but not habit. The Bayesian analytic approach demonstrated that the model was tenable with the model incorporating informative prior knowledge demonstrating better fit with the data and more precision for some of the parameter estimates. Overall, current results supported hypotheses relating to the first two premises of the trans- contextual model, that is, the premises specifying effects of perceived autonomy support on autonomous motivation in PE, and the trans-contextual effects of autonomous motivation across PE and leisure time context [5, 18]. PLOS ONE Similarly, there were also non-zero indirect effects of autonomous motivation in leisure time on intentions mediated by attitude (H31; β = 0.079, 95% CI [0.032, 0.131], p < .001), subjective norms (H32; β = 0.019, 95% CI [0.001, 0.044], p = .017), and per- ceived behavioral control (H33; β = 0.072, 95% CI [0.037, 0.115], p < .001). There were also non-zero indirect effects of autonomous motivation in leisure-time on intention (H35; β = 0.028, 95% CI [0.004, 0.064], p = .012) and physical activity participation (H40; β = 0.014, 95% CI [0.001, 0.033], p = .018) mediated by trait self-control, but indirect effects mediated by habit were no different from zero (H34, H39). However, indirect effects of autonomous motivation in the PE (H27−H30) and leisure-time (H36−H38) contexts through the social cognition constructs on leisure-time physical activity participation were no different from zero, primarily because the intention-behavior relationship was also no different from zero. The indirect effect of past behavior on physical activity participation mediated by habit (H41) was also no different from zero. Finally, effects of the intervention on leisure-time physical activity participation, and effects of age on model constructs, were no different from zero. However, we found non-zero PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 14 / 25 PLOS ONE Motivation and behavior in PE and leisure time effects of gender on autonomous motivation in PE (β = -.401, 95% CI [-0.717, -0.099], p = .004), with girls experiencing higher levels than boys, autonomous motivation in leisure time (β = 0.256, 95% CI [0.055, 0.458], p = .007), and attitudes (β = 0.210, 95% CI [0.016, 0.402], p = .017). PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 Discussion One possibility is that the current research was conducted in the context of an intervention. However, correlations of the intervention with key model constructs, particularly intentions and follow-up physical activity participation were no different from zero. In fact, the only effects of the intervention on variables from the current study were on perceived autonomy support and attitudes at baseline, and these effects were opposite to the predicted direction and were taken prior to the intervention. Furthermore, we also controlled for intervention effects in the current model, so reported effects were independent of intervention effects. This leaves the possibility of other extraneous constructs attenuating the intention-physical activity participation relationship in the current study. It is possible, for example, that students’ inten- tions were particularly unstable or inconsistent with their subsequent behavior, given research that has confirmed these intention properties moderate these relations [69]. However, this pos- sibility remains speculative as we have no data on intention stability or consistency, nor do we have any contextual or demographic information that would explain such inconsistencies. A third explanation may be that participation in leisure-time physical activity in the current sample of school students was largely determined by constructs that reflect individual-level non-conscious processes, that is, constructs that impact behavior directly independent of intentions. That the only determinants of leisure-time physical activity participation in the current study were past physical activity participation, attitude, and trait self-control is consis- tent with this interpretation. Focusing first on the direct effect of trait self-control on behavior, this construct is proposed to reflect non-conscious processes insofar as those endorsing it are purported to exhibit adaptive self-regulatory skills that assist in pursuing goal-directed behav- iors and help resist temptations to engage in alternative behaviors that may derail pursuit of the behavior [39, 43]. On the surface, such an effect implies that individuals applying such skills must engage in active, effortful decision making to ensure focus on the target behavior and manage distractions, a conscious process. This may be the case for behaviors with which the individual has little experience. However, where the individual has substantive experience and has engaged in such active deliberation over the management of the behavior and applica- tion of their skills, they are likely to have well-learned behavioral scripts or schemas stored in memory to manage distractions and maintain behavioral engagement, obviating the need for such conscious deliberation. Discussion It also provided support for the effects of autono- mous motivation in leisure time on intentions to participate in leisure-time physical activity mediated by the attitude, subjective norm, and perceived behavioral control constructs from the theory of planned behavior. However, there was scant evidence for the third premise, due to an intention-physical activity participation relationship that was no different from zero. These findings suggest that, in the current sample, the trans-contextual model is effective in identifying motivational and social cognition determinants of secondary school students’ intentions to participate in leisure-time physical activity, and the processes involved, but not their actual participation. We propose four possible interpretations of the current findings. First, results may raise questions on the effectiveness of the trans-contextual model in identify- ing the determinants of leisure-time physical activity participation. There have been occasions where studies on the motivational and social cognition constructs in multi-theory, integrated PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 15 / 25 PLOS ONE Motivation and behavior in PE and leisure time models have failed to yield non-zero effects for the primary predicted determinants of behavior [17, 20, 66]. Nevertheless, such occasions are rare, and are contrary to the substantive body of meta-analytic evidence applying the trans-contextual model [18] and other integrated models that have supported effects more broadly and in multiple populations and contexts [67, 68]. Therefore, it may be premature to use the current data as a basis for rejecting the trans-contex- tual model. A second interpretation may be that some of the hypothesized effects in the model were attenuated due to contextual factors that affected relations among constructs, particularly the intention-behavior relationship. It is important to note that the intention-behavior relation- ship is integral to the model as it is a key link in the ‘motivational sequence’ by which perceived autonomy support in PE and autonomous motivation in both contexts relates to leisure-time physical activity participation. An intention-behavior relationship that is no different from zero, therefore, suggests that the indirect effect of autonomy support and autonomous motiva- tion in both contexts on behavior, a key premise of the model, is not supported. This should not, however, invalidate the model. Rather it may signpost potential contextual or environ- mental factors that lead to effects in the model are attenuated. For example, research has shown that extraneous constructs moderate the intention-behavior relationship [69]. PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 Discussion This is consistent with research suggesting that individuals with good trait self-control are highly effective in managing their environment so as not to be encumbered by distractions and to ensure that the cues to their desired behavior are PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 16 / 25 PLOS ONE Motivation and behavior in PE and leisure time omnipresent [38]. While this mechanistic explanation is speculative, it may explain the direct effect of trait self-control on behavior in the current model and provides justification to explore the role of this constructs within the trans-contextual model. A fourth and final interpretation is that social environmental factors may have contributed to the weak intention-behavior relationship. The high availability of inactive highly-appealing pastimes available to young people (e.g., computer games) and social norms within families and peer groups to engage in inactive pastimes may have contributed to failure of students to engage in physical activity even if they had autonomous motives and intentions to do so. This is consistent with the current data in which students’ average intentions to engage in physical activity in their leisure time was above the scale mid-point (M = 5.651, SD = 1.282). The effects of peer norms are especially strong in this age group, so young people with intentions to be active may find that they are superseded by their need to conform. These premises are consis- tent with ecological models that stress environmental influences [e.g., 70], and research sug- gesting that such influences are important predictors of behavior beyond social cognition determinants [e.g., 71]. Analogously, if a child has low or no intention to participate in physical activity, they may still be compelled to spontaneously do so if their peer groups decides to have a ‘kick about’ with a football in their local park. The current study did not measure environ- mental influences, so such determinants cannot be empirically verified from the current data and should be considered speculative. Nevertheless, it points to the potential importance of incorporating constructs that reflect these environmental determinants within integrated models such as the trans-contextual model. Turning to the direct effect of attitude on leisure-time physical activity participation, cur- rent findings are consistent with previous research that has found a direct effect of attitude components on behavior in multiple health contexts [44, 45]. PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 Discussion Such effects might represent affective motives to engage in a behavior learned through positive or negative experiences that coincide with the behavior [72]. As a consequence, the anticipation of rewarding affective responses may be reasons why children and adolescents engage in physical activities outside of school without the need for reasoned decision making. Such an effect has not been identified in previous research adopting the trans-contextual model, but has been consistently identified in research applying the theory of planned behavior in health behavior contexts, including physical activity [45, 72]. With respect to the direct effect of past physical activity behavior, current findings corrobo- rate previous research reporting effects of past behavior on subsequent behavior in social cog- nition theories [e.g., 14, 25, 47, 73]. This research demonstrates that past behavior accounts for substantive variance in behavior and often attenuates effects of other constructs. The inclusion of past behavior in social cognition models is important as it provides an indication of the suf- ficiency of the theory [9, 73]. The absence of effects of theory constructs other than past behav- ior provides an indication that the theory may be inadequate as a means to explain behavior beyond the stability of the behavior itself. Although in the case of the current research, the exclusion of past behavior did not restore effects of other constructs such as intention on behavior. So, what might the large-sized effect of past behavior represent? Researchers have suggested that past behavior may model effects of unmeasured constructs in tests of these theories [9, 25]. Given social cognition theories incorporate constructs that reflect reasoned, deliberative processes, past behavior effects may model effects of constructs representing non-conscious processes such as habits and implicit beliefs. The substantive effect of past physical activity behavior on leisure-time physical activity participation in the current study suggests that lower secondary school students’ physical activity in their leisure time may be a function of these kinds of constructs. Current findings suggest, however, that habit may not be among these PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 17 / 25 PLOS ONE Motivation and behavior in PE and leisure time determinants, given that the independent effect of self-reported habit on leisure-time physical activity participation was no different from zero, and habit did not mediate effects of past behavior on physical activity participation. Discussion Although it must be stressed that the current mea- sure of habit focused exclusively on automaticity, one aspect of habit, and may not have suffi- ciently captured all habitual influences [e.g., 74]. The current study did not include measures that capture other aspects of habit such as context stability and accessibility of relevant cues to the behavior [26]. In addition, we did not measure other constructs that may reflect these non- conscious processes, such as implicitly held beliefs developed through past experiences of the behavior covarying with evaluations [24]. Research has suggested that measures of implicit beliefs predict behavior, including physical activity participation, independent of intentions [20] and may also mediate effects of past behavior on subsequent behavior [28]. The effects of past behavior in the current study may, therefore, indicate that physical activity behavior in lei- sure time may be a function of unmeasured constructs reflecting implicit processes, but such an inference is speculative and requires empirical verification. Finally, consistent with previous research on self-determination theory, we found an indirect effect of autonomous motivation in leisure-time on both intentions and behavior mediated by trait self-control. Research on self-determination theory suggests that autonomous motivation is associated with better self-regulatory capacity and resilience in the face of self-control resource depletion [40, 42]. These findings are an important augmentation of trans-contextual model as they provide an alternative process by which individuals enact leisure-time physical activity in the absence of the ‘motivational sequence’ outlined in the original model. This find- ing lends additional support to the ‘energizing’ effect of autonomous motivation–students in the current study were more likely to report greater self-control if they perceived their behavior to be autonomous. As self-control was measured as a trait and was not specific to physical activ- ity in the current study, the self-control-autonomous motivation relationship in the current study may, in fact, reflect a general tendency for autonomously motivated individuals to report greater self-control. This needs to be corroborated at the trait level, such as examining relations between causality orientations from self-determination theory and trait self-control, and exam- ine whether such individual differences are behaviorally relevant [75]. The current research also illustrates the value of adopting a Bayesian analytic approach to combine prior knowledge of the distributions of model effects with the observed distributions to produce precise estimates and variability among model constructs. Discussion This was demonstrated by the narrowing of the credibility intervals about some of the model parameters. Importantly, the data used for the informative priors was highly reliable given they were derived from meta- analyses of multiple studies with large samples sizes. It is, however, also important to note that although the informative priors for the trans-contextual model effects were a meta-analysis of studies on samples of school students with similar profile to the participants in the current study [18], priors for the effects of the additional variables, self-reported habit and trait self- control were derived from research from multiple populations and mostly adult samples [38, 46]. Therefore, the priors were not directly comparable to the current sample. Nevertheless, current findings may be of value as a source of informative priors for future applications of the extended trans-contextual model. Consistent with the Bayesian approach, the current study should form part of an ongoing iterative research process that yields increasingly precise esti- mates of effects in the model. Strengths, limitations and recommendations for future research Strengths of the current study include (1) a focus on the determinants of lower secondary school students’ leisure-time physical activity participation, a priority area of research; (2) the PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 18 / 25 PLOS ONE Motivation and behavior in PE and leisure time application of an extended trans-contextual model, an integrative multi-theory approach that provided a priori hypotheses on the relations among the determinants and leisure-time physi- cal activity participation; (3) adoption of a two-wave prospective design using validated mea- sures of model determinants and behavior; and (4) application of Bayesian analytic procedures that enabled utilization of prior knowledge to arrive at precise estimates of model effects. How- ever, it is also important to note limitations of the current research that may affect interpreta- tion of the findings and the extent to which they can be generalized. While we endeavored to incorporate additional constructs representing non-conscious deter- minants of leisure-time physical activity participation in the current study, our measures did not encompass a full range of candidate determinants. For example, the current study did not include measures of implicit cognition and motivation with respect to school students’ leisure-time physi- cal activity participation. Given that measures of constructs such as implicit beliefs and autono- mous motivation have been shown to predict behavior directly independent of intentions in adult samples [20, 28, 76], future tests of the extended trans-contextual model should consider incorporating measures of these constructs as predictors of leisure-time physical activity partici- pation. This is particularly important given the lack of effects of the intentional or motivational constructs on leisure-time physical activity participation in the current study, and inclusion of implicit beliefs may assist in providing an explanation of the effects of past behavior. We also did not include the beliefs that underpin the attitude and subjective norm con- structs [9]. Their effects on intentions and behavior are typically mediated by the direct atti- tude and subjective norm measures. Similarly, we did not include constructs related to socioecological environment that may determine behavior, and whose effects on behavior may be mediated by the social cognition constructs in the model [70]. There is precedence for the indirect effect of these beliefs and socio-ecological constructs in the model. Strengths, limitations and recommendations for future research Research has dem- onstrated that beliefs and socio-ecological factors relating to context and environment are related to the social cognition constructs that predict health behavior, and those constructs mediate the effects of the beliefs and socio-ecological factors on behavior [77, 78]. While the constructs in the current model are proposed to account for the effects of these variables, such influences need empirical verification and serve as an avenue for future research. In addition, current data are correlational, which limits the extent to which we could infer causal relations among the extended trans-contextual model constructs. As with many model tests, including those of the trans-contextual model, causal effects are inferred from theory not the data [18]. Future research should consider the adoption of panel designs that permit modeling of temporal change and direction among trans-contextual model constructs over time through cross-lagged effects [12]. Such designs should also consider examining measuring model con- structs over longer periods of time to test the capacity of the model to account for long-term change in its constructs and physical activity behavior, see Jacobs et al. [79] for an example. In addition, intervention and experimental designs that adopt appropriate behavior change tech- niques [80, 81] are needed to test the effect of manipulating the constructs found to have a direct effect on leisure-time physical activity participation [82]. For example, interventions targeting atti- tudes should seek to promote enjoyment and positive affect through positive experiences of physi- cal activity, and interventions targeting self-discipline should seek to provide self-regulatory skills that promote better control over impulses to spend excessive time on leisure-time alternatives to physical activity (e.g., video games, watching television) and identify and manage barriers. Acknowledgments We thank Pauliina Hietanen, Noora Kilpela¨inen, Sampsa Lo¨ppo¨nen, Stina Seppa¨nen, and Miika Tuominen for their assistance with data collection. We are grateful to Jari Villberg for his assistance with the data analysis. Jekaterina Schneider is now with the Centre for Appear- ance Research at University of the West of England, Bristol, UK. Conclusion The current research is the first to test an extended version of the trans-contextual model to identify determinants of leisure-time physical activity participation in lower secondary school 19 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 PLOS ONE Motivation and behavior in PE and leisure time students. Results indicate that the traditional motivational and social cognition constructs are effective in predicting leisure-time physical activity intentions, but not actual behavior. How- ever, we found direct effects of trait self-control and attitude on leisure-time physical activity participation, suggesting that students’ physical activity participation was determined by con- structs representing non-conscious processes. A further innovation of the current research is the application of a Bayesian analytic approach to update the effects and variability estimates of model parameters based on previous meta-analytic findings. Results raise questions over the effectiveness of the original trans-contextual model constructs in determining leisure-time physical activity participation, at least in the current societal context in which the physical environment may not support engagement in physical activity and offers various competing, non-active alternatives (e.g., video games). However, current findings highlight the potential of including additional constructs representing non-conscious processes. However, these data should not be considered unequivocal evidence to support rejection of the model as unmea- sured moderator variables may have affected model effects. Further replication of the extended trans-contextual model predictions in larger samples is warranted. References 1. Kurdaningsih SV, Sudargo T, Lusmilasari L. Physical activity and sedentary lifestyle towards teenagers’ overweight/obesity status. Int J Comm Med Pub Health. 2017; 3(3):630–5. https://doi.org/10.18203/ 2394-6040.ijcmph20160623 2. 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Methodology: Juho Polet, Jekaterina Schneider, Mary Hassandra, Taru Lintunen, Arto Lauk- kanen, Nelli Hankonen, Mirja Hirvensalo, Tuija H. Tammelin, Kyra Hamilton, Martin S. Hagger. Project administration: Juho Polet, Jekaterina Schneider, Mary Hassandra, Taru Lintunen, Arto Laukkanen, Martin S. Hagger. Writing – original draft: Juho Polet, Taru Lintunen, Martin S. Hagger. Writing – review & editing: Juho Polet, Jekaterina Schneider, Mary Hassandra, Taru Lintu- nen, Arto Laukkanen, Nelli Hankonen, Mirja Hirvensalo, Tuija H. Tammelin, Kyra Hamil- ton, Martin S. Hagger. 20 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0258829 November 12, 2021 PLOS ONE Motivation and behavior in PE and leisure time References Urban adolescents’ exercise intentions and behaviors: An exploratory study of a trans-contextual model. Contemp Educ Psychol. 2008; 33(4):841–58. https://doi.org/10. 1016/j.cedpsych.2007.09.002 16. 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New York, NY: Cambridge University Press; 2020. 25 / 25
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Preference Dependency Grammar and its Packed Shared Data Structure “Dependency Forest”
Shizen gengo shori
2,014
cc-by
27,297
Journal of Natural Language Processing Vol. 21 No. 4 Journal of Natural Language Processing Vol. 21 No. 4 Journal of Natural Language Processing Vol. 21 No. 4 translated from: Journal of Natural Language Processing Vol. 13, No. 3, pp. 37–90 Journal of Natural Language Processing Vol. 21 No. 4 translated from: Journal of Natural Language Processing Vol. 13, No. 3, pp. 37–90 Preference Dependency Grammar and its Packed Shared Data Structure “Dependency Forest” Hideki Hirakawa† Preference dependency grammar (PDG) is a framework for integrating morphological, syntactic, and semantic analyses. PDG provides packed shared data structures that can efficiently encompass all possible interpretations at each level of sentence analyses with preference scores. Using the structure, PDG can calculate a globally optimized interpretation for the target sentence. This paper first gives an overview of the PDG framework by describing the base model of PDG, which is a sentence analysis model, called a “multi-level packed shared data connection model.” Then this paper describes packed shared data structures, e.g., headed parse forests and dependency forests, adopted in PDG. Finally, the completeness and soundness of the mapping between the parse forest and the dependency forest are revealed. os ba Co po at o * This article has been partially revised for better understanding of overseas readers. Key Words: Sentence analysis, Phrase structure, Dependency structure, Packed shared data structure † Toshiba Corporation † Toshiba Corporation * Thi i l h b i ll i d f b d di f d 1 Introduction Natural language processing systems have very complicated and hard-to-formalize design issues because they have to treat some specific features of natural languages, such as variety and ambiguity, regularity and exceptionality, extensiveness and size, and time variance. A natural language analysis (NLA) system has to manage these features properly, according to the require- ment level specified by its application. One of the most important challenges in the design of NLA systems is the treatment of varieties and ambiguities in natural languages; i.e., how to find the correct interpretation of a sentence from among the myriad of ambiguities at the analysis levels of morphology, syntax, semantics, discourse, and pragmatics. More precisely, an NLA system computes structures for a sentence by generating a set of its possible interpretations (application of interpretation generation knowledge), rejecting impossible interpretations (application of constraint knowledge), and obtaining the preference order of pos- sible interpretations (application of preference knowledge). Figure 1 shows this sentence analysis September 2014 Vol. 21 No. 4 Journal of Natural Language Processing Fig. 1 Natural language analysis system model Fig. 1 Natural language analysis system model model.1 A set of interpretations of a sentence exists in the interpretation space prescribed by the interpretation description scheme. Each interpretation is either correct (◎), plausible (○), or implausible (×) with respect to the real-world situation. The interpretation generation knowl- edge generates a set of possible interpretations, constraint knowledge filters out any impossible interpretation, and preference knowledge provides the ordering of interpretations in the interpre- tation space. The output of an NLA system is the optimum interpretation extracted from among the remaining interpretations, according to the order of preference. All three knowledge types exist at each level of sentence analysis, i.e., morphology, syntax, semantics, etc. The compre- hensive ability of an NLA system is determined by the total of all knowledge at each language analysis level. Preference knowledge sometimes gives different suggestions for a sentence inter- pretation at different levels (Hirakawa and Amano 1989)．Such inconsistencies must be resolved appropriately in order to construct an accurate NLA system. Interpretation generation knowledge and constraint knowledge define the sentence coverage, i.e., a set of acceptable sentences in an NLA system. Therefore, these knowledge types correspond to a grammar in linguistics from the Chomskyan viewpoint. Many computational grammar frameworks have been proposed and studied, in which a variety of linguistic knowledge has been incorporated. 1 Constraint knowledge can be defined as a type of preference knowledge that does not provide any possibility. However, the application of constraint knowledge implies pruning in the computation, which is in clear contrast with the application of preference knowledge. 2 Some systems produce deep structures as an output. The classification here is based on the interpretation structure during the parsing. 3 3 Link grammar is not considered as an instance of dependency grammar by its creators, and it departs from the traditional view of dependency by using undirected links; however, the representations used in link grammar parsing are similar to the dependency representations in that they consist of words linked by binary relations (Nivre 2005). ( ) 4 Kaplan identified an idea to extend the LFG configuration to include more structures such as semantic and anaphoric structures. 1 Introduction Grammar frameworks are based on interpretation description formalisms that prescribe interpretation spaces such as phrase structure, dependency structure, semantic graph structure, and logical formula. In addition, considerable research on preference knowledge, such as preference semantics (Wilks 1975), has been conducted in linguistics. Preference knowledge has been widely adopted 734 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa in NLA systems through the use of a statistical method adapted from speech technology. As statistical methods extend their application scope from the N-gram model (word sequence) to context-free and dependency grammar, more NLA systems can benefit from the statistical power obtained using large-scale corpora. For example, statistical models are introduced to phrase-structure-based grammar frameworks such as CFG and lexical functional grammar (LFG) (Kaplan 1989; Riezler, King, Kaplan, Crouch, Maxwell III, and Johnson 2002)，head-driven phrase structure grammar (HPSG) (Pollard and Sag 1994; Tsuruoka, Miyao, and Tsujii 2004), combinatory categorical grammar (CCG) (Steedman 2000; Clark and Curran 2003),2 and to dependency-structure-based grammar frameworks such as probabilistic dependency grammar (Lee and Choi 1997), kakari-uke analysis (Shudo, Narahara, and Yoshida 1980; Ozeki 1994; Hirakawa 2001; Kudo and Matsumoto 2005; Kawahara and Kurohashi 2005), constraint dependency grammar (CDG) (Maruyama 1990; Wang and Harper 2004), and link grammar (Sleator and Temperley 1991; Lafferty, Sleator, and Temperley 1992).3 For example, statistical models are introduced to phrase-structure-based grammar frameworks such as CFG and lexical functional grammar (LFG) (Kaplan 1989; Riezler, King, Kaplan, Crouch, Maxwell III, and Johnson 2002)，head-driven phrase structure grammar (HPSG) (Pollard and Sag 1994; Tsuruoka, Miyao, and Tsujii 2004), combinatory categorical grammar (CCG) (Steedman 2000; Clark and Curran 2003),2 and to dependency-structure-based grammar frameworks such as probabilistic dependency grammar (Lee and Choi 1997), kakari-uke analysis (Shudo, Narahara, and Yoshida 1980; Ozeki 1994; Hirakawa 2001; Kudo and Matsumoto 2005; Kawahara and Kurohashi 2005), constraint dependency grammar (CDG) (Maruyama 1990; Wang and Harper 2004), and link grammar (Sleator and Temperley 1991; Lafferty, Sleator, and Temperley 1992).3 From the viewpoint of integrating multilevel knowledge, grammar frameworks are classified as either single-space or multi-space models. Simple CFG and simple dependency grammar are typical examples of a single-space framework without preference knowledge treatment, based on phrase structure and dependency structure schemes, respectively. y p p structure during the parsing. 1 Introduction DCG (Pereira and Warren 1980) and BUP (Matsumoto, Tanaka, Hirakawa, Miyoshi, and Yasukawa 1983) have developed a mechanism to extend the CFG framework to incorporate arbitrary extra conditions using Prolog codes, which, for example, can be used for introducing semantic constraints. This is a type of integration of multilevel constraint knowledge in the single-space model. CDG is a single-space grammar framework based on the dependency scheme with unary or binary constraints. These constraints are used for incorporating multilevel constraint knowledge (Maruyama 1990). On the other hand, LFG has at least two types of description schemes (or data structures), i.e., c-structure (phrase structure) and f-structure (functional structure).4 The functional scheme provides the constraints on the functional structures (Kaplan 1989). PCFG (Jelinek, Lafferty, and Mercer 1992) is an example of a single-space model with preference knowledge. A certain number of statistical CFG-based parsers incorporate statistical models that integrate the phrase From the viewpoint of integrating multilevel knowledge, grammar frameworks are classified as either single-space or multi-space models. Simple CFG and simple dependency grammar are typical examples of a single-space framework without preference knowledge treatment, based on phrase structure and dependency structure schemes, respectively. DCG (Pereira and Warren 1980) and BUP (Matsumoto, Tanaka, Hirakawa, Miyoshi, and Yasukawa 1983) have developed a mechanism to extend the CFG framework to incorporate arbitrary extra conditions using Prolog codes, which, for example, can be used for introducing semantic constraints. This is a type of integration of multilevel constraint knowledge in the single-space model. CDG is a single-space grammar framework based on the dependency scheme with unary or binary constraints. These constraints are used for incorporating multilevel constraint knowledge (Maruyama 1990). On the other hand, LFG has at least two types of description schemes (or data structures), i.e., c-structure (phrase structure) and f-structure (functional structure).4 The functional scheme provides the constraints on the functional structures (Kaplan 1989). PCFG (Jelinek, Lafferty, and Mercer 1992) is an example of a single-space model with preference knowledge. A certain number of statistical CFG-based parsers incorporate statistical models that integrate the phrase 735 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 structure information and the phrase head and dependency information; the integration of this information was proved to be effective in improving parse accuracy (Carroll and Charniak 1992; Eisner 1996; Collins 1999; Charniak 2000; Bikel 2004). 1 Introduction Probabilistic CDG (Wang and Harper 2004) is an example of a single-space model based on the dependency structure scheme. Preference dependency grammar (PDG) adopts a multi-space approach and integrates multilevel knowledge based on the packed shared data structures in multiple interpretation spaces, as described below. After providing PDG’s basic sentence analysis model and overview, this paper describes meth- ods for obtaining two main packed shared data structures of PDG, i.e., the “headed phrase struc- ture forest” and the “dependency forest,” and then gives proof of the completeness and soundness of the dependency forest. This paper also provides an experiment for analyzing prototypical am- biguous sentences, and discusses the mapping relations between the phrase structure tree(s), and the dependency tree(s), as well as the treatment of non-projective dependency structures in PDG. 2.1 Multilevel Packed Shared Data Connection Model PDG is a framework for the morphological, syntactic, and semantic analysis of natural lan- guage sentences, and is mainly designed for manipulating ambiguities in sentence interpretations (Hirakawa 2007). The targeted issues are as follows: (a) suppression of combinatorial explosion (a) suppression of combinatorial explosion (b) proper treatment of generation, preference, and constraint knowledge (c) modularity and integration of multilevel knowledge PDG adopts the “multilevel packed shared data connection (MPDC) model” shown in Figure 2 (c) modularity and integration of multilevel knowledge PDG adopts the “multilevel packed shared data connection (M Fig. 2 Multilevel packed shared data connection model Fig. 2 Multilevel packed shared data connection model 736 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa to achieve these targets. Each analysis level encompasses all possible sentence interpretations of its interpretation space on packed shared data structure. The input sentence side is called the lower level and the output side is called the upper level. Each interpretation at each level has a link called the “interpretation linkage” with its counterparts in the lower and, possibly, the upper levels (represented by the dotted line labeled “correspondence”). In the MPCD model, the interpretations at the lower level involve the corresponding interpretations at the upper level. The input sentence involves all interpretations at all levels. Each interpretation of a sentence in some interpretation space should have an interpretation linkage to its counterpart in the lower adjacent level of the interpretation space; however, the inverse is not necessarily true. For example, there can be a morphological interpretation of a sentence having no corresponding syntactic interpretations, but there cannot be a syntactic interpretation of a sentence having no corresponding morphological interpretations. Each level has its generation, preference, and constraint knowledge. The generation knowledge generates all possible interpretations from a set of its lower-level interpretations (7−→arrow in Figure 2). This is called externalization. The constraint knowledge restricts or rejects interpretations in the interpretation space. The preference knowledge gives the order of preference of the interpretations in the interpretation space. PDG aims to achieve the following effects by adopting this model. PDG aims to achieve the following effects by adopting this model. (a) Utilizing multiple types of linguistic data structures, such as phrase and dependency structures, to treat morphological, syntactic, and semantic knowledge with high module independence. 5 Word pos pair (WPP) is a pair of a word and a part of speech (POS). The word “time” has WPPs such as “time/n” and “time/v.” 2.1 Multilevel Packed Shared Data Connection Model (b) Utilizing packed shared data structures encompassing all ambiguities at each level of sen- tence analysis to suppress the combinatorial explosion without pruning. (c) Interpretation linkage from the input sentence to the uppermost level interpretations pro- vides a basis for the integration of multilevel preference knowledge (preference scores). (d) Incorporating an optimum tree search method for a packed shared dependency structure with arc co-occurrence constraints to compute the most plausible interpretation (depen- dency tree) of a sentence. Interpretations not rejected by the constraint knowledge are called well-formed interpretations of the sentence. The optimum well-formed interpretation is defined in each interpretation space, and is computed by the optimum solution procedure on the basis of preference and constraint knowledge. As described above, the application determines the requirement level for the NLA system. For example, machine translation systems between languages in the same family may require phrase structure output, whereas those between languages in different families may require 737 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 dependency structure output. The interpretation linkage can be used to search the optimum interpretation, based on upper-level information and not current-level information. This can be done by tracing back the linkage from the optimum interpretation at the upper level to the corresponding interpretation at the current level. For example, the tagger based on the optimum semantic analysis result is constructed naturally. 6 Constraints such as number agreements can be described as constraints at another level or can be described in more than one level in parallel. This is a design issue in actual grammar development. In general, constraint knowledge should be described at the lowest level possible, because it will improve the system efficiency by suppressing the generation of useless interpretations. 2.2 Implementation Model of the Preference Dependency Grammar Figure 3 shows an overview of the implementation model of PDG. PDG has three basic lin- guistic levels, i.e., morphology, syntax, and semantics with the syntax level further divided into two levels. In total, PDG has four levels of interpretation space, description scheme, and packed shared data structure. Four packed shared data structures, the “WPP trellis,”5 “headed phrase structure forest,” “functional dependency forest,” and “semantic dependency forest” encompass sets of sentence interpretations, i.e., WPP sequences, phrase structure trees, functional depen- dency trees, and semantic dependency trees, respectively. In this paper, the headed phrase struc- ture forest/tree and the functional dependency forest/tree are also called the phrase structure forest/tree and the dependency forest/tree, respectively. The processes for obtaining these data structures are called morphological analysis, syntactic analysis, dependency forest generation, and semantic expansion, respectively. Figure 4 briefly explains the data structures and examples of preference and constraint knowl- edge at each level. WPP trellis is a packed shared data structure encompassing all WPP sequences for a sentence. Morphological interpretations for a sentence are represented by sequences (or Fig. 3 PDG implementation model Fig. 3 PDG implementation model 5 Word pos pair (WPP) is a pair of a word and a part of speech (POS). The word “time” has WPPs such as “time/n” and “time/v.” 738 PDG and its Packed Shared Data Structure “Dependency Forest” PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Fig. 4 Packed shared data structures in PDG Fig. 4 Packed shared data structures in PDG strings) of WPP nodes, which represent the adjacency relations between words. The headed phrase structure forest encompasses a set of phrase structure trees that represent the sub- categorization (or adjacency) relations of phrases. Syntactic preference knowledge (e.g., phrase frequency) and constraint knowledge (e.g., number agreements) are described on top of the phrase structure.6 The functional dependency structure is another data structure at the syntax level of PDG. A functional dependency tree consists of WPP nodes and arcs labeled with functional dependency relations, such as subject and object. A set of functional dependency trees represent- ing the syntactic interpretations of a sentence is represented by a functional dependency forest. The dependency forest is a packed shared data structure that utilizes a dependency graph with a framework for describing the preference and constraint information for the arcs in the graph. Semantic interpretations for a sentence are represented by semantic dependency trees that consist of word POS concept triple (WPCT) nodes and arcs labeled with semantic dependency relations. A semantic dependency forest is used to represent a set of semantic dependency trees that show the semantic interpretations of a sentence. In the PDG model, preference scores provided by 739 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 preference knowledge at each level are integrated into the preference scores in the uppermost level data structure through the interpretation linkage from the input sentence to the uppermost level interpretations. The most plausible interpretation (dependency tree) of a sentence is calcu- lated by an optimum tree search method (Hirakawa 2005). Currently, an experimental syntactic analysis version of the PDG system has been implemented (Hirakawa 2007). This paper focuses on the syntactic level data structures in the PDG model. As described above, a characteristic feature of PDG is the integrated use of phrase and depen- dency structures as the intermediate syntactic data structures, which provides the advantage of a multilevel construction framework. For example, CDG, which adopts a single-level packed shared data model based on the dependency structure, generates all possible dependency relations for all word pairs in a sentence and applies constraints to obtain a set of well-formed interpretations for a sentence. This is called eliminative parsing. PDG and its Packed Shared Data Structure “Dependency Forest” This method has a superior feature in that it does not eliminate possible interpretations, but has a demerit in terms of efficiency in that it produces many useless hypotheses. Harper et al. proposed techniques for improving CDG, including the interpretation generation method (Harper, Hockema, and White 1999). However, PDG produces the dependency forest encompassing all possible dependency trees, which have their counterparts in the phrase structure forest, for a sentence. This indicates that generation and constraint knowledge at the phrase structure forest level, i.e., knowledge based on phrase structure representation,7 can suppress interpretations at the dependency forest level. Further- more, as described in Section 5.4, using the description capability of the phrase structure enables us to incorporate the controlled non-projectivity in the dependency forest (Hirakawa 2006, 2007). This indicates that the description capability of the dependency forest (dependency formalism) is enhanced using phrase structure formalism. 7 Extended CFG is used in PDG. 2.4.1 WPP Trellis and Packed Shared Phrase Structure Forest PDG utilizes WPP trellis as the basis for the morphological analysis level. WPP trellis is a packed shared data structure encompassing all WPP sequences for a sentence. The packed shared phrase structure forest, or simply, phrase structure forest, is a well-known packed shared data structure for encompassing all phrase structure trees (Tomita 1987). WPP trellis and packed shared phrase structure forest satisfy the interpretation linkage condition of the MPDC model because each phrase structure tree in the packed shared phrase structure forest corresponds to a WPP sequence in WPP trellis. 2.3 Prerequisites for Packed Shared Data Structures The following are prerequisites for the data structure at each level of the MPDC model. (a) no combinatorial explosion (b) a set of proportionate interpretations (c) proper basis for knowledge description (d) existence of interpretation linkages between the lower- and upper-level data structures (a) is a key issue with regard to constructing practical NLA systems. Enumerative treatment of interpretations leads to lack of time and space, or degrades the analytical capability due to (d) existence of interpretation linkages between the lower- and upper-level data structures (a) is a key issue with regard to constructing practical NLA systems. Enumerative treatment of interpretations leads to lack of time and space, or degrades the analytical capability due to 740 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa overpruning. (b) implies that the packed shared data structure at each level encompasses all possible solutions correctly, i.e., it assures no pruning of existing interpretations and no gener- ation of nonexistent interpretations originating from the packed shared data structures. If this requirement is assured, it is beneficial for an NLA system to be capable of introducing possible pruning (application of constraint knowledge) in the early stage of sentence analysis from the viewpoint of system performance. (c) is a prerequisite for the efficient and precise knowledge development an NLA system. The interpretation description scheme at each level should be an essential basis for generation, constraint, and preference knowledge (Hirakawa 2002). 2.4.2 Semantic Dependency Graph The “semantic dependency graph” (SDG) is a dependency graph representing ambiguities in word dependencies (structural ambiguities) and their semantic relations (semantic ambiguities) (Hirakawa 2002). SDG is generated from a kakari-uke structure produced by an ATN parser by expanding kakari-uke relations (dependency relations) to semantic dependency relations. The SDG method lacks in terms of generality in that it cannot handle backward dependency and multiple WPPs because it depends on some linguistic features peculiar to Japanese. PDG employs a dependency forest instead of an SDC. 8 The direction of dependency arc obeys the convention of the Japanese kakari-uke grammar (dependency grammar). The dependent node of an arc is the node located at the source of the arc. This is contrary to the convention in the syntactic graph, but not substantially different. Journal of Natural Language Processing Vol. 21 No. 4 Vol. 21 No. 4 2.4.3 Syntactic Graph Seo proposed the “syntactic graph,” which encompasses all dependency trees corresponding to phrase structure trees in the phrase structure forest for a sentence (Seo and Simmons 1989; Rim, Seo, and Simmons 1990). The syntactic graph is a promising candidate for a packed shared data structure in PDG, but cannot be adopted as it is due to its difficulty in satisfying prerequisite (d) in Section 2.3. 741 Journal of Natural Language Processing Vol. 21 No. 4 Vol. 21 No. 4 September 2014 The syntactic graph is a directed graph that consists of nodes representing WPPs and labeled arcs representing the syntactic relations between nodes. It defines a set of dependency trees (interpretations) for a sentence in combination with the “exclusion matrix,” which represents exclusive co-occurrence relations between arcs. The syntactic graph is a set of Triples, containing arc name and two nodes (containing WPP, surface position, etc.). Figure 5 shows the syntactic graph and exclusion matrix of the sentence “Time flies like an arrow.” The numbers in arcs are arc-IDs. Multiple arcs targeting one node represent modification ambiguities,8 and S corresponds to the starting symbol. The exclusion matrix is one whose rows and columns are a set of arcs in the syntactic graph that prescribes the co-occurrence relation between arcs. Setting “1” at position (i, j) of the exclusion matrix indicates that the i-th and j-th arcs must not co-occur in any dependency tree (interpretation) obtained from the syntactic graph. The syntactic graph and exclusion matrix are generated from a kind of packed shared phrase structure forest. PDG adopts the same data structure, called a headed phrase structure forest, Fig. 5 Syntactic graph and exclusion matrix for the example sentence 8 The direction of dependency arc obeys the convention of the Japanese kakari-uke grammar (dependency grammar). The dependent node of an arc is the node located at the source of the arc. This is contrary to the convention in the syntactic graph, but not substantially different. 742 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa described in Section 3. In the rest of this paper, phrase structure forest means headed phrase structure forest. The traditional phrase structure forest (packed shared phrase structure forest) is called a headless phrase structure forest. Seo discussed the completeness and soundness of the correspondence between the phrase structure forest and the syntactic graph (Seo and Simmons 1989). The completeness is satisfied if each phrase structure tree in each phrase structure forest has its counterpart(s) in a syntactic graph. The soundness is satisfied if each dependency tree in each syntactic graph has its coun- terpart(s) in a phrase structure forest. The completeness of a syntactic graph is shown in (Seo and Simmons 1989), but the soundness is not assured. All exclusion matrix cells are initially set to 1 (this means no two triples co-occur). Vol. 21 No. 4 Then, the cells for all triple pairs in the dependency tree generated from phrase structure trees are set to 0. Since the exclusion matrix prescribes the co-occurrence relations for all dependency trees in the dependency graph,9 the allowance of a co-occurrence of two triples (set 1 to the cell for two triples) is safe if and only if the restriction of these two triples is not necessary for all other interpretations (dependency trees). Appendix A shows an example in which the syntactic graph cannot satisfy the soundness. 3 Packed Shared Data Structures in PDG PDG adopts a phrase structure forest and dependency forest for the packed shared data structures for phrase structure and dependency structure representations, respectively. 9 The constraint in the exclusion matrix is, in a sense, global. 10 10 Phrase head is a WPP in PDG. 9 The constraint in the exclusion matrix is, in a sense, global. 9 The constraint in the exclusion matrix is, in a sense, global. 10 Phrase head is a WPP in PDG. 10 Phrase head is a WPP in PDG. 9 The constraint in the exclusion matrix is, in a sense, global. 10 Phrase head is a WPP in PDG. 3.2 Dependency Forest The dependency forest (DF) consists of a “dependency graph” (DG) and a “co-occurrence matrix” (CM, C-Matrix), and is expressed as DF = ⟨DG,CM⟩. 3.1 Headed Phrase Structure Forest The headed phrase structure forest is a type of packed shared parse forest, and consists of edges corresponding to rewriting rules in CFG. The sub-trees, which satisfy the following conditions, are packed and shared. Sub-trees have (a) the same nonterminal symbol (category) (b) the same coverage (phrase boundary) (c) the same phrase head10 (head constituent) Conditions (a) and (b) constitute the headless phrase structure forest (Schiehlen 1996). The phrase structure trees in the headed phrase structure forest have mapping to the phrase structure trees in the headless phrase structure forest. An example of the edges and the phrase structure forest in PDG, along with the parsing algorithm, is shown in Section 4. 743 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 Vol. 21 No. 4 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 11 Preference score represents the plausibility of the arc and is used for the computation of the optimum inter- pretation. 3.2.1 Dependency Graph and Co-occurrence Matrix Figure 6 shows a DG for the example sentence “Time flies like an arrow.” The DG consists of nodes and directed arcs, representing WPPs and the dependency relation between nodes, respectively. An arc has its ID and preference score,11 which are hidden in the figure. The dependency graph has one special node, called the “top node” (or sometimes called “root node,” for convenience), which is the root of all dependency trees in the DG. In practice, a DG is represented by a set of “dependency pieces.” A dependency piece consists of one arc and its “dependent (or modifier) node” and “governor (or modificand) node.” Since the dependency piece and the arc have a one-to-one correspondence, a dependency piece is referred to as an arc in this paper. The number of arcs in a DG is called the “size of the dependency forest.” Fig. 6 Initial dependency forest for the example sentence Fig. 6 Initial dependency forest for the example sentence 11 Preference score represents the plausibility of the arc and is used for the computation of the optimum inter- pretation. 744 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa A “dependency tree” is a subset of the DG that forms a tree. Dependency trees represent interpretations of sentences or phrases at dependency relation level. A “dependency tree” is a subset of the DG that forms a tree. Dependency trees represent interpretations of sentences or phrases at dependency relation level. A CM is a matrix whose rows and columns are a set of arcs in a DG that prescribes the co-occurrence relation between arcs. Only when CM(i, j) is ○, arci and arcj are co-occurable in one dependency tree. The co-occurrence relation is symmetric, and CM is a symmetric matrix. Definition 1 [Well-formed dependency tree] well-formed dependency tree constraint well-formed dependency tree constraint (a) Each input word has a corresponding node in the DT (coverage constraint). (b) No two nodes in the DT occupy the same input position (single role constraint). (c) Each arc pair in the DT has a co-occurrence relation in the CM (arc co-occurrence constraint). (c) Each arc pair in the DT has a co-occurrence relation in the CM (arc co-occurrence constraint). (a) and (b) are collectively referred to as the “covering constraint.” A dependency tree satisfying the covering constraint is called a “well-covered dependency tree.” A dependency tree satisfying (c) is called a “well-co-occurred dependency tree.” A set of well-formed dependency trees is the set of possible interpretations for an input sentence. The DF in Figure 6 has four well-formed dependency trees. In PDG, a set of one WPP node is considered to be a special case of a dependency tree with no arcs, which satisfies the well-formed dependency tree constraint. (a) and (b) are collectively referred to as the “covering constraint.” A dependency tree satisfying the covering constraint is called a “well-covered dependency tree.” A dependency tree satisfying (c) is called a “well-co-occurred dependency tree.” A set of well-formed dependency trees is the set of possible interpretations for an input sentence. The DF in Figure 6 has four well-formed dependency trees. In PDG, a set of one WPP node is considered to be a special case of a dependency tree with no arcs, which satisfies the well-formed dependency tree constraint. 3.2.2 Well-formed Dependency Tree The “well-formed dependency tree” is a dependency tree DT in the DF that satisfies the following conditions, called the “well-formed dependency tree constraint.” Definition 1 [Well-formed dependency tree] Definition 1 [Well-formed dependency tree] 4 Generation of the Phrase Structure Forest and the Initial Dependency Forest PDG generates a dependency forest from an input sentence through four processes, i.e., morphological analysis, syntactic analysis, phrase structure/dependency forest generation, and dependency forest reduction. The explanation of the morphological analysis process is beyond the scope of this study. 12 The dependency structure is a set of arcs, but is represented by a list format using [ ]. In this paper, sets are sometimes represented by [ ] in program codes. 3.2.3 Initial Dependency Forest and Reduced Dependency Forest There can be more than one different-sized DF encompassing the equivalent set of dependency trees with respect to the degree of arc sharing. PDG treats the “initial dependency forest” and the “reduced dependency forest,” which is obtained from the initial dependency forest. The initial dependency forest consists of the “initial dependency graph” and the “initial C-matrix.” The reduced dependency forest is simply called a dependency forest in this paper. The dependency graph of the initial dependency forest in Figure 6 is different from the syntactic graph in Figure 5, in terms of the number of arcs between “fly/n” and “time/v.” 745 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 4.1 Grammar Rules Grammar rules in PDG are extended CFG rules, which define possible phrase structures and mapping from the phrase structures to the corresponding dependency structures. Grammar rules are written in the following format. y/Y →x1/X1, . . . , xn/Xn : [arc(arcname1, Xi, Xj), . . . , arc(arcnamen−1, Xk, Xl)] (0< i, j, k, l≤n) y/Y →x1/X1, . . . , xn/Xn : [arc(arcname1, Xi, Xj), . . . , arc(arcnamen−1, Xk, Xl)] (0< i, j, k, l≤n) e.g., vp/V →v/V , np/NP, pp/PP : [arc(obj, NP, V ), arc(vpp, PP, V )] (0< i, j, k, l≤n) e.g., vp/V →v/V , np/NP, pp/PP : [arc(obj, NP, V ), arc(vpp, PP, V )] e.g., vp/V →v/V , np/NP, pp/PP : [arc(obj, NP, V ), arc(vpp, PP, V )] A grammar rule consists of two parts separated by “:”, the rewriting rule part and the dependency structure part. The left side of the rewriting rule “y/Y ” and constituent “xi/Xi” mean “syntactic category/variable.” Y is a head constituent, called the “phrase head,” and is the same as one of the variables “X1. . .Xn” in the “rule body.” The dependency structure part is a set of arcs in the form “arc(arcname, variable1, variable2).”12 A variable is bound to a WPP node, which is a phrase head of a constituent in the rewriting rule. In the example above, the dependency structure shows where dependents NP and PP are connected to the governor phrase head V by means of the obj arc and the vpp arc, respectively. The dependency structure part constitutes a partial dependency tree, which satisfies the following “partial dependency structure conditions.” Definition 2 [Partial dependency structure condition] Definition 2 [Partial dependency structure condition] (a) A partial dependency structure constitutes a tree structure whose top node is a phrase head of the head constituent Y . Phrase heads of non-head constituents are the dependents of phrase heads of other constituents. (b) The phrase heads of the constituents in the rule body have one-to-one correspondence with variables in the partial dependency structure. 746 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Fig. 7 Grammar and lexicon for the example sentence Fig. 7 Grammar and lexicon for the example sentence Figure 7 shows the grammar rules and lexicons for analyzing the example sentence: “Time flies like an arrow.” Rule (R0), whose rule head and rule body are “root” (predefined special symbol) and “s”(starting symbol) as rule body, is a special rule for creating a “root edge” of the phrase structure forest and a “top node” [root]-x of the dependency forest.13 4.2 Parsing Algorithm The syntactic analysis of PDG is implemented by extending the bottom-up chart-parsing algorithm to generate a dependency structure. 13 This rule is introduced for convenience in the treatment of data structures. 13 This rule is introduced for convenience in the treatment of data structures 4.2.1 The Structure of Edge An ordinal chart parser utilizes edges composed of five elements ⟨FP, TP, C, FCS, RCS⟩, i.e., the from-position (FP), the to-position (TP), the category (C), the found constituent sequence (FCS), and the remaining constituent sequence (RCS). The head of the grammar rule corresponds to C. The body of the grammar rule corresponds to both FCS and RCS and is partitioned by a dot (・), which shows the boundary of FCS and RCS, as shown in the following edge written in diagrammatic form. ⟨0, 1, s →np ・vp pp⟩ This edge is generated from the grammar rule “s →np vp pp” and has elements FP=0, TP=1, C=s, FCS=[np], and RCS=[vp, pp]. The result of the dictionary look-up for an input word is an inactive edge whose category is the POS of the word, and whose found constituent sequence is a word list, as follows: ⟨0, 1, n →[time]・⟩ ⟨0, 1, n →[time]・⟩ ⟨0, 1, n →[time]・⟩ 747 Vol. 21 No. 4 Journal of Natural Language Processing Vol. 21 No. 4 Journal of Natural Language Processing Vol. 21 No. 4 September 20 September 2014 The parsing algorithm of PDG has two extensions, i.e., the treatment for the dependency structure part in a grammar rule and the construction of the packed shared data structure. The edge for PDG parsing has two additional elements, i.e., the phrase head (PH) and the dependency structure (DS), as follows: andard edge: ⟨0, 1, s →np ・vp pp⟩ Standard edge: ⟨0, 1, s →np ・vp pp⟩ Standard edge: ⟨0, 1, s →np ・vp pp⟩ PDG edge: ⟨0, 1, s/PH →np/n1 ・vp/PH pp/PP : DS⟩ PDG edge: ⟨0, 1, s/PH →np/n1 ・vp/PH pp/PP : DS⟩ As described in Section 4.1, PH and DS represent a phrase head (node) and dependency structure (a set of arcs), respectively. The symbol n1 shows a node (WPP) that is the head of the np phrase. PDG utilizes another data structure called “packed edge,” which is obtained by packing inactive edges into one. The packed edge has the list of FCS and of DS, instead of the FCS and the DS in the PDG edge. The PDG edge with FSC and DS is called “single edge,” in contrast to packed edge. The packed edge is equivalent to a set of single edges. The following shows the relation between single edge and packed edge, diagrammatically. Fig. 8 Structure of edge have correspondence and constitute two CSDS pairs, i.e., ([103, 169] {obj-25})14 and ([103, 119, 165] {obj-4, vpp-20}). Edge @E5 is an example of an edge generated from a dictionary look- up operation, called “lexical edge.” The data structure of a lexical edge is a set of one node corresponding to the consulted word. The lexical edge is explicitly represented by adding “@.” 14 The obj-25 is the abbreviation of arc(obj-25, [flies]-n-1, [time]-v-0). 4.2.1 The Structure of Edge Single edge: ⟨0, 5, s/n2 →np/n1 vp/n2 pp/n3・: DS1⟩ ⟨0, 5, s/n2 →np/n1 vp/n2 ・: DS2⟩ Packed edge: ⟨0, 5, s/n2 →[[np/n1 vp/n2 pp/n3], [np/n1 vp/n2]] ・: [DS1, DS2]⟩ Packed edge: ⟨0, 5, s/n2 →[[np/n1 vp/n2 pp/n3], [np/n1 vp/n2]] ・: [DS1, DS2]⟩ n1 to n3 are nodes (WPPs) and n2 is a phrase head. [np/n1 vp/n2 pp/n3] and [np/n1 vp/n2] are constituent sequences with their phrase head (nodes). DS1 and DS2 are dependency structures (partial dependency trees). For convenience, a packed edge is represented in the form “E,” “⟨E . . .⟩,” or “edge E,” and a single edge is represented as “e,” “⟨e . . .⟩,” or “edge e.” When it is not ambiguous, “edge” is used for representing “packed edge” or “single edge.” Inactive edges are represented by adding “*” at the top of the edge symbol. Edges *E and *e are an inactive packed edge and an inactive single edge, respectively. The syntactic parsing of PDG described below utilizes packed edges. Figure 8 shows the formal constitution of a packed edge. A packed edge consists of eight elements. FCSL and DSL are lists (or sequences) with the same length. The pair (FCSi, DSi) obtained by extracting the i-th elements of FCSL and DSL is called the “CSDS pair.” The CSDS pair corresponds to the single edge described above. The edges E1 to *E3 in Figure 8 show the growth of the edge generated from a grammar rule for a noun phrase. Edge *E3 is the inactive edge showing the interpretation that the input words “an arrow” constitute a noun phrase, and its dependency structure is {arc(det-14, [an]- det-3, [arrow]-n-4)}. The complex expression [arrow]-n-4 consists of three parts: the first part indicates a node for the word “arrow”, the second pos “n”, and the third word position 4. Edge *E4 is an edge with more than one interpretation. Each of the two elements in FCSL and DSL 748 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa irakawa PDG and its Packed Shared Data Structure “Dependency Forest” Fig. 8 Structure of edge rakawa PDG and its Packed Shared Data Structure “Dependency Forest” Fig. 8 Structure of edge irakawa PDG and its Packed Shared Data Structure “Dependency Forest” Fig. 8 Structure of edge 4.2.2 Parsing Algorithm Since only inactive edges are merged, no active edge has more than one CSDS pair in this algorithm. If the whole sentence is parsed successfully, the chart has one inactive edge with phrase head [root]-x covering the whole sentence. This edge is called “root edge,” and is described as *Eroot. Edge *E3 (edge#160) in Figure 8 is an edge generated from the grammar rule “np →det n.” The edges in the constituent sequence [153, 156] in edge#160, i.e., edge#153 and edge#156, have the phrase category “det” and “n,” respectively. Edge#153 and edge#156 are called reachable from edge#160. This “reachable” relation is associative. Edges with more than one CSDS pair, like *E4 in Figure 8, are generated by merge csds(Figure 9 (d)). Since only inactive edges are merged, no active edge has more than one CSDS pair in this algorithm. If the whole sentence is parsed successfully, the chart has one inactive edge with phrase head [root]-x covering the whole sentence. This edge is called “root edge,” and is described as *Eroot. 4.2.2 Parsing Algorithm Figure 9 shows the parsing algorithm of PDG, which is basically a standard, bottom-up, chart-parsing algorithm using an agenda (Winograd 1983). This algorithm inputs words from left to right, one by one, adds lexical edges generated from the input words to the agenda (Figure 9 (a), (b)), and combines the edges in the agenda with inactive edges in the chart or in the grammar rules until the agenda becomes empty (Figure 9 (e), (f)). Packed edges are generated by checking whether each edge in the agenda is mergeable ((c), (j)), and then merging it to the existing edge if possible. A detailed explanation of the algorithm is omitted. The following part explains the construction of the data structure, which is peculiar to PDG parsing. 749 Vol. 21 No. 4 September 2014 p chart parsing algorithm Fig. 9 PDG bottom-up chart parsing algorithm The PDG parser creates dependency structures in parallel with edge generation. This is done by binding variables in the dependency structures in an edge. Variable binding is performed by bind var, which binds the phrase head (node) of the inactive edge to the variable in the first constituent of the remaining constituent sequence of the edge, when a new edge is created, either from a grammar rule (Figure 9 (g)) or an active edge in the agenda (Figure 9 (h)). If this binding generates an arc whose dependent and governor are bound, add arcid generates a unique arc-ID and is attached to the arc (Figure 9 (i)). The arc with a bound dependent and governor is called a “fixed arc.” Edge *E3 in Figure 8 is generated by binding the variable $2 in E2 to the node [arrow]-n-4. Edges are associated through edge-IDs. The lower edge can be traced from the upper edge. Edges are associated through edge-IDs. The lower edge can be traced from the u 750 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Hirakawa Edge *E3 (edge#160) in Figure 8 is an edge generated from the grammar rule “np →det n.” The edges in the constituent sequence [153, 156] in edge#160, i.e., edge#153 and edge#156, have the phrase category “det” and “n,” respectively. Edge#153 and edge#156 are called reachable from edge#160. This “reachable” relation is associative. Edges with more than one CSDS pair, like *E4 in Figure 8, are generated by merge csds(Figure 9 (d)). Fig. 11 Phrase structure forest for “time flies like an arrow” 4.3 Generation of Phrase Structure Forest and Initial Dependency Forest When parsing is finished, the chart has active and inactive edges. The phrase structure forest for an inactive edge *E, hpf(*E), is defined as a set of edges reachable from edge *E. The phrase structure forest PF is defined as hpf(*Eroot). PF is a subset of the inactive edges in the chart, because there are inactive edges that are unreachable from *Eroot. The initial dependency graph IDG is a set of arcs in the dependency structures of the edges in PF. Figure 10 shows the algorithm Fig. 10 Algorithm for computing phrase Fig. 10 Algorithm for computing phrase structure forest and initial dependency forest Fig. 10 Algorithm for computing phrase structure forest and initial dependency forest 751 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 to compute PF, IDG, and the initial C-Matrix (ICM) from *Eroot. Figure 11 shows PF for the example sentence computed by the algorithm using the grammar in Figure 7. All RCSLs of the edges in PF are [ ], and are not shown in Figure 11. The number of edges in the phrase structure forest is called the “size of the phrase structure forest.” The size of the headed phrase structure forest is more than or equal to that of the headless phrase structure forest, because the edge merge condition of the headed phrase structure forest (Figure 9 (j)) is more strict compared with that of the headless phrase structure forest. The algorithm in Figure 10 traverses the chart by using three mutually recursive functions, try edge, try FCSL, and try CS which compute PF, IDG, and ICM for their arguments, i.e., the packed edge, the constituent sequence list, and the constituent, respectively. try edge calls try FCSL (Figure 10 (d)), try FCSL calls try CS (Figure 10 (h)), and try CS calls try edge (Figure 10 (j)). The arc in the arc sets returned from try edge(E), try FCSL(FCSL), and try CS(CS) are called the arc governed by E, FCSL, and CS, respectively. The algorithm starts from Figure 10 (a) by calling try edge(*Eroot). try edge judges whether the argument is already computed at (b). If it has already been computed, try edge simply returns the set of arcs recorded in TER. The registration of a set of arcs is performed in (g) when Fig. 4.3 Generation of Phrase Structure Forest and Initial Dependency Forest 11 Phrase structure forest for “time flies like an arrow” 752 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa a new result is obtained. At (c) and (e), new edges are added to PF. As shown at (f), the arcs governed by edge E are the union of the DSL in E and those governed by FCSL. try FCSL processes a set of CSDS pairs, and try CS processes one CSDS pair in it. As shown at (i), the set of arcs governed by FCSL is the union of the arcs governed by the CSs in the FCSL. As shown at (k), the set of arcs governed by CS is the union of the arcs governed by the packed edges in the CS. Figure 12 shows the execution process of E#170 in Figure 11. The (c#) shows a function call and (r#) shows the return value, i.e., the set of arcs of the function, and (c1) to (c4) correspond to the function calls (j), (d), and (h) in Figure 10, respectively. The (c4) returns {} at (r4) because E#103 is a lexical edge. The function call (c4) returns result (r4). Then, the second CSDS pair ([103, 119, 165], {obj-4, vpp-20}) is processed by function call (c5). The second time execution for “try edge(E#103)” occurs at (c6). This time, the execution result stored in TER at Figure 10 (b) is searched and returned. Finally, the set of arcs at (r1) is obtained. The generation of the CM is performed on the basis of the following C-Matrix setting condi- tions, which work to allow co-occurrence between all arcs in the set of edges constituting a phrase structure tree in the phrase structure forest. Definition 3 [C-Matrix setting conditions] C-Matrix setting condition” is any of the following three conditions: The “C-Matrix setting condition” is any of the following three conditions: The “C-Matrix setting condition” is any of the following three conditions: (CM1) The arcs in the same DS co-occur with each other. (CM2) Given a CSDS pair (CS, DS), the arcs in the DS co-occur with those governed by the CS. (CM1) The arcs in the same DS co-occur with each other. (CM2) Given a CSDS pair (CS, DS), the arcs in the DS co-occur with those governed by the CS. arcs in the same DS co-occur with each other. (CM3) The arcs governed by one CS co-occur with one another. (CM3) The arcs governed by one CS co-occur with one another. (CM3) The arcs governed by one CS co-occur with one another. (CM1) to (CM3) correspond to the CM processing (1) to (3) in Figure 10. In processing E#170, set CM ({obj-4, vpp-20}, {obj-4, vpp-20}) (set CM is defined in Figure 10) is executed at the CM processing (1) because the arcs in the second CSDS pair ([103, 119, 165], {obj-4, vpp-20}) satisfy the co-occurrence setting condition (CM1). At CM processing (2), A CS has been set to the set of arcs shown at (r5) in Figure 12 and set CM ({obj-4, vpp-20}, {pre-15, det-14}) is executed, due to (CM2). In processing try CS([103, 119, 165]), the CM processing (3) set CM from among the arcs governed by E#103, E#119, and E#165, due to (CM3). The outputs PF and IDF are shown in Figure 11 and 6, respectively. E#181, E#176, and E#174 have the same category “s” and the same coverage (from 0 to 5), but are not shared because their phrase heads are different. PP, V)] if the description schema {} for optional elements is introduced. This grammar rule does not generate the equivalent arcs for obj relation. 15 (R9) and (R10) are merged into one grammar rule vp/V→v/V, np/NP, ｛pp/PP｝:[arc(obj, NP, V),arc(vpp, PP, V)] if the description schema {} for optional elements is introduced. This grammar rule does not generate the equivalent arcs for obj relation. R10) are merged into one grammar rule vp/V→v/V, np/NP, ｛pp/PP｝:[arc(obj, NP, V),arc(vpp, {} 4.3 Generation of Phrase Structure Forest and Initial Dependency Forest (CM2) Given a CSDS pair (CS, DS), the arcs in the DS co-occur with those governed by the CS. Fig. 12 Example of algorithm execution Fig. 12 Example of algorithm execution 753 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 15 (R9) and (R10) are merged into one grammar rule vp/V→v/V, np/NP, ｛pp/PP｝:[arc(obj, N 4.4 Generation of the Reduced Dependency Forest The two arcs obj4 and obj25 in the IDF in Figure 6 have the same structure except for their arc-IDs. IDFs may contain arcs of this kind, called “equivalent arcs,” which are sometimes generated, from one grammar rule and sometimes from different grammar rules. For example, obj4 and obj25 are generated from the constituent sequence “vp np” in (R9) and (R10) in Figure 7. In fact, (R9) and (R10) differ in terms of the existence of the prepositional phrase, but the interpretation of the “obj” relations in (R9) and (R10) is considered to be the same.15 Here, some definitions are given for treating equivalent arcs. The “generalized arc” is an arc with arc-ID ‘?’. Arcs with number IDs are called IDed arcs. The generalized arc for an IDed arc is obtained by simply replacing the arc-ID in the IDed arc with ‘?’. A dependency tree consisting of generalized arcs is called a “generalized dependency tree.” A dependency tree consisting of IDed arcs is called an “IDed dependency tree.” The generalized arc for an IDed arc X is written as ?X. The generalized tree for an IDed tree DT is written as ?DT. Two dependency trees that have the same generalized tree are called “equivalent.” The reduced dependency forest is obtained by reducing the initial dependency forest. The reduction of the dependency forest is an operation in that more than one equivalent arc is merged into another arc without increasing the number of generalized dependency trees in the dependency forests. The reduced dependency has a smaller size, compared with the original dependency forest before the merge operation. 754 PDG and its Packed Shared Data Structure “Dependency Forest” PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa 16 This is equivalent to sharing all equivalent arcs during the parsing process 4.4.1 Merge Operation for Equivalent Arcs The merge operation for the equivalent arcs X, Y (written in equiv(X, Y )) is defined as follows: Definition 4 [Arc merge operation] Definition 4 [Arc merge operation] (1) Compute a new dependency graph DG’ by removing Y from DG. (DG’=DG−{ (2) Generate a new C-Matrix CM’ from CM by applying set CM(X, I) for arc I(I ∈DG, I ̸= X, I ̸= Y , CM(Y , I)=○) The merge operation generates a new dependency forest ⟨DG’, CM’⟩. Figure 13 shows a diagrammatic example of a merge operation. In the following sections, changes in various values are discussed; for example, the number of generalized dependency trees in the dependency forest before and after the merge operation. To make this distinction, the expression “wrt ⟨DG, CM⟩” or “wrt DF” (wrt: with respect to) is used. For example, the set of arcs that co-occurs with an arc A is defined as co(A). Then, “co(A) wrt ⟨DG, CM⟩” and “co(A) wrt ⟨DG’, CM’⟩” represent the set of arcs before and after the merge operation. “co(A) wrt ⟨DG, CM⟩= co(A) wrt ⟨DG’, CM’⟩” means the set of arcs is not changed by the merge operation. In order to simplify the description, “wrt ⟨DG, CM⟩” is not shown in default. Merging all equivalent arcs in CM16 makes the structure equivalent to a syntactic graph. This operation possibly impairs the soundness of the data structure. Fig. 13 Merge operation for the equivalent arc pair (X, Y) Fig. 13 Merge operation for the equivalent arc pair (X, Y) 755 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 4.4.2 Merge Condition for Equivalent Arcs From the definition, the condition of the dependency forest reduction is to preserve the sound- ness, i.e., no new generalized dependency tree (interpretation) increase by the merge operation. [Merge Condition for Equivalent Arcs] When the dependency forest DF’ is generated from DF by merging arc Y with X in the dependency graph of DF, following is the condition for forest reduction: “a set of generalized dependency trees in DF = a set of generalized dependency trees in DF’.” “a set of generalized dependency trees in DF = a set of generalized dependency trees in DF’.” This condition is verifiable by searching a new generalized dependency tree in DF’. The condition for the existence of a generalized dependency tree not in DF but in DF’ is called an “increase con- dition for generalized dependency trees” (ICG) in this paper. The merge condition is represented as “ICG is not satisfied.” A dependency forest is a set of IDed arcs, and prescribes the set of IDed dependency trees and that of generalized dependency trees. The condition for the existence of an IDed dependency tree not in DF but in DF’ (this kind of tree is called a “new” dependency tree) is called an “increase condition for IDed dependency trees” (ICI). Obviously, if no IDed dependency tree increases by the merge operation, no generalized dependency tree increases. Moreover, even if there exist new IDed dependency trees in DF’, no generalized dependency trees increase when the generalized dependency trees of the new IDed dependency trees are equivalent to those in DF. This indicates that ICI is a necessary condition for ICG. In the following, ICI is discussed first, and then ICG is verified to obtain a detailed merge condition for equivalent arcs. 4.4.3 Increase Condition for IDed Dependency Trees Increase of a new IDed dependency tree is caused by allowing a new co-occurrence relation between arcs caused by a merge of equivalent arcs. The allowance of the co-occurrence relation in CM, i.e., the change from CM(U, V)̸= ○to CM(U, V)= ○for arcs U, V, is called an “allowance of the arc pair (U, V).” The following lemma is established. Lemma 4.1 (The allowance of arc pair and the increase of dependency trees) If a new well-formed dependency tree increases by the allowance of the arc pair (U, V), the de- pendency tree includes U and V. Proof This lemma is obvious because well-formed dependency trees in DG’, which include neither U nor V, exist in DG. 756 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Here, uniq and diffare the sets of arcs defined for the equivalent arcs X and Y, as follows: uniq(X, Y)={I | CM(X, I)=○, CM(Y, I)̸= ○, I∈DG} diff(X, Y)={(I, J) | I ∈uniq(X, Y), J ∈uniq(Y, X)} For arcs X, Y in Figure 13, uniq(X, Y)={j, n}, uniq(Y, X)={k}, and diff(X, Y)={(j, k), (n, k)}. The following lemma is established. The following lemma is established. Lemma 4.2 (Arcs in a new well-formed dependency tree) In the case that a well-formed dependency tree is generated by the merge of equivalent arcs X, Y, the new tree includes at least two arcs A, B, such that (A, B) ∈diff(X, Y). Lemma 4.2 (Arcs in a new well-formed dependency tree) In the case that a well-formed dependency tree is generated by the merge of equivalent arcs X, Y, the new tree includes at least two arcs A, B, such that (A, B) ∈diff(X, Y). Proof Let DF and DF’ be the dependency forests before and after the merge of X and Y. Assume that a new dependency tree DTx is obtained by the allowance of the arc pair (X, Bi) caused by the merge of X and Y. X and Bi are included in DTx, according to Lemma 4.1. Here, let R=DTx−{X, Bi}. CM(X, U)= ○wrt DF’, CM(Bi, U)=○wrt DF’ for U∈R, because DTx is a well-formed dependency tree. Assuming that there is no arc U such that CM(Y, U)̸= ○wrt DF (i.e., CM(Y, U)= ○wrt DF, U∈R), DTy={Y, Bi}+R is a well-formed dependency tree in DF. 4.4.3 Increase Condition for IDed Dependency Trees DTx is not a new generalized dependency tree because DTx and DTy differ only in equivalent arc X and Y, i.e., DTx and DTy are equivalent. Therefore, DTx must include at least one Ui, such that CM(Y, Ui)̸= ○wrt DF is a new generalized dependency tree. This lemma is established because (Bi, Ui)∈diff(X, Y). The following theorem is derived from Lemma 4.2. Journal of Natural Language Processing Vol. 21 No. 4 In the case of (RC3), the existence of a well-formed dependency tree that includes {X, A, B}, i.e., dts with arcs(X, A, B) wrt ⟨DG’, CM’⟩={}, should be checked for ICI. Journal of Natural Language Processing Vol. 21 No. 4 Vol. 21 No. 4 September 2014 B} is a new well-formed IDed dependency tree. Some functions and notations are introduced for discussion on the increase condition of depen- dency trees. same position(U, V ): The positions of dependent nodes of U and V are the same same position(U, V ): The positions of dependent nodes of U and V are the same. same position(U, V ): The positions of dependent nodes of U and V are the same. dts(S) wrt ⟨DG, CM ⟩: a set of IDed well-formed dependency trees that consist of arcs in arc set S⊂DG and satisfy the arc co-occurrence constraint co(U) wrt ⟨DG, CM ⟩: a set of arcs that co-occur with arc U including U, i.e., {X | X=U or CM(X, U)=○, X∈DG} dts with arcs(A1, A2, . . . , An) wrt ⟨DG, CM ⟩: a set of well-formed dependency trees in ⟨DG, CM ⟩that include arcs A1, A2, . . . , An, i.e., dts(co(A1)∪· · ·∪co(An)) wrt ⟨DG, CM ⟩ same position(U, V ): The positions of dependent nodes of U and V are the same. dts(S) wrt ⟨DG, CM ⟩: a set of IDed well-formed dependency trees that consist of arcs in arc set S⊂DG and satisfy the arc co-occurrence constraint co(U) wrt ⟨DG, CM ⟩: a set of arcs that co-occur with arc U including U, i.e., {X | X=U or CM(X, U)=○, X∈DG} dts with arcs(A1, A2, . . . , An) wrt ⟨DG, CM ⟩: a set of well-formed dependency trees in ⟨DG, CM ⟩that include arcs A1, A2, . . . , An, i.e., dts(co(A1)∪· · ·∪co(An)) wrt ⟨DG, CM ⟩ dts(S) wrt ⟨DG, CM ⟩: a set of IDed well-formed dependency trees that consist of arcs in arc set S⊂DG and satisfy the arc co-occurrence constraint ICI with respect to the arc pair (A, B) ∈diff(X, Y), equiv(X, Y) can be checked by searching the existence of a well-formed dependency tree including {X, A, B} in ⟨DG’, CM’⟩, according to Theorem 4.1. To make this search process more efficient, the following three cases with respect to arcs X, A, and B are considered. In cases (RC1) and (RC2), no well-formed dependency trees that include {X, A, B} exist in ⟨DG’, CM’⟩because of the existence of the single role constraint and the co-occurrence constraint, respectively. Theorem 4.1 (The increase condition for IDed dependency tree) Let arc pair (A, B)∈diff(X, Y) for equivalent arcs X, Y in DG of the dependency forest ⟨DG, CM⟩. The IDed dependency trees increase if and only if ⟨DG’, CM’⟩obtained by the merge of Y to X has an IDed dependency tree NDT that includes {X, A, B}. Let arc pair (A, B)∈diff(X, Y) for equivalent arcs X, Y in DG of the dependency forest ⟨DG, CM⟩. The IDed dependency trees increase if and only if ⟨DG’, CM’⟩obtained by the merge of Y to X has an IDed dependency tree NDT that includes {X, A, B}. Proof This theorem is proved by showing that a new well-formed IDed dependency tree in- cludes {X, A, B} and a well-formed IDed dependency tree that includes {X, A, B} is a new well-formed IDed dependency tree. Assuming that NDT is a new IDed dependency tree existing in ⟨DG’, CM’⟩, there exists at least one arc pair (Ai, Bi)∈diff(X, Y), Ai ∈NDT, Bi ∈NDT. On the other hand, X∈NDT is true, due to Lemma 4.1. Therefore, a new well-formed IDed dependency tree includes {X, A, B}. Moreover, no IDed well-formed dependency trees exist in ⟨DG, CM⟩ because (A, B)∈diff(X, Y). Therefore, an IDed well-formed dependency tree that includes {X, A, 757 Journal of Natural Language Processing Vol. 21 No. 4 Journal of Natural Language Processing Vol. 21 No. 4 As described above, ICI is a necessary condition for ICG. Therefore, ICG is defined as follows: [The increase condition for generalized dependency tree] As described above, ICI is a necessary condition for ICG. Therefore, ICG is defined as follows: [The increase condition for generalized dependency tree] As described above, ICI is a necessary condition for ICG. Therefore, ICG is defined as follows: [The increase condition for generalized dependency tree] Let DF’ be a dependency forest generated from dependency forest DF by merging arc Y to X, where X and Y are equivalent arcs, and let DTnew be the set of IDed dependency trees not in DF but in DF’. There exists at least one IDed dependency tree DT ∈DTnew, such that the generalized dependency tree ?DT is not included in DF. The merge condition for equivalent arcs is the negation of ICG for the arcs. 758 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Fig. 14 Algorithm for reduction of dependency forest 4.4.5 Dependency Forest Reduction Algorithm Figure 14 shows the dependency forest reduction algorithm for ⟨DG, CM⟩, based on the merge condition for equivalent arcs (i.e., the condition for forest reduction) described in the previous section. In this algorithm, CM is represented as a set of co-occurable arc pairs. Arcs X and Y are co-occurable if ⟨X,Y⟩∈CM. Figure 14 (a) selects a pair of equivalent arcs X, Y in DG, and the part from (b) to (h) checks Figure 14 (a) selects a pair of equivalent arcs X, Y in DG, and the part from (b) to (h) checks 759 Journal of Natural Language Processing Vol. 21 No. 4 Vol. 21 No. 4 September 2014 if a new generalized dependency tree is generated in the dependency forest when arc pair (A, B) is allowed. If all arc pairs in diff(X, Y) fail to generate any new generalized dependency tree, the forest reduction is performed at (i). The availability of the allowance of (A, B) is determined by checking ICG after checking ICI. At Figure 14 (b), the conditions (RC1) and (RC2) in Section 4.4.3 are checked. If either of the conditions is satisfied, the processing proceeds to the next arc pair in diff(X, Y) because the allowance of (A, B) generates no new IDed dependency trees. If not, the processing proceeds to the check of ICI. At (c), ⟨DG’, CM’⟩is generated by merging Y to X. Since the existence check of a new IDed tree is basically performed by tree search for ⟨DG’, CM’⟩, the reduction of search space improves the efficiency. Based on Theorem 4.1, the search space is reduced from DG’ to DG XAB(=co(X)∩co(A)∩co(B)) at (d). Then, search dt at (e) searches a new IDed dependency tree for DG XAB. If no dependency tree is obtained, the allowance of (A, B) satisfies the merge condition for equivalent arcs. If a dependency tree DT is obtained, DT is an IDed dependency tree that includes {X, A, B}. new generalized dt(DT, CM, DG) at (f) checks whether the generalized dependency tree ?DT is new by searching ?DT for ⟨DG,CM⟩. The detailed explanation of new generalized dt is omitted here, but it realizes the search for the generalized dependency tree by limiting the arc set DG X so that it has only the equivalent arcs of the arcs in DT by add equiv arcs at (q). 4.4.5 Dependency Forest Reduction Algorithm When ?DT is a new generalized dependency tree, the merging of X and Y is not available. Processing for arcs X and Y is terminated by (g) and (h), and proceeds to the check of the next equivalent arc pair. If ?DT is not new, the merging of X and Y, i.e., the forest reduction, is performed at (i). Furthermore, when ?DT is proved to be not new at (f), the search of other dependency trees for DG XAB is performed at (e). search dt searches a dependency tree that satisfies the co-occurrence condition in depth-first manner with respect to input position P. search dt selects one arc from the arc set arcs at(DG, P) that is a set of arcs with position P. search dt searches all possible dependency trees by selecting another arc at (k) when there are no solutions for arcs, from P + 1 to the end position at (m). 4.4.6 Execution Example of the Dependency Forest Reduction Algorithm This section explains the execution process of the algorithm in Figure 14 for the example sentence “Tokyo taxi driver call center” in Appendix A. The reduced dependency forest for this example has equivalent arcs. Figure 15 (a) shows the initial dependency forest for the example sentence. It has four sets of equivalent arcs, (1, 2), (5, 7), (13, 15), (25, 26, 27), which are surrounded with double lines. The forest reduction is performed along with the algorithm in Figure 14. The first equivalent 760 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Fig. 15 Initial dependency forest and its reduction Fig. 15 Initial dependency forest and its reduction arc pair (1, 2) is selected to set X = 1, Y = 2. diff(X, Y ) is computed as {(5, 14), (5, 15), (5, 27), . . . } by combining the elements in uniq(X, Y ) = {5, 24, 25} and uniq(Y, X) = {14, 15, 27}. The first arc pair (5, 14) is skipped by the condition check for (RC1) at Figure 14 (b) because same position(5, 14) is true. The second arc pair (5, 15) is tried. (5, 15) does not match the conditions at (b), so CM’ and DG’ are generated at (c). CM′ = CM + {⟨1, 14⟩, ⟨1, 15⟩, ⟨1, 27⟩}, as shown in Figure 15 (b). Then, DG XAB is computed at (d). X = 1, A = 5, and B = 15 result in DG XAB = co(1) ∩co(5) ∩co(15) wrt ⟨DG’, CM’⟩= {1, 28}. The allowance of (5, 15) generates no new dependency trees because the search of the dependency tree for DG XAB by search dt at (e) fails. The processing proceeds to the check of the next arc pair (5, 27). Similarly, all arc pairs in diff(1, 2) are assured to generate no new dependency trees, and then DG’ and CM’ are set to DG and CM at (i), respectively. Figure 15 (c) shows the reduced dependency forest finally obtained by the algorithm. It has three equivalent arcs 25, 16, and 27. The processing of the algorithm for this dependency forest is described. Let X = 25, Y = 26. Then, uniq(X, Y ) = {1, 24}, uniq(Y, X) = {6, 13}, diff(X, Y ) = {(1, 6), (1, 13), (24, 6), (24, 23)}. The arc pair (1, 6) is skipped by the condition check for (RC1). 4.4.6 Execution Example of the Dependency Forest Reduction Algorithm Therefore, the merging of X = 25 and Y = 26 is not performed. The dependency forest in Figure 15 (c) includes the dependency trees in Figure 26 (a) to (c), and retains the soundness. DG XAB = co(25)∩co(1)∩co(13) wrt ⟨DG’, CM’⟩= {25, 1, 13, 5, 28}. A new IDed dependency tree {25, 1, 13, 5, 28} is obtained by search dt at (e) for DG XAB.17 Then, new generalized dt at (f) is called, and add equiv arcs at (q) computes a set of arcs DG X, where the equivalent arcs of the arcs in DT are added. In the case of Figure 15 (c), arc 25 has equivalent arcs 26 and 27. Addition of these arcs results in DG X = {25, 26, 27, 1, 13, 5, 28}. search dt at Figure 14 (r) tries to get a dependency tree for DG X, but fails because all equivalent arcs 25, 26, and 27 have inconsistent arcs in DG X, i.e., ⟨25, 13⟩, ⟨26, 1⟩, and ⟨27, 5⟩are not in CM. As a result, new generalized dt at (f) becomes true, that is, an increase of the generalized dependency tree occurs. Therefore, the merging of X = 25 and Y = 26 is not performed. The dependency forest in Figure 15 (c) includes the dependency trees in Figure 26 (a) to (c), and retains the soundness. The above algorithm does not assure generation of one reduced dependency forest. The output dependency forest may vary by the application order of the merge operations for equivalent arcs. There exist different dependency forests containing the same three generalized dependency trees for the above example. The algorithm in Figure 14 does not assure that it generates the minimum dependency forest. In fact, there exists a dependency forest smaller than that in Figure 15 (c). Moreover, there is room for improving the computational amount in the above algorithm. The construction of the smallest reduced dependency forest and improvement in algorithm performance are future tasks.18 The above algorithm does not assure generation of one reduced dependency forest. The output dependency forest may vary by the application order of the merge operations for equivalent arcs. There exist different dependency forests containing the same three generalized dependency trees for the above example. The algorithm in Figure 14 does not assure that it generates the minimum dependency forest. ( ) 18 PDG allows arbitrary mapping between the constituent sequences and the partial dependency trees defined in grammar rules. Therefore, any dependency structure can be assigned for any constituent sequence, provided that they satisfy the partial dependency structure condition. This feature suggests that, not only the opti- mization techniques in the general algorithm, but also the techniques based on the structural analysis of the grammar rules are effective. 4.4.6 Execution Example of the Dependency Forest Reduction Algorithm In fact, there exists a dependency forest smaller than that in Figure 15 (c). Moreover, there is room for improving the computational amount in the above algorithm. The construction of the smallest reduced dependency forest and improvement in algorithm performance are future tasks.18 17 This tree corresponds to the tree in Figure 26 (d). 7 This tree corresponds to the tree in Figure 26 (d). 4.4.6 Execution Example of the Dependency Forest Reduction Algorithm The arc pair (1, 13) is not skipped by the condition check for (RC1) and (RC2). Then, DG XAB is computed at Figure 14 (d). X = 25, A = 1, and B = 13 result in 761 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 DG XAB = co(25)∩co(1)∩co(13) wrt ⟨DG’, CM’⟩= {25, 1, 13, 5, 28}. A new IDed dependency tree {25, 1, 13, 5, 28} is obtained by search dt at (e) for DG XAB.17 Then, new generalized dt at (f) is called, and add equiv arcs at (q) computes a set of arcs DG X, where the equivalent arcs of the arcs in DT are added. In the case of Figure 15 (c), arc 25 has equivalent arcs 26 and 27. Addition of these arcs results in DG X = {25, 26, 27, 1, 13, 5, 28}. search dt at Figure 14 (r) tries to get a dependency tree for DG X, but fails because all equivalent arcs 25, 26, and 27 have inconsistent arcs in DG X, i.e., ⟨25, 13⟩, ⟨26, 1⟩, and ⟨27, 5⟩are not in CM. As a result, new generalized dt at (f) becomes true, that is, an increase of the generalized dependency tree occurs. Therefore, the merging of X = 25 and Y = 26 is not performed. The dependency forest in Figure 15 (c) includes the dependency trees in Figure 26 (a) to (c), and retains the soundness. DG XAB = co(25)∩co(1)∩co(13) wrt ⟨DG’, CM’⟩= {25, 1, 13, 5, 28}. A new IDed dependency tree {25, 1, 13, 5, 28} is obtained by search dt at (e) for DG XAB.17 Then, new generalized dt at (f) is called, and add equiv arcs at (q) computes a set of arcs DG X, where the equivalent arcs of the arcs in DT are added. In the case of Figure 15 (c), arc 25 has equivalent arcs 26 and 27. Addition of these arcs results in DG X = {25, 26, 27, 1, 13, 5, 28}. search dt at Figure 14 (r) tries to get a dependency tree for DG X, but fails because all equivalent arcs 25, 26, and 27 have inconsistent arcs in DG X, i.e., ⟨25, 13⟩, ⟨26, 1⟩, and ⟨27, 5⟩are not in CM. As a result, new generalized dt at (f) becomes true, that is, an increase of the generalized dependency tree occurs. sponds to the tree in Figure 26 (d). p g ( ) arbitrary mapping between the constituent sequences and the partial dependency trees defined in 4.5 Correspondence between Phrase Structure Forest and Dependency Forest Appendix B shows that the initial dependency forest satisfies the completeness and soundness with respect to the phrase structure forest. Since the reduced dependency forest has the same set of generalized dependency trees as the initial dependency forest, the soundness and completeness between the (reduced) dependency forest and the phrase structure forest are assured. The cor- respondences between the phrase structure trees (phrase structure trees) in the phrase structure forest and the dependency trees in the dependency forest are not necessarily simple one-to-one relations. One phrase structure tree may correspond to more than one dependency tree, whereas more than one phrase structure tree may correspond to one dependency tree. Considering the va- 762 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa riety (one meaning can be expressed by more than one expression) and ambiguity (one expression expresses more than one meaning) encoded in natural languages, these multiple correspondences may be natural. The correspondences between phrase structure trees and dependency trees are discussed in the next section by referring to the experiments for sentence analysis using a PDG prototype system. 5 Experiment for Analysis of Example Sentences One of the design targets of PDG is the suppression of the combinatorial explosion caused by a variety of ambiguities using packed shared data structures. This section describes the experiment for analyzing typical ambiguous sentences, which contain various kinds of ambiguities, using PDG grammar rules. This section also discusses the relation between the phrase structure forest and the dependency forest, and the generation of non-projective dependency trees on the basis of real analysis examples. The performance of the algorithm is an important factor from a practical viewpoint. The algorithms for parsing, generation of phrase structure forest, and initial dependency forest and dependency forest reduction described in this paper are implemented for verifying the PDG analysis. The practical implementation and its evaluation are subjects for future work. The following experiment utilizes a prototype PDG system, implemented in Prolog. 5.1 PDG Grammar Rules for Example Sentences Figure 16 shows PDG grammar rules used for analyzing the example sentences containing various prototypical ambiguities. The POSs in the grammar rules are determiner(det), n(noun), be-verb(be), present particle of verb (ving), verb (v), preposition (pre), adverb (adv), and rela- tional phrase (relp). The grammar rules are not for linguistic analysis, but for experiments with respect to the PDG framework and algorithms. The grammar rules include the following types of ambiguities. PP attachment: Two kinds of attachment ambiguities in R6 (noun attachment) and R10, R17 (verb attachment) Coordination scope:]R11 (and) and R12 (or) are coordination noun phrase rules Coordination scope:]R11 (and) and R12 (or) are coordination noun phrase rules Be-verb interpretation: Two structural ambiguities caused by be-verb interpretations, i.e., R15 (present progressive) and R16 (copula) Be-verb interpretation: Two structural ambiguities caused by be-verb interpretations, i.e., R15 (present progressive) and R16 (copula) Interpretation of the present progressive form of verb: the following three ambiguities of the present progressive form of verb are described (a) adjective usage where the modified noun occupies the subject role (R7) (a) adjective usage where the modified noun occupies the subject role (R7) 763 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 Fig. 16 Grammar for the example sentences Journal of Natural Language Processing Vol. 21 No. 4 September 2014 (b) adjective usage where the modified noun occupies the object role (R8) R8 and R9 are similar since both rules prescribe the relation between a noun and verb as an object. However, they have different interpretations in the phrase head and generate different structures of dependency trees. The grammar contains declarative form (R1) and imperative sentence form (R2) to produce structural ambiguities in combination with POS ambiguities of words for sentences like “Time flies like an arrow.” (R19) is a rule for generating a non-projective dependency structure. PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa 19 Preference scores show the degree of preference of arcs (Hirakawa 2006, 2007). The preference score is not used in this paper. 20 In judging the equivalence of the phrase structure trees, phase heads (head nodes) are taken into consideration. 5.2 Analysis of Prototypical Ambiguous Sentences The example for an ambiguous sentence with POS ambiguities has already been shown in the previous sections in detail. The examples in the following sections show prototypical syntactic ambiguities, i.e., PP attachment ambiguity, coordination scope ambiguity, and ambiguities in structural interpretations. 764 PDG and its Packed Shared Data Structure “Dependency Forest” 5.2.1 PP attachment Ambiguity The above equivalent arcs are generated from the combination with edges np(ID:126), np(ID:163), and np(ID:203), which correspond to noun phrases with different coverage. The reduced dependency forest has no equivalent arcs because all equivalent arcs in the initial dependency forest are merged. 21 The packed edge whose category is v and edge-ID is 109. 5.2.1 PP attachment Ambiguity Figure 17 shows the dependency forest for the sentence “I saw a girl with a telescope in the forest,” which has PP attachment ambiguities. Each arc in the dependency graph has an arc name attached by arc-ID and preference score.19 The table in Figure 17 shows the POS and position information of each node. This sentence has no POS ambiguities, but has PP attachment ambiguities for prepositions “with” (two ambiguities: npp13, vpp14) and “in” (three ambiguities: npp23, npp25, vpp26). CM in Figure 17 inhibits some of the combinations of these ambiguous arcs. npp13 and vpp14 (or npp23, npp25, and vpp26) have no co-occurrence relation because they have the same position (the single role constraint). The co-occurrence between vpp14 and npp25 is also inhibited. If this co-occurrence constraint does not exist, the dependency forest has six interpretations caused by two PP attachment ambiguities (2 ∗3 = 6). CM(14, 25)̸= ○, which corresponds to the projectivity constraint, excludes a non-projective dependency tree from a set of well-formed dependency trees in the dependency forest. This dependency forest has five well-formed dependency trees, which are possible interpretations for the example sentence. The sizes of the phrase structure forest, the initial dependency forest, and the reduced depen- dency forest for this example are 25, 18, and 13, respectively. The phrase structure forest contains five phrase structure trees20 corresponding to the five interpretations of the sentence. The initial Fig. 17 DF for the example sentence including PP attachments Fig. 17 DF for the example sentence including PP attachments 765 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 dependency forest and the reduced dependency forest have five IDed dependency trees, which correspond to the five generalized dependency trees. obj5, npp13, vpp14, and pre11 have 2, 1, 1, and 1 equivalent arcs in the initial dependency forest, respectively. For example, obj5 and its equivalent arcs are generated from the edges, shown below in diagrammatic form, originated in the grammar rule (R14). ⟨1, 4, vp/([saw]-v-1) →v(ID:109) np(ID:126) ・, {arc(obj-5, [girl]-n-3, [saw]-v-1)}⟩ ⟨1, 7, vp/([saw]-v-1) →v(ID:109) np(ID:163) ・, {arc(obj-15, [girl]-n-3, [saw]-v-1)}⟩ ⟨1, 10, vp/([saw]-v-1) →v(ID:109) np(ID:203) ・, {arc(obj-28, [girl]-n-3, [saw]-v-1)}⟩ The first edge has coverage from 1 to 4 (“saw a girl”), phrase head [saw]-v-1, constituent sequence v(ID:109)21 np(ID:126), and the obj arc with arc-ID 5. 5.2.2 Coordination Scope Ambiguity Figure 18 shows the dependency forest for the sentence “Earth and Moon or Jupiter and Ganymede,” which has coordination scope ambiguities. “Earth” and “Moon” have two and three outgoing arcs, respectively, corresponding to coordination scope ambiguities. CM(22, 12)̸= ○, which corresponds to the projectivity constraint, excludes a non-projective dependency tree from Fig. 18 DF for the example sentence, including conjunctions Fig. 18 DF for the example sentence, including conjunctions 766 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa a set of well-formed dependency trees in the dependency forest. This dependency forest has five well-formed dependency trees, which are possible interpretations for the example sentence. The sizes of the phrase structure forest, the initial dependency forest, and the reduced de- pendency forest for this example are 18, 17, and 10, respectively. The phrase structure forest contains five phrase structure trees corresponding to the five interpretations of the sentence. The initial dependency forest and the reduced dependency forest have five IDed dependency trees, which correspond to the five generalized dependency trees. or22, or9, cnj6, and18, and cnj14 have 1, 1, 1, 2, and 2 equivalent arcs in the initial dependency forest, respectively. The reduced dependency forest has no equivalent arcs because all equivalent arcs in the initial dependency forest are merged. The coordination scope ambiguity is similar to the PP attachment ambiguity in the previous section, but is different from the PP attachment ambiguity because it has the modification scope problem described below. Vol. 21 No. 4 Vol. 21 No. 4 September 2014 The sizes of the phrase structure forest, the initial dependency forest, and the reduced de- pendency forest for this example are 23, 24, and 16, respectively. The phrase structure forest contains eight phrase structure trees, corresponding to 10 interpretations of the sentence. The initial dependency forest and the reduced dependency forest have 10 IDed dependency trees, which correspond to the 10 generalized dependency trees. dsc9, dsc8, obj5, npp21, and vpp22 have 2, 2, 5, 2, and 2 equivalent arcs in the initial dependency forest, respectively. In contrast to the example in Section 5.2.1, the equivalent arcs are generated from more than one grammar rule. For example, the equivalent arcs of obj5 are generated from the edges, shown below, originated in grammar rules (R9), (R10), and (R15) (R9) ⇒⟨3, 5, np/([watching]-ving-3) →ving(ID:121) np(ID:130)・, {arc(obj-5, [birds]-n-4, [watching]-ving-3)}⟩ (R9) ⇒⟨3, 5, np/([watching]-ving-3) →ving(ID:121) np(ID:130)・, {arc(obj-5, [birds]-n-4, [watching]-ving-3)}⟩ (R10) ⇒⟨3, 7, np/([watching]-ving-3) →ving(ID:121) np(ID:130) pp(ID:176)・, (R10) ⇒⟨3, 7, np/([watching]-ving-3) →ving(ID:121) np(ID:130) pp(ID:176)・, {arc(obj-6 [birds]-n-4 [watching]-ving-3) arc(vpp-22 [with]-pre-5 [watching]-vin (R10) ⇒⟨3, 7, np/([watching]-ving-3) →ving(ID:121) np(ID:130) pp(ID:176)・, {arc(obj-6, [birds]-n-4, [watching]-ving-3), arc(vpp-22, [with]-pre-5, [watching]-ving-3)}⟩ (R15) ⇒⟨2, 5, vp/([watching]-ving-3) →be(ID:117) ving(ID:121) np(ID:130)・, {arc(prg-2, [is]-be-2, [watching]-ving-3), arc(obj-7, [birds]-n-4, [watching]-ving-3)}⟩ {arc(obj-6, [birds]-n-4, [watching]-ving-3), arc(vpp-22, [with]-pre-5, [watching]-v (R15) ⇒⟨2, 5, vp/([watching]-ving-3) →be(ID:117) ving(ID:121) np(ID:130)・, {arc(prg-2, [is]-be-2, [watching]-ving-3), arc(obj-7, [birds]-n-4, [watching]-ving-3 j-6, [birds]-n-4, [watching]-ving-3), arc(vpp-22, [with]-pre-5, [watching]-ving-3)}⟩ ⟨2, 5, vp/([watching]-ving-3) →be(ID:117) ving(ID:121) np(ID:130)・, (prg-2, [is]-be-2, [watching]-ving-3), arc(obj-7, [birds]-n-4, [watching]-ving-3)}⟩ The reduced dependency forest has no equivalent arcs. In this example, the number of phrase structure trees in the phrase structure forest is smaller than that of generalized dependency trees in the dependency forest. One phrase structure tree corresponds to more than one dependency tree. The following section discusses the correspondence between phrase structure trees and dependency trees. 5.3 1 to N / N to 1 Correspondences between Phrase Structure Trees and Dependency Trees The correspondence between a phrase structure tree and a dependency tree is assured in PDG, but sometimes, one phrase structure tree has more than one corresponding dependency tree, and more than one phrase structure tree has one corresponding dependency tree. 5.2.3 Ambiguity in Structural Interpretation Figure 19 shows the dependency forest for the sentence “My hobby is watching birds with a telescope,” which has ambiguities such as the interpretation of be-verb (present progressive form or copula), the interpretation of “watching birds” (adjs3, adjo4, obj5), and PP attachment (npp21, vpp22, npp24, vpp25). This sentence has 10 interpretations. Fig. 19 DF for the example sentence, including structural ambiguities 767 ournal of Natural Language Processing Vol. 21 No. 4 Sep Journal of Natural Language Processing Vol. 21 No. 4 5.3.1 1 to N Correspondence from Phrase Structure Tree to Dependency Trees When one phrase structure has more than one interpretation, one phrase structure tree may correspond to more than one dependency tree. For example, when “watching birds” is assigned to one phrasal structure where the verb in present particle form modifies the noun, two depen- dency structures, “watching subj −−−→birds” and “watching obj −−→birds,” are assigned to the phrasal structure. This happens when there exist more than one grammar rule having the same rewriting rule but different dependency structure parts. This is the case for (R7) and (R8) in Figure 16, 768 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa which are arbitrary rules, introduced for verifying the dependency forest. Two kinds of ambi- guities in 1 to N mapping from phrase structure trees to dependency trees are considered, i.e., the ambiguities in functional relation and semantic relation. The former means the ambiguities in functional assignments (subject, object, etc.) for phrase structures. The functional structures and phrase structures have close relation, and the difference in functional structures can be re- flected by the difference in phrase structure.22 Therefore, it seems to be unusual for more than one functional structure to be assigned to one rewriting rule. In contrast, the assignment of more than one semantic structure seems to be quite a general phenomenon. However, introduction of 1 to N mapping (from phrase structure to dependency structures) into grammar rules may cause problems in terms of system performance and grammar rule maintenance. In general, sen- tence analysis approaches, which treat syntactic analysis and semantic analysis independently, are widely proposed and utilized. PDG introduces a semantic analysis level utilizing an SDG, which represents lexical concepts and semantic relations between them. The framework itself, for mapping one phrase structure tree to more than one dependency tree, is independent of the linguistic discussion here. Rules with mappings to semantic structures can be utilized properly with respect to the requirements from the design and development of the grammar. The example in Section 5.2.3 (Figure 19) contains a phrase structure tree that corresponds to two dependency structures generated from (R7) and (R8). Figure 20 shows this phrase structure tree and dependency trees. Fig. 20 Mapping from one phrase structure tree to two dependency trees Fig. 22 For example, the category in the rewriting rule is segmented into more detailed categories, reflecting the difference in functional assignments. This segmentation works to remove ambiguous mapping from one phrase structure to more than one functional structure. 5.3.1 1 to N Correspondence from Phrase Structure Tree to Dependency Trees 20 Mapping from one phrase structure tree to two dependency trees 22 For example, the category in the rewriting rule is segmented into more detailed categories, reflecting the difference in functional assignments. This segmentation works to remove ambiguous mapping from one phrase structure to more than one functional structure. 769 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 23 For example, “softly knock twice” has two equivalent semantic interpretations softly(twice(knock)) and twice(softly(knock)), whereas “intentionally knock twice” has two different semantic interpretations inten- tionally(twice(knock)) and twice(intentionally(knock)). y( ( )) ( y( )) 24 The model theoretic ambiguities as observed in, “Three men bought ten cups,” cannot be distinguished by standard phrase structure and dependency structure representations. 23 For example, “softly knock twice” has two equivalent semantic interpretations softly(twice(knock)) and twice(softly(knock)), whereas “intentionally knock twice” has two different semantic interpretations inten- tionally(twice(knock)) and twice(intentionally(knock)). 24 The model theoretic ambiguities as observed in, “Three men bought ten cups,” cannot be distinguished by t d d h t t d d d t t t ti 5.3.2 N to 1 Correspondence from Phrase Structure Trees to One Dependency Tree Spurious ambiguity (Noro, Okazaki, Tokunaga, and Tanaka 2002) is an example of N to 1 mapping from phrase structures to dependency structure. A real ambiguity is one where the difference in syntactic structures represents the difference in semantic interpretations. Spurious ambiguity is one where the difference in syntactic structures does not represent the difference in semantic interpretations, or is an ambiguity caused by linguistically illegal syntactic structures, generated by incomplete grammar rules. Spurious ambiguity is an important issue in grammar development from corpora (Noro, Hashimoto, Tokunaga, and Tanaka 2005). Although it is not CFG, CCG has many spurious ambiguities because of the flexibility of rule application. A method for obtaining one normal form tree is proposed (Eisner 1996), which ensures that one phrase structure tree among the phrase structure trees in one semantic class is obtained on the basis of the definition that phrase structure trees having the same set of leaf CCG category have the same meaning. In PDG, phrase structure trees corresponding to the same generalized dependency tree (interpretation of a sentence) can be classified into one semantic class. Figure 21 shows the dependency forest for the example “She curiously saw a cat in the forest” using the example grammar having a spurious ambiguity. There is only one co-occurrence con- straint between npp17 and vpp18 that corresponds to the single role constraint. The dependency forest has two dependency trees, which have different governors for the part “in the forest.” The phrase structure forest has three phrase structure trees. The initial dependency forest has three IDed dependency trees and two generalized dependency trees and the reduced dependency forest Fig. 21 DF containing the mapping from N phrase structure trees to 1 dependency tree (spurious ambiguity) Fig. 21 DF containing the mapping from N phrase structure trees to 1 dependency tree (spurious ambiguity) Fig. 21 DF containing the mapping from N phrase structure trees to 1 dependency tree (spurious ambiguity) 770 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa twice(softly(knock)), whereas “intentionally knock twice” has two different semantic interpretations inten- tionally(twice(knock)) and twice(intentionally(knock)). 24 The model theoretic ambiguities as observed in, “Three men bought ten cups,” cannot be distinguished by standard phrase structure and dependency structure representations. Hirakawa has two IDed dependency trees and two generalized dependency trees. The spurious ambigui- ties are generated from the difference in rule application order of (R17) and (R18) for attaching a modification phrase to a verb phrase. Figure 22 shows the phrase structure trees and the dependency tree. Obviously, the method of identifying the semantic class based on the dependency tree does not capture all semantic aspects in natural languages. For example, the subtle semantic differ- ence (Eisner 1996)23 and the ambiguities in number/quantifier scope24 have to be considered in discussing the equivalent semantic class. The treatment of differences in semantic interpretation requires further study. Mel’cuk discussed some linguistic structures where an ordinary depen- dency structure fails to express the interpretations of a sentence (Mel’cuk 1988). These structures Fig. 22 Example of mapping from N phrase structure trees to 1 dependency tree (spurious ambiguity) Fig. 22 Example of mapping from N phrase structure trees to 1 dependency tree (spurious ambiguity) Fig. 22 Example of mapping from N phrase structure trees to 1 dependency tree (spurious ambiguity) Fig. 22 Example of mapping from N phrase structure trees to 1 dependency tree (spurious ambiguity) Fig. 22 Example of mapping from N phrase structure trees to 1 dependency tree (spurious ambiguity) 771 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 are classified into two categories: the structures, which cannot be treated by phrase structure properly, and the others. The former includes the model theoretic interpretation of a sentence. The latter is observed when a dependency structure has a head word that has dependents located at the right- and left-hand sides of the headword. In this case, the dependency structure has ambiguities in modification scope, i.e., the right-hand modifier modifies only the headword or the phrase including the left-hand modifier. This problem is called the “modification scope problem” in this paper. Figure 23 shows the dependency forest for “Earth and Jupiter in Solar System.” This sentence has two interpretations, i.e., the prepositional phrase modifies only the headword “Jupiter” or the phrase “Earth and Jupiter.” The phrase structure forest has two phrase structure trees corresponding to these two interpretations. On the other hand, the initial dependency forest has two IDed dependency trees and one generalized dependency tree, and the reduced dependency forest has one IDed dependency tree corresponding to one generalized dependency tree. 25 A special node called a “scoping node” is introduced to specify the scope of a dependency modification as required. Hirakawa The two phrase structure trees and one dependency tree are shown in Figure 24. Mel’cuk proposes introducing a concept called Grouping into the dependency structure frame- work to solve the modification scope problem (Mel’cuk 1988). Grouping is theoretically equivalent to phrase, in the sense that it specifies the word coverage information. However, grouping infor- mation is not attached to every part of the structure, but to some specific structures including the “conjoined structure” and “operator word,” such as “not” and “only.” The grammar framework for a machine translation system (Amano, Hirakawa, Nogami, and Kumano 1989) incorporates a mechanism similar to the grouping.25 In this grammar development for a real-world application, Fig. 23 DF containing the mapping from N phrase structure trees to 1 dependency tree (real ambiguity) Fig. 23 DF containing the mapping from N phrase structure trees to 1 dependency tree (real ambiguity) 25 A special node called a “scoping node” is introduced to specify the scope of a dependency modification as required. 772 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Fig. 24 Example of mapping from N phrase structure trees to 1 dependency tree (real ambiguity) Fig. 24 Example of mapping from N phrase structure trees to 1 dependency tree (rea Fig. 24 Example of mapping from N phrase structure trees to 1 dependency tree (real ambiguity) the scope nodes are used only for conjoined structures.26 This experience suggests the limitation of the application scope of grouping proposed by Mel’cuk is reasonable. Moreover, the treatment of the modification scope ambiguity differs from language to language. According to (Mel’cuk 1988), some modification scope ambiguities are distinguishable by lexical or syntactic marking in Russian. Japanese does not inherently have the modification scope problem because it has the basic grammatical constraint that modifiers should be located at the left-hand side of their mod- ificand. In the PDG framework, equivalent arcs represent the difference of modification scope, as shown in the previous section. Therefore, the modification scope problem may be avoided by introducing grouping into the treatment of equivalent arcs. This is a future task. pp p g g g g g 27 The projectivity condition consists of two conditions, i.e., “no cross dependency exits” and “no dependency covers the top node.” The second condition is unnecessary when a special top node is introduced at the top or end of a sentence. 26 This is the case for an English-to-Japanese system. The requirement level may differ, according to the language pairs. For example, translation between languages in the same family may not require a grouping mechanism because the modification scope ambiguities are avoided by bypassing, i.e., an ambiguous source language 26 This is the case for an English-to-Japanese system. The requirement level may differ, according to the language pairs. For example, translation between languages in the same family may not require a grouping mechanism because the modification scope ambiguities are avoided by bypassing, i.e., an ambiguous source language structure is mapped to the corresponding target language structure without disambiguation. 27 The projectivity condition consists of two conditions, i.e., “no cross dependency exits” and “no dependency covers the top node.” The second condition is unnecessary when a special top node is introduced at the top or end of a sentence. p g y yp g, , g structure is mapped to the corresponding target language structure without disambiguation. 27 28 This is an artificial example only for showing the rule applicability. 5.4 Generation of Non-projective Dependency Tree The projectivity constraint27 is a basic constraint adopted by many dependency analysis sys- tems, and these parsers are called projective parsers. Projective parsers fail to analyze sentences 773 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 with non-projective structures. Almost all sentences in many languages are projective, but some types of non-projective sentences exist (Mel’cuk 1988). For example, “John saw a dog yesterday which was a Yorkshire Terrier” in English and “私は本を東京に買いに昨日行きました。” (I went to Tokyo to buy a book yesterday) in Japanese have projective dependency structures. McDonald reported that the non-projective parser outperformed the projective parser in overall accuracy for the analysis of Czech, which has a high degree of word order freedom compared with English (McDonald, Crammer, and Pereira 2005). However, the simple introduction of non-projective interpretation may deteriorate the accuracy of the parsers for other languages. This seems to be the reason why the majority of dependency parsers adopt the projectivity constraint. As described in Section 4.1, the mapping between the constituent sequence (the body of grammar rule) and the partial dependency tree (the dependency structure of the grammar rule) is defined in the grammar rule in PDG. This framework, in combination with the description ability of the C-Matrix, enables a controlled non-projectivity instead of all-or-nothing non-projectivity. Controlled non-projectivity means that the non-projective structures are defined by some rules, which prescribe the well-formedness conditions. The (R19) in Figure 16 is a grammar rule for a phrase pattern where an adverb is inserted before a relative clause and produces a well-formed non-projective dependency structure. Figure 25 shows the dependency tree for “She saw the cat curiously which was Persian,”28 obtained by the example PDG grammar. The dependency forest has one non-projective dependency tree. Fig. 25 Example of non-projective dependency tree generation 774 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa 6 Concluding Remarks This paper describes the MPDC model, which is the basic analysis model adopted by PDG, and explains two packed shared data structures of PDG, i.e., the phrase structure forest and the dependency forest. The completeness and soundness of the correspondence between the phrase structure forest and the dependency forest are assured. This means that the sentence interpretations represented in a packed shared phrase structure and the sentence interpretations represented in a packed shared dependency structure have linkages. This paper also describes the experimental results for analyzing some typical ambiguous sentences using an example PDG grammar. The current implementation of the PDG system focuses on a feasibility study of the PDG framework. The practical PDG system and its performance evaluation are future tasks. 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Semantic and Syntactic Preference to Guide the Parsing.” In IJCNLP-04 Work- nd Shallow Analyses—Formalisms and Statistical Modeling for Deep Analyses. Harper, M. P. (2004). “A Statistical Constraint Dependency Grammar (CDG) Wang, W. and Harper, M. P. (2004). “A Statistical Constraint Dependency Grammar (CDG) Parser.” In Workshop on Incremental Parsing: Bringing Engineering and Cognition Together (ACL), pp. 42–49. Wilks, Y. A. (1975). “An Intelligent Analyzer and Under-stander of English.” Communications of the A.C.M., 18, pp. 264–274. Winograd, T. (1983). Language as a Cognitive Process: Volume 1 Syntax. Addison-Wesley Publishing. 778 PDG and its Packed Shared Data Structure “Dependency Forest” PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa B Proof of the Completeness and Soundness of the Dependency Forest The phrase structure forest PF and the dependency forest DF = ⟨DG, CM⟩is assumed in the following proof. Before showing the proof, some relations between the phrase structure forest and the dependency forest generated from the algorithms explained in Section 4.2, and some lemmas required for the proof, are described. 29 The partial dependency tree is represented in {} because it is a set of arcs. A Problem in the Syntactic Graph Consider the parsing of “Tokyo taxi driver call center” using the following grammar rules and lexicons. [Grammar Rules] np/NP →npc/NP : [] npc/Nb →np1/NP1,n/Na,n/Nb : [arc(nj,NP1,Nb),arc(nc,Na,Nb)] npc/Na →np2/NP2,n/Na : [arc(nc,NP2,Na)] npc/Na →np3/NP3,n/Na : [arc(nc,NP3,Na)] np1/Nc →n/Na,n/Nb,n/Nc : [arc(nc,Na,Nb),arc(nc,Nb,Nc)] np2/Nd →n/Na,n/Nb,n/Nc,n/Nd : [arc(nj,Na,Nc),arc(nc,Nb,Nc),arc(nc,Nc,Nd)] np3/Nd →n/Na,n/Nb,n/Nc,n/Nd : [arc(nc,Na,Nb),arc(nj,Nb,Nd),arc(nc,Nc,Nd)] This example sentence has three well-formed dependency trees shown in Figure 26 (a), (b), Fig. 26 Problem in the syntactic graph/exclusion matrix Fig. 26 Problem in the syntactic graph/exclusion matrix 779 Journal of Natural Language Processing Vol. 21 No. 4 Vol. 21 No. 4 September 2014 and (c). The boxes np1, np2, and np3 in the dependency trees are given only for showing the correspondences between the phrase structures and dependency structures. Since nc-1 and nc-2 in (a), nc-2 and nc-3 in (b), and nc-3 and nc-1 in (c) have co-occurrence relations, respectively, the values of the exclusion matrix for these three pairs are 0 (“ ” in the figure). This allows the existence of dependency tree (d), which has no corresponding phrase structure tree in the phrase structure forest in the syntactic graph/exclusion matrix. Therefore, the syntactic graph violates the soundness condition. Edge and its elements cs(X): The constituent sequence of single edge X. cs(X): The constituent sequence of single edge X. For example, cs(*e1) =[103, 169] where 103 and 169 are packed edge-IDs. ds(X): The dependency structure DS of single edge X or the node of single lexical edge X. ds(X): The dependency structure DS of single edge X or the node of single lexical edge X. edge X. For example, ds(*e1)={arc(obj-25, [flies]-n-1, [time]-v-0)}, ds(@e3)={[arrow]-n-4} For example, ds(*e1)={arc(obj-25, [flies]-n-1, [time]-v-0)}, ds(@e3)={[arrow]-n-4} Arcs in the dependency forest and edge: The arcs in a single edge X mean a∈ds(X). An arc in a packed edge Y means a∈ds(X), X∈Y . An arc in the phrase structure forest means a∈ds(X), X∈Y , Y ∈PF. Relations between arcs and nodes Relations between arcs and nodes gov(X): The governor node of arc X. gov(X): The governor node of arc X. For example, gov(arc(obj-25, [flies]-n-1, [time]-v-0)) = [time]-v-0 or example, dep(arc(obj-25, [flies]-n-1, [time]-v-0) = [flies]-n-1 For example, dep(arc(obj-25, [flies]-n-1, [time]-v-0) = [flies]-n-1 top node(X): The top node of dependency tree X (the node that is not a dependent of any arcs in X). top node(X): The top node of dependency tree X (the node that is not a dependent of any arcs in X). Relations between arcs X, Y in dependency tree DT Relations between arcs X, Y in dependency tree DT sib(X, Y ): gov(X)=gov(Y ). X and Y are called sibling arcs. X 1 −−→ DT Y : dep(X)=gov(Y ). X is a parent of Y and Y is a child of X. This relation is called a parent relation. X + −−→ DT Y : There is a parent relation chain from X to Y . X is an ancestor arc of Y and Y is a descendant arc of X. X ∗ −−→ DT Y : X = Y or X + −−→ DT Y [Packed Edge and Single Edge] For example, cs(*e1) =[103, 169] where 103 and 169 are packed edge-IDs. ds(X): The dependency structure DS of single edge X or the node of single lexical edge X. For example, ds(*e1)={arc(obj-25, [flies]-n-1, [time]-v-0)}, ds(@e3)={[arrow]-n-4} Arcs in the dependency forest and edge: The arcs in a single edge X mean a∈ds(X). An arc in a packed edge Y means a∈ds(X), X∈Y . An arc in the phrase structure forest means a∈ds(X), X∈Y , Y ∈PF. Relations between arcs and nodes gov(X): The governor node of arc X. For example, gov(arc(obj-25, [flies]-n-1, [time]-v-0)) = [time]-v-0 dep(X): The dependent node of arc X. For example, dep(arc(obj-25, [flies]-n-1, [time]-v-0) = [flies]-n-1 top node(X): The top node of dependency tree X (the node that is not a dependent of any arcs in X). Relations between arcs X, Y in dependency tree DT sib(X, Y ): gov(X)=gov(Y ). X and Y are called sibling arcs. X 1 −−→ DT Y : dep(X)=gov(Y ). X is a parent of Y and Y is a child of X. This relation is called a parent relation. X + −−→ DT Y : There is a parent relation chain from X to Y . X is an ancestor arc of Y and Y is a descendant arc of X. X ∗ −−→ DT Y : X = Y or X + −−→ DT Y [Packed Edge and Single Edge] The phrase structure forest is a set of packed edges. As described in Section 4.2.1, a packed edge is equivalent to a set of single edges. In this proof, packed edges are treated as a set of single edges. The following packed edge is shown in Figure 8. Packed edge: ⟨ID, FP, TP, C, PH, FCSL, RCS, DSL⟩ where FCSL=[CS1, . . . , CSn], DSL=[DS1, . . . , DSn] is equal to the following set of single edges. Packed edge: ⟨ID, FP, TP, C, PH, FCSL, RCS, DSL⟩ where FCSL=[CS1, . . . , CSn], DSL=[DS1, . . . , DSn] is equal to the following set of single edges. is equal to the following set of single edges. *e1: ⟨ID-1, FP, TP, C, PH, (CS1 DS1), RCS⟩ *en: ⟨ID-n, FP, TP, C, PH, (CSn DSn), RCS⟩ For example, *E4 in Figure 8 is a set of arcs *e1, *e229 *en: ⟨ID-n, FP, TP, C, PH, (CSn DSn), RCS⟩ For example, *E4 in Figure 8 is a set of arcs *e1, *e229 Single edge *e1: ⟨170-1, 0, 5, vp, [time]-v-0, [103, 169], [], {arc(obj-25, [flies]-n-1, [time]-v-0)}⟩ Single edge *e2: ⟨170-2, 0, 5, vp, [time]-v-0, [103, 119, 165], [], 8em{arc(obj-4, [flies]-n-1, [time]-v-0), arc(vpp-20, [like]-pre-2, [time]-v-0)}⟩ Each edge is identified in the phrase structure forest by the packed edge-ID and the position in the CSDS pair of the packed edge. For example, *e1 is identified by 170-1. The lexical edge is Single edge *e1: ⟨170-1, 0, 5, vp, [time]-v-0, [103, 169], [], {arc(obj-25, [flies]-n-1, [time]-v-0)}⟩ Single edge *e2: ⟨170-2, 0, 5, vp, [time]-v-0, [103, 119, 165], [], 8em{arc(obj-4, [flies]-n-1, [time]-v-0), arc(vpp-20, [like]-pre-2, [time]-v-0)}⟩ 8em{arc(obj-4, [flies]-n-1, [time]-v-0), arc(vpp-20, [like]-pre-2, [time]-v-0)}⟩ Each edge is identified in the phrase structure forest by the packed edge-ID and the position in the CSDS pair of the packed edge. For example, *e1 is identified by 170-1. The lexical edge is 780 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa treated as a set consisting of a single lexical edge. @E5 in Figure 8 is equal to the set @e3. Single edge @e3: ⟨156-1, 4, 5, n, [arrow]-n-4, [lex([arrow]-n)], {[[arrow]-n-4]}⟩ Various elements included in a packed edge and a single edge have correspondences with one another. The following shows the definitions of terms and relations. Edge and its elements Edge and its elements cs(X): The constituent sequence of single edge X. [Edges and Phrase Structure Trees in the Phrase Structure Forest] The phrase structure forest PF is a directed acyclic graph consisting of packed edges, where the root is the root edge *Eroot and the leaves are lexical edges. The “path in PF” is defined as follows: 781 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 Definition 5 [Path in the phrase structure forest] Definition 5 [Path in the phrase structure forest] Definition 5 [Path in the phrase structure forest] Definition 5 [Path in the phrase structure forest] Definition 5 [Path in the phrase structure forest] A path in the phrase structure forest is a sequence consisting of packed edges and single edges obtained by tracing a packed edge and a single edge one after another by selecting one single edge from a packed edge (a set of single edges) and selecting one packed edge from the constituent sequence (a sequence of packed edges) of a single edge. Now, let *E0, *E1, *E2 · · · in the phrase structure forest as follows: *E0 ={*e1, *e2}, *E1 ={*e3}, *E2 ={*e4, *e5}, *E3 ={*e6, *e7} . . . cs(*e1)=[*E1, *E2], cs(*e2)=[*E3], cs(*e3)=[*E4, *E5] . . . The following are examples of paths *E0 ={*e1, *e2}, *E1 ={*e3}, *E2 ={*e4, *e5}, *E3 ={*e6, *e7} . . . cs(*e1)=[*E1, *E2], cs(*e2)=[*E3], cs(*e3)=[*E4, *E5] . . . The following are examples of paths. The following are examples of paths. [*E0, *e1, *E2, *e5], [*E0, *e1, *E2], [*e1, *E1, *e3, *E5], [*e1, *E1, *e3] The following shows the definitions of terms and relations used in the latter p [*E0, *e1, *E2, *e5], [*E0, *e1, *E2], [*e1, *E1, *e3, *E5], [*e1, *E1, *e3] The following shows the definitions of terms and relations used in the latter p [*E0, *e1, *E2, *e5], [*E0, *e1, *E2], [*e1, *E1, *e3, *E5], [*e1, *E1, *e3] The following shows the definitions of terms and relations used in the latter part. Terms and relations related to the phrase structure forest Terms and relations related to the phrase structure forest X + −−→ PF Y : There is a path [X, . . ., Y ] from a packed or single edge X to a packed or single edge Y . X is an ancestor of Y . X ∗ −−→ PF Y : X = Y or X + −−→ PF Y is true for single or packed edges X and Y . Y is called “reachable” from X. X ↙[P F ]↘Y : X ̸= Y , ¬(X ∗ −−→ PF Y ), ¬(Y ∗ −−→ PF X) are true for single or packed edges X and Y , and there exists at least one single or packed edge Z in the phrase structure forest PF such that Z ∗ −−→ PF X and Z ∗ −−→ PF Y . Definition 8 [All phrase structure trees] ps trees(*E) is a set of all phrase structure trees for *E. Definition 5 [Path in the phrase structure forest] Arcs governed by an edge: Arc X is governed by packed edge *E if X ∈ds(*e), *E ∗ −−→ PF *e is true. From the definition of the phrase structure forest, there exists a path from the root packed edge *Eroot to every single or packed edge in the phrase structure forest. Using the definition above, the C-Matrix setting conditions in Section 4.3 are defined as follows: efinition 6 [The C-Matrix setting conditions] Definition 6 [The C-Matrix setting conditions] Arcs X, Y are co-occurable if any of the following conditions is satisfied. (C1) There exists a single edge *e, such that X, Y ∈ds(*e), *e∈*E, *E∈PF (C2) There exist *ex and *ey, such that X∈ds(*ex), Y ∈ds(*ey), *ex + −−→ PF *ey or *ey + −−→ PF *ex. (C3) There exist *ex and *ey, such that X∈ds(*ex), Y ∈ds(*ey), *ex ↙[P F ]↘*ey. Definition 6 [The C-Matrix setting conditions] Arcs X, Y are co-occurable if any of the following conditions is satisfied. (C1) There exists a single edge *e, such that X, Y ∈ds(*e), *e∈*E, *E∈PF (C2) There exist *ex and *ey, such that X∈ds(*ex), Y ∈ds(*ey), *ex + −−→ PF *ey or *ey + −−→ PF *ex. (C3) There exist *ex and *ey, such that X∈ds(*ex), Y ∈ds(*ey), *ex ↙[P F ]↘*ey. Arcs X, Y are co-occurable if any of the following conditions is satisfied. X, Y are co-occurable if any of the following conditions is satisfied. Arcs X, Y are co occurable if any of the following conditions is satisfied. (C1) There exists a single edge *e, such that X, Y ∈ds(*e), *e∈*E, *E∈PF (C2) There exist *ex and *ey, such that X∈ds(*ex), Y ∈ds(*ey), *ex + −−→ PF *ey or *ey + −−→ PF *ex. (C3) There exist *ex and *ey, such that X∈ds(*ex), Y ∈ds(*ey), *ex ↙[P F ]↘*ey. A phrase structure tree is defined as follows: A phrase structure tree is defined as follows: 782 PDG and its Packed Shared Data Structure “Dependency Forest” PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Fig. 27 Algorithm for obtaining a phrase structure tree Fig. 27 Algorithm for obtaining a phrase structure tree Fig. 27 Algorithm for obtaining a phrase structure tree 30 The parsing algorithm utilizes packed edges because another algorithm, which shares active edges, can be constructed. Definition 7 [Phrase structure tree] A phrase structure tree for a packed edge *E is a set of single arcs obtained by a recursive procedure get tree(*E) defined in Figure 27. In the figure, select(RPE) selects one arbitrary arc included in a packed edge RPE. If SE is a lexical edge, lexical edge(SE) is true. A phrase structure tree covers the words from the from-position to the to-position of edge *E. Definition 8 [All phrase structure trees] [Relations between Edges and Arcs/Partial Dependency Trees] Although the parsing algorithm is constructed using packed edges as basic data structures, the packing of edges is performed only when inactive edges are generated (Figure 9 (c), (d)). Therefore, every active packed edge has one single edge,30 and one active packed edge corresponds to one single active edge and vice versa. In the following discussion, the word “edge” is used for representing a packed edge. Parsing proceeds by generating new edges by combining an inactive edge to an active edge. Using a diagrammatic expression, as described in Section 4.2.1, a combination of two arcs gen- erates a new edge by moving ‘・’ in the active edge to the right neighbor position, and binding the variable for the constituent at ‘・’ to the phrase head (node) of the inactive edge to combine. Figure 28 is a tree called an “edge combination tree,” which represents the generation process of inactive edges from a grammar rule by edge combinations. The inactive edges located at the leaf 783 atural Language Processing Vol. 21 No. 4 September 2014 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 of the edge combination tree are generated from a grammar rule located at the root of the tree31 through the active edges in between. The grammar rule is as follows: y/Xh →x1/X1 · · · xh/Xh · · · xn/Xn : {A1, A2, . . . , An−1} Ai is an arc in the form of arc(ai, Xk, Xl), where ai is an arcname, 1≤k≤n, 1≤l≤n, k̸=l. A set of arcs {A1, . . . , An−1} satisfies the partial dependency structure condition in Section 4.1. Ai is an arc in the form of arc(ai, Xk, Xl), where ai is an arcname, 1≤k≤n, 1≤l≤n, k̸=l. A set of arcs {A1, . . . , An−1} satisfies the partial dependency structure condition in Section 4.1. The edges in the edge combination tree are expressed in diagrammatic form using ‘・’, ne- glecting the from and to positions. The arrows between edges represent the edge combinations where the edge at the source of the arrow is combined with the inactive edge attached to the arrow to generate the resulting edge at the target position of the arrow. For example, E11 (Figure 28 (a)) is combined with ⟨*E x2/n21 →. . .⟩((b)) to generate E21 ((c)). 31 The grammar rule is written in edge form in the edge combination tree. This edge is not generated in the real parsing process, but is introduced for convenience of explanation. [Relations between Edges and Arcs/Partial Dependency Trees] Since ‘・’ moves to the right neighbor position, the depth of the tree, i.e., the number of arcs from E0 to each inactive edge, is equal to n, i.e., the number of elements in the rule body in the grammar rule. The phrase head (node) is bound to a variable in the active edge during edge combination. The variable bindings are shown at the right of the edge by { }. For example, the combination Fig. 28 Edge combination tree Fig. 28 Edge combination tree 784 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa of E0 and the edge (d) whose phrase head is n11 generates E11 which is then combined with the edge (b), whose phrase head is n21 to produce E21. As a result, the variable bindings of E21 are {X1:=n11, X2:=n21}.32 An arc whose governor and dependent are bound is called a “fixed arc” and has a new unique arc-ID generated by add arcid at Figure 9 (i). Fixed arcs are represented by a small letter such as a1 in Figure 28 (e). One variable binding may generate zero or more fixed arcs. The generated fixed arc has one unique variable binding corresponding to one edge combination. This edge combination generates one unique edge. This unique edge is called the edge, which generated the fixed arc, or simply “source edge” of the fixed arc, and is referred to as src Edge(a), where ’a’ is a fixed arc. For example, in the edge combination between (e) and (f) in Figure 28, provided that the binding of the node nim (let it [like]-pre-3) to the variable Xi generates the fixed arc ai (let it arc(pre-28, [like]-pre-3, [time]-v-0)) from the unfixed arc Ai (arc(pre, Xi, [time]-v-0)), the edge that generated the fixed arc ai, i.e., src Edge(ai) is Eim in Figure 28 (g). Each inactive edge (leaf of the combination tree) (e.g., Figure 28 (h)) has only fixed arcs because all variables including phrase head variable in the edge are bound, due to the partial dependency structure condition. An inactive edge represents a result of a sequence of variable bindings, caused by the edge bindings from the root to the leaf of the edge combination tree. The following shows the definitions of terms and relations related to the edge combination tree. 32 Scope of a variable is within edge. Lemma B.1 (Relation between arcs in one partial dependency tree) Let ai and aj be fixed arcs ai, aj ∈ds(e), where e is a single edge. Their source edges src Edge(ai) and src Edge(aj) are in origin relation. Lemma B.1 (Relation between arcs in one partial dependency tree) Let ai and aj be fixed arcs ai, aj ∈ds(e), where e is a single edge. Their source edges src Edge(ai) and src Edge(aj) are in origin relation. A fixed arc in edge e in the edge combination tree is in the edges that have e as their origins. For example, fixed arc ai generated at (g) is contained in (h). Lemma B.2 (Relation between an arc and the edge that generated the arc) Suppose that fixed arcs ai, aj satisfy src Edge(ai) ∗ −−→ CT src Edge(aj). Then aj ∈ds(*e) implies ai ∈ds(*e) for every single arc *e in PF (*e∈*E, *E∈PF). Lemma B.2 (Relation between an arc and the edge that generated the arc) Suppose that fixed arcs ai, aj satisfy src Edge(ai) ∗ −−→ CT src Edge(aj). Then aj ∈ds(*e) implies ai ∈ds(*e) for every single arc *e in PF (*e∈*E, *E∈PF). Since a unique arc-ID is assigned to each arc, all inactive single edges in the inactive packed edges in an arc combination tree, i.e., the leaves of the tree *En1 · · · *Eno . . . *Enw in Figure 28, have different partial dependency trees. Therefore, ds(*ei)̸=ds(*ej) for arbitrary arcs *ei, *ej(*ei ̸=*ej) in PF. A single inactive edge and a partial dependency tree have one-to-one mapping. In the parsing process, inactive edges that satisfy the conditions shown in Figure 9 (j) are merged into one, and this merged edge becomes an element of the phrase structure forest. The one-to-one mapping relation between a single edge and a partial dependency tree is assured in the phrase structure forest because this merge operation does not change the dependency structures in the single edges. Moreover, the following lemma related to the phrase structure forest is established with respect to the C-Matrix setting condition (C2). Lemma B.3 (Constraints with respect to the arcs in the edges included in a path) Suppose arcs ai ∈ds(ei), aj ∈ds(ej). If ei + −−→ PF ej, then dep(ai)̸=dep(aj) is true. Inversely, if dep(ai)=dep(aj), then ¬(ei + −−→ PF ej) is true. [Relations between Edges and Arcs/Partial Dependency Trees] fixed arc: Arc whose governor and dependent nodes are fixed by the variable bindings caused by edge combinations. fixed arc: Arc whose governor and dependent nodes are fixed by the variable bindings caused by edge combinations. src Edge(a) (source edge): The active or inactive edge that generated a fixed arc a. Mapping from a fixed arc to its corresponding edge is one to one, whereas the reverse is 1 to 0 - many. src Edge(a) (source edge): The active or inactive edge that generated a fixed arc a. Mapping from a fixed arc to its corresponding edge is one to one, whereas the reverse is 1 to 0 - many. X ∗ −−→ CT Y (origin): Edge X is located on a route from the top node to edge Y or X = Y . X is called an origin of Y . X ∗ −−→ CT Y (origin): Edge X is located on a route from the top node to edge Y or X = Y . X is called an origin of Y . origin relation: Edges X, Y in an edge combination tree CT are said to be in origin relation if X ∗ −−→ CT Y or Y ∗ −−→ CT X is true. CT CT edge(a, DT) (corresponding edge): The corresponding edge for a fixed arc a and a well- formed dependency tree DT is a single edge e that satisfies the following condition (defined in Lemma B.4): ( ) ( ) formed dependency tree DT is a single edge e that satisfies the following condition (defined in Lemma B.4): DT⊇ds(e), a∈ds(e) 32 Scope of a variable is within edge. 785 Vol. 21 No. 4 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 According to the structure of the edge combination tree described above, two fixed arcs ai and aj have the following relations. Lemma B.3 (Constraints with respect to the arcs in the edges included in a path) Suppose arcs ai ∈ds(ei), aj ∈ds(ej). If ei + −−→ PF ej, then dep(ai)̸=dep(aj) is true. Inversely, if dep(ai)=dep(aj), then ¬(ei + −−→ PF ej) is true. Proof This lemma is established because data structures in the single edges are trees whose top nodes are phrase heads, according to the partial dependency structure condition in 4.1. Lemma B.4 (Existence of corresponding edge) Suppose an arc ai in a well-formed de- pendency tree DT (ai ∈DT). There exists only one packed edge E∈PF and single edge e∈E that satisfy the following condition: DT ⊇ds(e), ai ∈ds(e) 786 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Proof Let the number of nodes in DT be n (Number of arcs is n −1). Divide arc set DT into the following two arc sets IN ARCS, OUT ARCS with respect to ai. Proof Let the number of nodes in DT be n (Number of arcs is n −1). Divide arc set DT into the following two arc sets IN ARCS, OUT ARCS with respect to ai. Proof Let the number of nodes in DT be n (Number of arcs is n −1). Divide arc set DT into the following two arc sets IN ARCS, OUT ARCS with respect to ai. IN ARCS = { aj | src Edge(ai) ∗ −−→ CT src Edge(aj) or src Edge(aj) ∗ −−→ CT src Edge(ai)} OUT ARCS = DT−IN ARCS Let SRC EDGES be a set of source edges corresponding to the arcs in IN ARCS. SRC EDGES {E | E Ed ( ) IN ARCS} Let SRC EDGES be a set of source edges corresponding to the arcs in IN ARCS. SRC EDGES = {E | E=src Edge(a), a∈IN ARCS} SRC EDGES = {E | E=src Edge(a), a∈IN ARCS} First, the following statement is to be established. “Arbitrary edges in SRC EDGES are in origin relation” (A) (A) Suppose edges U, V ∈SRC EDGES. U and V are in origin relation by definition and in one o the following three cases. Suppose edges U, V ∈SRC EDGES. U and V are in origin relation by definition and in one of the following three cases. (a) One arc is an origin of Ei, and Ei is an origin of the other arc. U ∗ −−→ CT Ei, Ei ∗ −−→ CT V (a) One arc is an origin of Ei, and Ei is an origin of the other arc. U ∗ −−→ CT Ei, Ei ∗ ∗ (b) Both arcs are origins of Ei. Lemma B.3 (Constraints with respect to the arcs in the edges included in a path) Suppose arcs ai ∈ds(ei), aj ∈ds(ej). If ei + −−→ PF ej, then dep(ai)̸=dep(aj) is true. Inversely, if dep(ai)=dep(aj), then ¬(ei + −−→ PF ej) is true. 4 September 2014 September 2014 contradicts the C-Matrix setting conditions between au and av. Therefore, statement (A) is true. Now, let Elast be the last edge connected from Ei, i.e., the edge that satisfies the following conditions: contradicts the C-Matrix setting conditions between au and av. Therefore, statement (A) is true. Now, let Elast be the last edge connected from Ei, i.e., the edge that satisfies the following conditions: Ei ∗ −−→ CT Elast Elast is the only edge Ej such that Elast ∗ −−→ CT Ej (Ej ∈SRC EDGES) Figure 29 shows the relation between IN ARCS and SRC EDGES diagrammatically. Estart corresponds to a grammar rule. The grammar rule is as follows: Elast is the only edge Ej such that Elast ∗ −−→ CT Ej (Ej ∈SRC EDGES) Elast is the only edge Ej such that Elast ∗ −−→ CT Ej (Ej ∈SRC EDGES) Figure 29 shows the relation between IN ARCS and SRC EDGES diagrammatically. Estart corresponds to a grammar rule. The grammar rule is as follows: y/X →x /X x /X :{A A } rresponds to a grammar rule. The grammar rule is as follows: y/Xh →x1/X1 · · · xz/Xz :{A1, . . ., Az−1} SRC EDGES constitutes a route on the edge combination tree CT with root Estart, containing E1, . . ., Elast. The source edge Ei for ai exists somewhere on this route. There exists at least one arc alast that is generated by Elast in IN ARCS. Elast is either an active edge or an inactive edge. Figure 29 shows a case where Elast is an active edge. Elast is proved to be an inactive edge as follows: Suppose that Elast is an active edge. As shown in Figure 30, Elast (Figure 30 (a)) has at least one remaining constituent xu+1 (Figure 30 (b). Variable for the constituent is not shown). Let s1 and t1 be a from-position and a to-position of Elast, respectively. From the premise alast∈DT, there exists at least one inactive edge *Ex (Figure 20 (c)) that has Elast, the source edge of alast, as its origin in the phrase structure forest PF. As shown in the figure, *Ex has the from-position equal to the from-position s1 of Elast and the to-position greater than the to-position t1 of Elast. Consider the node nt1+1 at the position t1+1 (Figure 30 (d)).33 DT has one arc Fig. 33 One nt1+1 exists due to the well-formedness condition of DT. Lemma B.3 (Constraints with respect to the arcs in the edges included in a path) Suppose arcs ai ∈ds(ei), aj ∈ds(ej). If ei + −−→ PF ej, then dep(ai)̸=dep(aj) is true. Inversely, if dep(ai)=dep(aj), then ¬(ei + −−→ PF ej) is true. U ∗ −−→ CT Ei, V ∗ −−→ CT Ei (b) Both arcs are origins of Ei. U ∗ −−→ CT Ei, V ∗ −−→ CT Ei (c) Ei is an origin of both arcs. Ei ∗ −−→ CT U, Ei ∗ −−→ CT V In cases (a) and (b), statement (A) obviously holds, according to the structure of the edge combination tree. The following shows that a contradiction is derived from the assumption that U and V are not in origin relation in case (c). Suppose that U and V are not in origin relation. There exist arcs au, av ∈DT such that U=src Edge(au), V =src Edge(av), according to the premise. Since all arcs in DT are in the packed shared forest, there exist inactive edges that have U and V as their origin, respectively. Let *Eu and *Ev be origins of U and V , respectively. Let eu in *Eu and *ev in *Ev be edges such that au ∈*eu, av ∈*ev. From Lemma B.2, both *eu and *ev contain ai. Arcs au and av in the well-formed dependency tree DT satisfy any of the C-Matrix setting conditions (C1) to (C3). However, au and av do not satisfy (C1) because the contradiction for the assumption that U and V are not in origin relation is deduced from (C1), i.e., the existence of e such that au, av ∈ds(e), according to Lemma B.1. (C2) indicates that *eu + −−→ PF *ev is true (The reverse case is shown in the same way). Nodes included in arcs in ds(*eu) are phrase heads of the constituents in cs(*eu), according to the partial dependency structure condition. This implies that either dep(ai) or gov(ai) of ai ∈ds(*eu) is a node that locates outside the coverage of *ev. On the other hand, both dep(ai) and gov(ai) must be in the coverage of *ev because ai ∈ds(*ev) is true. From this contradiction, au and av do not satisfy (C2). The (C3), i.e., *eu ↙[P F ]↘*ev , is not satisfied by au and av because the coverage of *eu and *ev have to be overlapped, due to the premise that ai is in both *eu and *ev. From the above, the supposition that U and V are not in origin relation 787 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 Journal of Natural Language Processing Vol. 21 No. 34 If am =anext, src Edge(anext) and Ei are in origin relation. This contradicts the premise anext∈OUT ARCS. Lemma B.3 (Constraints with respect to the arcs in the edges included in a path) Suppose arcs ai ∈ds(ei), aj ∈ds(ej). If ei + −−→ PF ej, then dep(ai)̸=dep(aj) is true. Inversely, if dep(ai)=dep(aj), then ¬(ei + −−→ PF ej) is true. 29 Edges corresponding to arcs in DT Fig. 29 Edges corresponding to arcs in DT 788 PDG and its Packed Shared Data Structure “Dependency Forest” PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Fig. 30 Existence of corresponding edge Fig. 30 Existence of corresponding edge anext(dep(anext) = nt1+1 (Figure 30 (e)). anext is an element of OUT ARCS, as per the defi- nition of alast. anext∈DT implies that at least one inactive edge *Ey whose origin is Enext, the source edge of anext, exists in the phrase structure forest PF. Since Enext has anext within its cov- erage, Enext has the from-position s1 less than or equal to t1 (Figure 30 (f)), and the to-position t2 is greater than or equal to t1+1 (Figure 30 (g)). Therefore, the from-position of *Ey is less than or equal to t1. From the above, *Ex, which has its origin Elast, overlaps *Ey, which has its origin Enext at the t1 position. anext(dep(anext) = nt1+1 (Figure 30 (e)). anext is an element of OUT ARCS, as per the defi- nition of alast. anext∈DT implies that at least one inactive edge *Ey whose origin is Enext, the source edge of anext, exists in the phrase structure forest PF. Since Enext has anext within its cov- erage, Enext has the from-position s1 less than or equal to t1 (Figure 30 (f)), and the to-position t2 is greater than or equal to t1+1 (Figure 30 (g)). Therefore, the from-position of *Ey is less than or equal to t1. From the above, *Ex, which has its origin Elast, overlaps *Ey, which has its origin Enext at the t1 position. Now, no co-occurrence setting conditions for alast and anext hold: Arcs alast and anext do not satisfy (C3) because *Ex and *Ey overlap, as explained above. (C1) is not satisfied from the premise *Ex̸=*Ey. Consider (C2) meaning that *Ex + −−→ PF *Ey is true (the reverse case is shown in the same way). Let am be the arc in *Ex whose dependent node is nt1+1. am ̸=anext.34 According to Lemma B.3, if *Ex + −−→ PF *Ey is not true, then (C2) is not satisfied. From the above, alast and anext satisfy no co-occurrence setting conditions. This contradicts the premise that DT is a well-formed dependency tree. Therefore, Elast is not an active edge. Let Elast be an inactive edge *Elast. lation between Connected Arcs and their Corresponding Edge Arcs ai, aj(ai 1 −−→ DT aj or sib(ai, aj)) are called “connected arcs.” The following two lemmas are established with respect to connected arcs in a well-formed dependency tree DT. Journal of Natural Language Processing Vol. 21 No. 4 September 2014 (b) *Elast∈PF is true, due to alast∈DT. (b) *Elast∈PF is true, due to alast∈DT. (c) According to the premise alast∈DT and Lemma B.2, DT⊇ds(*elast) is true. Lemma B.2 is true due to (a) to (c). (c) According to the premise alast∈DT and Lemma B.2, DT⊇ds(*elast) is true. Lemma B.2 is true due to (a) to (c). Lemma B.3 (Constraints with respect to the arcs in the edges included in a path) Suppose arcs ai ∈ds(ei), aj ∈ds(ej). If ei + −−→ PF ej, then dep(ai)̸=dep(aj) is true. Inversely, if dep(ai)=dep(aj), then ¬(ei + −−→ PF ej) is true. Let Elast be an inactive edge *Elast. (a) If *Elast, the source edge of alast, is an inactive edge (a leaf of the edge combination tree), then no other packed edges contain alast. Moreover, there exists only one *elast such that alast∈ds(*elast), *elast∈*Elast. (a) If *Elast, the source edge of alast, is an inactive edge (a leaf of the edge combination tree), then no other packed edges contain alast. Moreover, there exists only one *elast such that alast∈ds(*elast), *elast∈*Elast. alast∈ds(*elast), *elast∈*Elast. 789 Journal of Natural Language Processing Vol. 21 No. 4 Lemma B.7 (Relation between top edge(DT) and edge(aj, DT)) *et ∗ −−→ PF *ej is true for *et=top edge(DT) and *ej=edge(aj, DT) (aj ∈DT). Lemma B.7 (Relation between top edge(DT) and edge(aj, DT)) *et ∗ −−→ PF *ej is true for *et=top edge(DT) and *ej=edge(aj, DT) (aj ∈DT). Proof If aj is an arc just under the top node of DT, i.e., gov(aj)=top node(DT), *et ∗ −−→ PF *ej, is true, according to Lemma B.5 and the definition of top edge. If not, aj is a descendant of one of the arcs just under the top node of DT. *et ∗ −−→ PF *ej is true, according to Lemma B.6. [Division of Well-formed Dependency Tree] The “division of a well-formed dependency tree DT” means the creation of a set of partial dependency trees DT1, . . ., DTm by removing a set of arcs in ds(top edge(DT)) from DT, where m is the number of nodes in ds(top edge(DT)). Nodes isolated from all other nodes by this operation are dependency trees that consist of one node. For example, suppose that ds(top edge(DT))={as, at, au, aw} in Figure 31, DT is divided into partial dependency trees DTs, DTt, DTu, DTv, DTw whose top nodes are ns, nt, nu, nv, nw, respectively. Since nodes ns, nw are isolated from other nodes, DTs and DTw are dependency trees consisting of a single node, i.e., {ns} and {nw}, respectively. The phrase heads of the packed edges in cs(e) of a single edge e have one-to-one correspondence with the nodes in the partial dependency tree df(e), due to the partial dependency structure condition in Section 4.1. Therefore, there exists one packed edge *Ei (1≤i≤m) whose phrase head is a top node ni of DTi(1≤i≤m). *Ei is called a “root packed edge” for DTi and is referred to as root Edge(DTi). Lemma B.5 (Connected arcs and their corresponding edges) Suppose *ei = edge(ai, DT) *ej = edge(aj DT) for connected arcs ai aj in DT At least one of (a) (b) (c) is true Lemma B.5 (Connected arcs and their corresponding edges) Suppose *ei = edge(ai, DT)， *ej = edge(aj, DT) for connected arcs ai, aj in DT. At least one of (a), (b), (c) is true. *ej = edge(aj, DT) for connected arcs ai, aj in DT. At least one of (a), (b), (c) is t (a) *ei =*ej (b) *ei + −−→ PF *ej (c) *ej + −−→ PF *ei (a) *ei =*ej (b) *ei + −−→ PF *ej (c) *ej + −−→ PF *ei Lemma B.5 means that if two arcs in DT are connected, one of their corresponding edges is reachable from another edge in PF. Proof *ei and *ej satisfy at least one of the C-Matrix setting conditions (r1) to (r3), because *ei and *ej co-occur in DT. (r1) *ei=*ej (r2) *ei + −−→ PF *ej or *ej + −−→ PF *ei (r3) *ei ↙[P F ]↘*ej (r1) *ei=*ej (r2) *ei + −−→ PF *ej or *ej + −−→ PF *ei (r3) *ei ↙[P F ]↘*ej Let n be a node shared by the connected arcs ai and aj. Both *ei and *ej cover n. Therefore, (r3) is not satisfied by *ei and *ej. *ei and *ej have to satisfy (r1) or (r2). Lemma B.6 (Ancestor-descendant arcs and their corresponding arcs) Suppose *ei = edge(ai, DT)，*ej = edge(aj, DT) for ai, aj(ai + −−→ DT aj). DT *ei ∗ −−→ PF *ej Proof In the case that dep(ai)=gov(aj), one of (a), (b), or (c) in Lemma B.5 is true. From the node positioning relation prescribed by the partial dependency structure condition in Section 4.1, (c) is not satisfied by ai and aj because ¬(*ej + −−→ PF *ei) is true for *ej and *ei. Therefore, parent-child arcs satisfy either (a) or (b). Lemma B.6 is established for ai ∗ −−→ DT aj, due to the associativity of the relation ∗ −−→ PF . 790 PDG and its Packed Shared Data Structure “Dependency Forest” PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa [Top single edge top edge(DT)] Definition 9 [Top single edge] Definition 9 [Top single edge] A “top single edge” for DT top edge(DT) is the single edge that locates at the topmost position in PF among the edges that correspond to the arc just under the top node of DT. That is, top edge(DT) is edge ai satisfying the following conditions. top node(DT)=gov(ai) edge(ai) ∗ −−→ PF edge(aj) for all aj such that top node(DT)=gov(aj) A “top single edge” for DT top edge(DT) is the single edge that locates at the topmost position in PF among the edges that correspond to the arc just under the top node of DT. That is, top edge(DT) is edge ai satisfying the following conditions. edge(ai) ∗ −−→ PF edge(aj) for all aj such that top node(DT)=gov(aj) If DT is a tree consisting of one node, top node(DT) is the single lexical edge corresponding to the node. Definition 10 [Root packed edge] Suppose DTi and its top node ni (1≤i≤m) is obtained by the division of DT. A root Edge(DTi) is a packed edge that is an element of cs(top edge(DT)) and whose phrase head is top node(DTi). 791 Vol. 21 No. 4 September 2014 For example, in Figure 31, *ev is top edge(DT) and has the constituent sequence *Es, *Et, *Eu, *Ev, *Ew, whose phrase heads are ns, nt, nu, nv, nw, respectively. Then, root Edge(DTt) is *Et. The following two lemmas are explained with reference to Figure 32. Lemma B.9 (Well-formedness of partial trees obtained by division of DT Lemma B.9 (Well-formedness of partial trees obtained by division of DT) Suppose partial dependency trees DTi and its root packed edge *Ei with the from-position spi and to- position tpi are obtained from the division of the well-formed dependency tree DT. DTi is a well-formed dependency tree that covers from spi to tpi. Proof DTi is a well-formed dependency tree if it satisfies the co-occurrence constraint and the well-covering constraint. Obviously DTi satisfies the co-occurrence constraint because DT satisfies the co-occurrence constraint. The following part shows the well-covering constraint. In the case that DT is a tree consisting of a single node, it satisfies the well-covering constraint from the definition. Consider the case where DT is a tree consisting of arcs. Let ni and nj be the top node of DT and one of any other nodes in DT (nj ̸=ni). There exists arc aj such that nj = dep(aj) in DT. Moreover, there exists ak ∈DTi such that gov(ak)=ni, ak ∗ −−→ DT aj for aj ∈DTi. Let *ej and *ek be *ej =edge(aj, DT), *ek =edge(ak, DT). *ek ∗ −−→ PF *ej is true due to Lemma B.6, because ak is equal to aj or ak is an ancestor of aj. Now, let *et be the top single node of DT, *et ∗ −−→ PF *ek is true according to Lemma B.7. *ek is reachable from one of the packed edges in cs(*et). *Ei ∗ −−→ PF *ek is true because the phrase head of *ek is ni. From the above, *Ei ∗ −−→ PF *ek ∗ −−→ PF *ej is true and nj is in the coverage of *Ei; that is, all nodes in DTi are in the coverage from spi to tpi. Furthermore, the nodes in DTk(k̸=i) are not in the coverage from spi to tpi. Since DT satisfies the well-covering constraint, all nodes in DTi occupy whole positions from spi to tpi. Lemma B.8 (Relation between root packed edge and top single edge) Suppose partial Lemma B.8 (Relation between root packed edge and top single edge) Suppose partial dependency trees DTi, obtained from the division of the well-formed dependency tree DT. *Ei + −−→ PF *eo is true for the root packed edge *Ei =root Edge(DTi) and the top single edge *eo =top edge(DTi). o =top edge(DTi). Fig. 31 Division of well-formed DT Fig. 32 Well-formed DTi obtained from division of DT Fig. 31 Division of well-formed DT Fig. 31 Division of well-formed DT Fig. 32 Well-formed DTi obtained from division of DT Fig. 32 Well-formed DTi obtained from division of DT 792 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Proof Let *et be top edge(DT) and ni be the top node of DTi (Figure 32). Consider the case where DT is a tree consisting of a single node, i.e., DTi ={ni}. *eo is the single lexical edge corresponding to ni. *Ei + −−→ PF *eo is true because the phrase head of *Ei is ni. Consider another case where DT is a tree consisting of arcs. *et ∗ −−→ PF *eo is true, according to Lemma B.6. Therefore, X ∗ −−→ PF *eo is true for some packed edge X in cs(*et). X=*Ei is true because *Ei ∈cs(*et) from the definition and the phrase heads of *Ei and *eo are the same. [Proof of the Completeness and Soundness of the Dependency Forest] [Proof of the Completeness and Soundness of the Dependency Forest] A corresponding dependency tree dependency tree(PT) for a phrase structure tree PT = {*e1, . . ., *em} is defined as follows: A corresponding dependency tree dependency tree(PT) for a phrase structure tree PT = {*e1, . . ., *em} is defined as follows: 793 Language Processing Vol. 21 No. 4 September 2014 Journal of Natural Language Processing Vol. 21 No. 4 September 2014 ournal of Natural Language Processing Vol. 21 No. 4 Sept Journal of Natural Language Processing Vol. 21 No. 4 September 2014 September 2014 Definition 11 [Dependency tree for a phrase structure tree PT] dependency tree(PT) = ds(*e1) ⊎· · · ⊎ds(*em) The operator ⊎is similar to the union operator ∪, which is introduced to manage the union of dependency structures that may be either a set of arcs or a set of a node. ⊎removes nodes from the union of dependency structures if it has at least one arc. The following are examples of ⊎, where ni and ai represent node and arc, respectively. {n1}⊎{a1, a2} = {a1, a2} {a1}⊎{a2, a3} = {a1, a2, a3} {n1}⊎{} = {n1} {n1}⊎{a1, a2} = {a1, a2} {a1}⊎{a2, a3} = {a1, a2, a3} {n1}⊎{} = {n1} {n1}⊎{a1, a2} = {a1, a2} {a1}⊎{a2, a3} = {a1, a2, a3} {n1}⊎{} = {n1} dependency tree(PT) is a tree because it is consructed by combining each partial dependency tree ds(*ei). dependency tree(PT) is a tree because it is consructed by combining each partial dependency tree ds(*ei). Theorem B.1 (The completeness of the dependency forest) Let PT be a phrase structure tree in the phrase structure forest PF. DT=dependency tree(PT) is a well-formed dependency tree in the dependency forest DF. Proof From the definition of the dependency forest, DT is included in DG. Nodes contained in DT and PT have a one-to-one relation, according to the partial dependency structure condition. Since PT covers the whole sentence, DT is a well-covered dependency tree. According to the C- Matrix setting conditions, every two arcs in DT satisfy the co-occurrence constraint. Therefore, dependency tree(PT) is a well-covering and well-co-occurred dependency tree in DF. Theorem B.2 (The soundness of the dependency forest) A-Step3 (Division of DT): Divide DT by removing edges in ds(top edge(DT)) to get a set of partial dependency trees DTi(1≤i≤m) and root packed edges *Ei=root Edge(DTi). A-Step4 (Computation of Partial Phrase Structure Trees): Apply the phrase structure tree generation algorithm to each DTi and *Ei(1≤i≤m) and compute each PTi. A-Step5 (Construction of Phrase Structure Tree): Returns PT=PATH ∪{*et} ∪PT1 ∪ · · · ∪PTm as a phrase structure tree for DT, *Er. n the case that DT is a set of a node (DT={n}): N-Step1 (Identification of Lexical Edge): Identify the lexical edge @elex that generated node n. N-Step2 (Identification of a Path): Identify a path from *Er to elex and return a set of single edges in the path as a phrase structure tree for DT, *Er. [Phrase Structure Tree Generation Algorithm] In the case that DT is a set of arcs: A-Step1(Identification of the Top Single Edge): Let *et be the top single edge top edge(DT). A-Step2 (Identification of a Path): Identify a path from *Er to *et. Let PATH be a set of single edges in the path except for *et. A-Step3 (Division of DT): Divide DT by removing edges in ds(top edge(DT)) to get a set of partial dependency trees DTi(1≤i≤m) and root packed edges *Ei=root Edge(DTi). A-Step4 (Computation of Partial Phrase Structure Trees): Apply the phrase structure tree generation algorithm to each DTi and *Ei(1≤i≤m) and compute each PTi. A-Step5 (Construction of Phrase Structure Tree): Returns PT=PATH ∪{*et} ∪PT1 ∪ · · · ∪PTm as a phrase structure tree for DT, *Er. In the case that DT is a set of a node (DT={n}): N-Step1 (Identification of Lexical Edge): Identify the lexical edge @elex that generated node n. N-Step2 (Identification of a Path): Identify a path from *Er to elex and return a set of single edges in the path as a phrase structure tree for DT, *Er. A-Step2 (Identification of a Path): Identify a path from *Er to *et. Let PATH be single edges in the path except for *et. A-Step3 (Division of DT): Divide DT by removing edges in ds(top edge(DT)) to get a set of partial dependency trees DTi(1≤i≤m) and root packed edges *Ei=root Edge(DTi). A-Step4 (Computation of Partial Phrase Structure Trees): Apply the phrase structure tree generation algorithm to each DTi and *Ei(1≤i≤m) and compute each PTi. Theorem B.2 (The soundness of the dependency forest) Let DT be a well-formed dependency tree in the dependency forest DT. There exists a phrase structure tree PT in the phrase structure forest PF such that DT=dependency tree(PT). Proof The existence of a phrase structure tree PT that satisfies PT∈ps trees(*Eroot) and dependency tree(PT)=DT is shown below. Let n be a number of input words. The following is an algorithm, called the phrase struc- ture tree generation algorithm, which generates a phrase structure tree from a packed edge *Er with from-position spr and to-position tpr (1≤spr <tpr≤n) and a well-formed dependency tree DT, which covers from spr to tpr. The proof that the phrase structure tree generation algo- rithm generates a phrase structure tree that satisfies the above conditions is shown below using mathematical induction for the number of arcs in the dependency tree. 794 PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Phrase Structure Tree Generation Algorithm] In the case that DT is a set of arcs: A-Step1(Identification of the Top Single Edge): Let *et be the top single edge top edge(DT). A-Step2 (Identification of a Path): Identify a path from *Er to *et. Let PATH be a set of single edges in the path except for *et. A-Step3 (Division of DT): Divide DT by removing edges in ds(top edge(DT)) to get a set of partial dependency trees DTi(1≤i≤m) and root packed edges *Ei=root Edge(DTi). A-Step4 (Computation of Partial Phrase Structure Trees): Apply the phrase structure tree generation algorithm to each DTi and *Ei(1≤i≤m) and compute each PTi. A-Step5 (Construction of Phrase Structure Tree): Returns PT=PATH ∪{*et} ∪PT1 ∪ · · · ∪PTm as a phrase structure tree for DT, *Er. In the case that DT is a set of a node (DT={n}): N-Step1 (Identification of Lexical Edge): Identify the lexical edge @elex that generated node n. N-Step2 (Identification of a Path): Identify a path from *Er to elex and return a set of single edges in the path as a phrase structure tree for DT, *Er. Phrase Structure Tree Generation Algorithm] n the case that DT is a set of arcs: A-Step1(Identification of the Top Single Edge): Let *et be the top single edge top edge(DT). A-Step2 (Identification of a Path): Identify a path from *Er to *et. Let PATH be a set of single edges in the path except for *et. Theorem B.2 (The soundness of the dependency forest) A-Step5 (Construction of Phrase Structure Tree): Returns PT=PATH ∪{*et} ∪PT1 ∪ · · · ∪PTm as a phrase structure tree for DT, *Er. In the case that DT is a set of a node (DT={n}): N-Step1 (Identification of Lexical Edge): Identify the lexical edge @elex that generated node n. N-Step1 (Identification of Lexical Edge): Identify the lexical edge @elex that generated node n. N-Step2 (Identification of a Path): Identify a path from *Er to elex and return a set of single edges in the path as a phrase structure tree for DT, *Er. N-Step2 (Identification of a Path): Identify a path from *Er to elex and return a set of single edges in the path as a phrase structure tree for DT, *Er. When DT is s a set of arcs, the phrase structure tree PT is constructed through A-Step1 to A-Step5. Figure 33 shows the behavior of the algorithm diagrammatically. A-Step1 computes the top single edge *et(=top edge(DT)) (Figure 33 (S1)). A-Step2 identifies a path from *Er to *et and computes PATH, a set of single edges. The existence of this path (*Er + −−→ PF *et) is assured as follows: … … Fig. 33 Generation of PT from DT and *Er Fig. 33 Generation of PT from DT and *Er 795 Journal of Natural Language Processing Vol. 21 No. 4 Journal of Natural Language Processing Vol. 21 No. 4 Vol. 21 No. 4 September 2014 September 2014 In the case that *Er is *Eroot, it is obvious. In the case that *Er is obtained by the division of a dependency graph (*Ei in A-Step4), it is assured by Lemma B.8. The dependency structure parts of the single edges in PATH are {}. This is obvious because *Er and *et have the same coverage since all nodes in DT are in the coverage of *et. dependency tree(PATH) = {} (A) (A) A-Step3 performs the division of DT and generates DTi, *Ei(1≤i≤m), as shown in Figure 33 (S3). According to Lemma B.9, DTi is a well-formed dependency tree covering the coverage of *Ei and the phrase structure tree generation algorithm is applicable recursively at A-Step4. A-Step5 computes the phrase structure tree PT (Figure 33 (S5)). From the definition of the phrase structure tree, it is obvious that PT is a phrase structure tree if each PTi is a phrase structure tree. When DT is a set of a node (DT={n}), N-Step1 and N-Step2 compute a phrase structure tree PT. The existence of a path from *Er to elex is assured for the same reason described in the explanation of A-Step1. The phrase structure tree PT generated by the phrase structure tree generation algorithm satisfying DT=dependency tree(PT) is shown as follows: In the case that DT={nr}, the algorithm generates PT at N-Step2. PT is a phrase structure tree containing one node nr. From the definition of dependency tree, dependency tree(PT)={nr} is true. In the case that DT is a dependency tree that consists of a set of arcs, the phrase structure tree generation algorithm, the definition of dependency tree, and (A) give the following equation. dependency tree(PT) dependency tree(PT) dependency tree(PT) = dependency tree(PATH ∪{∗et} ∪PT1 ∪· · · ∪PTm) = dependency tree(PATH ∪{∗et} ∪PT1 ∪· · · ∪PTm) = dependency tree(PATH) ⊎dependency tree({∗et})⊎dependency tree(PT1) = dependency tree(PATH) ⊎dependency tree({∗et})⊎dependency tree(PT1) ⊎· · · ⊎dependency tree(PTm) = dependency tree({∗et}) ⊎dependency tree(PT1) ⊎· · · ⊎dependency tree(PTm) Assume that PTi corresponding to DTi, *Ei in A-Step4 satisfies the following. dependency tree(PTi)=DTi (1≤i≤m) 796 PDG and its Packed Shared Data Structure “Dependency Forest” PDG and its Packed Shared Data Structure “Dependency Forest” Hirakawa Hirakawa Now, PT generated at A-Step5 generates DT as shown below. dependency tree(PT) = dst ⊎DT1 ⊎· · · ⊎DTm = dst ⊎DT1 ⊎· · · ⊎DTm = DT Hideki Hirakawa: Hideki Hirakawa received the B.E. and M.E. degrees in elec- trical engineering from Kyoto University, Kyoto, Japan, in 1978 and 1980, respectively. He received the PhD degree of Information and Science Technol- ogy from Tokyo University, Tokyo, Japan, in 2007. In April 1980, he joined the Toshiba Corp, Kawasaki, Japan. From 1982 to 1984, he was a researcher at Institute for New Generation Computer Technology (ICOT), Tokyo, Japan. From 1994 to 1995, he worked as a research affiliate of the MIT Media Labo- ratory, Boston, U.S.A. He is currently a Chief Specialist of the Government & External Relations Office, Toshiba Corp., Tokyo, Japan. He is a member of the Association for Natural Language Processing of Japan, the Information Pro- cessing Society of Japan, and the Japanese Society for Artificial Intelligence. (Received October 31, 2005) (Accepted February 4, 2006) 797
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Islam and Modernity in Crosshairs of History
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Islam and Modernity in Crosshairs of History Dr Ghulam Shabbir Assistant Professor(Humanities), Comsats Institute of Information Technology,Park Road, Chack Shehzad, Islamabad. Email: shabbir1world@yahoo.com Published: 10-07-2021 Accepted: 26-05-2021 Received: 25-04-2021 Saleem Nawaz Assistant Professor,Head, Islamic Studies Department Army Burn Hall College for Boys,Abbottabad Email: saleem.scholar@gmail.com Received: 25-04-2021 OPEN ACCESS S Al-Duhaa ﴾﴾﴾﴾Journal of Islamic Studies ﴿﴿﴿﴿ ISSN (print): 2710-0812 ISSN (online): 2710-3617 www.alduhaa.com OPEN ACCESS S Al-Duhaa ﴾﴾﴾﴾Journal of Islamic Studies ﴿﴿﴿﴿ ISSN (print): 2710-0812 ISSN (online): 2710-3617 www.alduhaa.com Al-duhaa, Vol.:2, Issue: 1, Jan- June 2021 DOI: 10.51665 /al-duhaa.002.01.0041 , PP: 01-24 Al-duhaa, Vol.:2, Issue: 1, Jan- June 2021 DOI: 10.51665 /al-duhaa.002.01.0041 , PP: 01-24 Abstract Static view of religion is a cause of concern in all religious communities because it not only gnaws at their dynamism, more often it tends to swing history back to the old life patterns which have lost their validity and moral force. On the other hand history moves forward and seeks its direction intuitively. So, traditional view of religion collides with the forces of history in futile effort to cease the torrential stream of time. Resultantly, time or history crush them or throw them in the yokes of slavery of others who entertain ever fresh and dynamic view of history and religion. Same went with Islam. With an advent of modernity after European renaissance when Europe collided horns with Islam, once all the Muslim world submerged in European colonialism.it forced Muslim intelligentsia on serious soul search and brought forth three schools of thought the traditionalist who strictly cling to tradition even if it had relevance to the emerging realities of history or not; the revivalists who seek assuage in pristine Islam and turn the tide of history back while modernists sensing new realities harness materieux of history for moral cause and go hand in hand with history. Modernity is also a departure from mythos to the rationale so the most crucial question of todays’ scholarship is whether Islam and modernity are compatible or poles apart in context of history. Keywords: Islam, modernity, history, modernists, revivalists, traditionalists, civilizations, monotheism, moral order, orthodoxy, weltanschauung, socio-economic cum political order, multiplicity of religions, Asabiyya (group solidarity). Religious communities the world over, be it the monotheistic traditions of Judaism, Christianity and Islam or other major religions like Hinduism are abhorrent of modernity. To a common observer, instead of successfully assimilating, rejecting, adapting and adjusting themselves to the currents of modern thoughts or accommodating the same in their folds they preferred to lock horns with modernity. Unoriginal and none creative response of religious orthodoxy provoked the hubris of modern man to such 1 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 an extent that with a greater vengeance he said that God is dead1; modernity has sealed the fate of religion. Abstract Now, it is a global feel that while the unsettling modernity has outpaced socio-religious sciences, the attempt and response of religious community is like an old confused woman with a cottage on the coasts of Atlantic, busy in futile efforts of sweeping back the tidal waves of the grand ocean. While modernity has swung humanity to the threshold of unprecedented power, peace and prosperity, the supreme objects of man’s desire and religious scriptures too instruct him how to manage and wield these precious blessings, then why religion and modernity instead of mutual harmony have become hornet’s nest to each other.Mutual harmony is the ultimate destiny of religion and modernity; for who says “ my religion is inconsistent with science has passed judgment on the falsity of his religion.Now, when “human intellect has about outgrown its fundamental categories of time, space and causality3”; cognitive revolutions suggest new ways of looking at the problem common to both religion and science. Standing at the precipice of utter extinction modern man is entrapped in cobweb of his own intellect. This reality unfolds itself in Noam Chomsky while he says that piles of nuclear arsenals and climatic challenges are clear signs that man has proved to be an evolutionary error- either melting glaciers would destroy us or a trigger of nuclear war would suffice to erase this unlucky creature from earth. He (man) was indeed unjust and foolish (33: 72). Man “has certainly not yet fulfilled God’s [primordial] command” (80:23). Recipient and donor of al-amana (Trust(establishment of moral social order on earth visit again 80:23)) both took unique risk, but man forgot that rather than being only unique privilege it is an obligation too. Man is bestowed with faculty of creative knowledge (2:30 -35) and religion and science had to walk hand in hand but religious academic orthodoxy failed to touch the pace of modernity and as a corollary both parted ways. Abstract Karen Armstrong did a commendable job by evaluating the historic developments in monotheistic religions of Judaism, Christianity and Islam viz a viz modernity more or less on same patterns but conclusion of her work seem to portray as if religion be an art work like other fine art rather than a transcendental source of human guidance by which the unseen tables and statutes of the law are revealed from the other world, together with several of the sciences of the Realms Celestial and Terrestrial and preeminently Theology4. Still credit or discredit of Karen’s thought goes to the rich secular academic milieu which shaped her mindset. However, in this paper I have to evaluate the historicity of Islam and modernity- whether Islam and modernity are poles apart or obverse and converse of the same coin? Fi t t I l i t t f d it ld h t an extent that with a greater vengeance he said that God is dead1; modernity has sealed the fate of religion. Now, it is a global feel that while the unsettling modernity has outpaced socio-religious sciences, the attempt and response of religious community is like an old confused woman with a cottage on the coasts of Atlantic, busy in futile efforts of sweeping back the tidal waves of the grand ocean. While modernity has swung humanity to the threshold of unprecedented power, peace and prosperity, the supreme objects of man’s desire and religious scriptures too instruct him how to manage and wield these precious blessings, then why religion and modernity instead of mutual harmony have become hornet’s nest to each other.Mutual harmony is the ultimate destiny of religion and modernity; for who says “ my religion is inconsistent with science has passed judgment on the falsity of his religion.Now, when “human intellect has about outgrown its fundamental categories of time, space and causality3”; cognitive revolutions suggest new ways of looking at the problem common to both religion and science. Standing at the precipice of utter extinction modern man is entrapped in cobweb of his own intellect. This reality unfolds itself in Noam Chomsky while he says that piles of nuclear arsenals and climatic challenges are clear signs that man has proved to be an evolutionary error- either melting glaciers would destroy us or a trigger of nuclear war would suffice to erase this unlucky creature from earth. Abstract He (man) was indeed unjust and foolish (33: 72). Man “has certainly not yet fulfilled God’s [primordial] command” (80:23). Recipient and donor of al-amana (Trust(establishment of moral social order on earth visit again 80:23)) both took unique risk, but man forgot that rather than being only unique privilege it is an obligation too. Man is bestowed with faculty of creative knowledge (2:30 -35) and religion and science had to walk hand in hand but religious academic orthodoxy failed to touch the pace of modernity and as a corollary both parted ways. Karen Armstrong did a commendable job by evaluating the historic developments in monotheistic religions of Judaism, Christianity and Islam viz a viz modernity more or less on same patterns but conclusion of her work seem to portray as if religion be an art work like other fine art rather than a transcendental source of human guidance by which the unseen tables and statutes of the law are revealed from the other world, together with several of the sciences of the Realms Celestial and Terrestrial and preeminently Theology4. Still credit or discredit of Karen’s thought goes to the rich secular academic milieu which shaped her mindset. However, in this paper I have to evaluate the historicity of Islam and modernity- whether Islam and modernity are poles apart or obverse and converse of the same coin? First to see Islam in context ofmodernity, we would have to understand the difference between Din and religion to grasp the nettle firmly. Though on different occasions Quran define Din as belief in God, angles, revealed books, prophets and Day of Judgment, still in its simplest form Din is belief in God and righteous deeds(2:62; 5:69); and anything that helps in 2 Islam and Modernity in Crosshairs of History establishing justice and equity in society will be considered part of Din5; for élan of alamana (33:73) is the establishment of just moral order on the earth.Message of all the prophets was the same; they advocated the same Din but Sharia have been many, dictated by differences of time and place6. Messages of all the Prophets gush out from a same single source “the Mother of the Book” (43:4; 13:39), “Preserved Tablet” or “the Hidden Book” (56:78). Abstract Hence God’s Din is indivisible; as a corollary prophethood is also indivisible: “The same Religion (Din)has he established for you as that which he enjoined on Noah—that which we have sent by inspiration to thee and that which enjoined upon Abraham, Moses, and Jesus: Namely that you should remain steadfast in religion and make no divisions therein…. (42:13; also see 4:163). That is because Prophet felt obligated to believe in prophethood of all prophets whether named in Quran or not, for “and there is no nation wherein warner has not come (35:24) and “for every people a guide has been provided” (13:7). In a same vein there is no distinction between prophets (3:84; 2:285; 4:150-152), still some have superiority over others which is functional, earned rather than inherent or built-in for that goes against the justice of God.In nutshell, Din walks parallel to history (historic phenomenon) while if a certain set of values get the status of finality or permanence at a particular moment of history assumes the form of religion. So Din is dynamic while religion is static. Orthodox behaviors resist change while Din accommodates it. Issue is, the prophets’ messages act as watersheds, so after their departure their followers are liable to deviate or split from the main, for divisive forces are perhaps part of Divine Mystery (10:19; 11:18).Hence the people of Book who had been split or deviated from the Din e Hanif (“Straight”, upright”, upright monotheist who does not follow divisive forces) in initial phase of revelation are addressed as sectarians, partisans, the people who are divisive of the unity of Religion. It happens, when teaching of Din ossifies, different parties and stakeholders emerge within the folds of same religion. In early Meccan era, these Hizbs (sects) or Shiyyas (groups) are invited to join the same Din which starts from Adam and culminates in personality of Muhammad (PBUH) so that One God one Humanity paradigm might be materialized, when it falters, it sets him (PBUH)a theological problem of first order which from the start of his prophetic career goes well into the last phase of prophets life. But Divine Mystery (Mashiyyah) resolves it by acknowledging others as communities like Judaism and Christianity by dropping the terms “Sectarians” and “partisans” and the Muslims are declared as a separate community after eighteen months of Hijra in Madina. Abstract [Thi i ] th it f f f th Ab h h l d establishing justice and equity in society will be considered part of Din5; for élan of alamana (33:73) is the establishment of just moral order on the earth.Message of all the prophets was the same; they advocated the same Din but Sharia have been many, dictated by differences of time and place6. Messages of all the Prophets gush out from a same single source “the Mother of the Book” (43:4; 13:39), “Preserved Tablet” or “the Hidden Book” (56:78). Hence God’s Din is indivisible; as a corollary prophethood is also indivisible: “The same Religion (Din)has he established for you as that which he enjoined on Noah—that which we have sent by inspiration to thee and that which enjoined upon Abraham, Moses, and Jesus: Namely that you should remain steadfast in religion and make no divisions therein…. (42:13; also see 4:163). That is because Prophet felt obligated to believe in prophethood of all prophets whether named in Quran or not, for “and there is no nation wherein warner has not come (35:24) and “for every people a guide has been provided” (13:7). In a same vein there is no distinction between prophets (3:84; 2:285; 4:150-152), still some have superiority over others which is functional, earned rather than inherent or built-in for that goes against the justice of God.In nutshell, Din walks parallel to history (historic phenomenon) while if a certain set of values get the status of finality or permanence at a particular moment of history assumes the form of religion. So Din is dynamic while religion is static. Orthodox behaviors resist change while Din accommodates it. Issue is, the prophets’ messages act as watersheds, so after their departure their followers are liable to deviate or split from the main, for divisive forces are perhaps part of Divine Mystery (10:19; 11:18).Hence the people of Book who had been split or deviated from the Din e Hanif (“Straight”, upright”, upright monotheist who does not follow divisive forces) in initial phase of revelation are addressed as sectarians, partisans, the people who are divisive of the unity of Religion. It happens, when teaching of Din ossifies, different parties and stakeholders emerge within the folds of same religion. Abstract In early Meccan era, these Hizbs (sects) or Shiyyas (groups) are invited to join the same Din which starts from Adam and culminates in personality of Muhammad (PBUH) so that One God one Humanity paradigm might be materialized, when it falters, it sets him (PBUH)a theological problem of first order which from the start of his prophetic career goes well into the last phase of prophets life. But Divine Mystery (Mashiyyah) resolves it by acknowledging others as communities like Judaism and Christianity by dropping the terms “Sectarians” and “partisans” and the Muslims are declared as a separate community after eighteen months of Hijra in Madina. [This is] the community of your forefather Abraham, who already named you Muslim before this; let the messenger [Muhammad] be a witness over you in this regard and let you be witnesses over mankind. So establish prayer, pay zakat and hold fast to God who is your protector-what an excellent protector and what an excellent helper! (22:78). After it, Jews and Christians as sectsab initioare recognized as 3 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 communities per se. Now, the issue of the multiplicity of religions gets center stage and is equally addressed. Here lies the rub of the problem. Each religion entertains actual power and strength (asabiya), at mass level, but it proves disastrous or catastrophe when it is at the cost of truthfulness. Stance of Islam is very clear, if we remove potholes of the ancient past and modern mines of the future. To secure and justify the multiplicity of religions Quran explain goodness at universal plane rather than clothing it into particular rituals or customs. “it is not righteousness(Universal Goodness) that you turn your faces Towards East or West; but it is righteousness to believe in God and the Last Day, and the Angles and the Book and the messengers ; to spend of your substance , out of love for Him, for your kin, for orphans, for the needy, for the wayfarer , for those who ask, and for the ransom of slaves; to be steadfast in prayer, and practice regular charity; to fulfill the contracts which you made; and to be firm and patient in pain and adversity and throughout all periods of panic. Abstract Such are the people of Truth; the God fearing (2:177).So crux of Goodness is, to believe in God and to be on the right side of history.Now, proceed forward to understand what grand great divine mystery works in multiplicity of religions. In fact, at the outset, man would live in the paradise of divine guidance, and pay heed to the engravings on his heart (91:7-10). Then a flash of consciousness sparked on his “self” and sense of possession was enticed in him that caused his ouster from that state of mind and due to greed and diverging benefits this unlucky creature got divided into various groups. Humanity was a single community then they got divided into groups(10:19; 2:213). Was it a result of sheer selfishness or egotism on part of man or divine mystery prevails here?“If your Lord had so willed, He would have made mankind one community, but they continue to remain divided (11:118). So whether traits of divisions or disputations are intrinsic to man or earned one, in any case are best suited to divine scheme. By it a competitive world of goodness come to fore. “ and We have sent down to you the Book in truth, confirming the books that existed already before it and protecting it….for each one of you[several communities]We have appointed a Law and a Way of Conduct [While the essence of religion is identical]. If God had so willed, He would have made all of you one community; so compete in goodness. To God shall you all return and He will tell you [the truth] about what you have been disputing (5:51).Hence the positive value or raison detre of multiplicity of religion is to compete in goodness, in this regard no community is God’s darling; Muslims are warned of their substitute if fail to deliver (9:38). Abraham on being appointed as leader of men asked the fate of his progeny, simple reply was “We do not extend promise to the unjust ones” (2:114).Noah was rebuked over the fate of his son. In Divine scheme justice prevails. Be it an individual or community, in competition law of struggle does matter. In the world of unity of Din and plurality of Sharia (42:13) only those prevail who protect and enhance the fruits of power, peace and prosperity. If all the mankind were on the same religion, how insipid the communities per se. Abstract Now, the issue of the multiplicity of religions gets center stage and is equally addressed. Here lies the rub of the problem. Each religion entertains actual power and strength (asabiya), at mass level, but it proves disastrous or catastrophe when it is at the cost of truthfulness. Stance of Islam is very clear, if we remove potholes of the ancient past and modern mines of the future. To secure and justify the multiplicity of religions Quran explain goodness at universal plane rather than clothing it into particular rituals or customs. “it is not righteousness(Universal Goodness) that you turn your faces Towards East or West; but it is righteousness to believe in God and the Last Day, and the Angles and the Book and the messengers ; to spend of your substance , out of love for Him, for your kin, for orphans, for the needy, for the wayfarer , for those who ask, and for the ransom of slaves; to be steadfast in prayer, and practice regular charity; to fulfill the contracts which you made; and to be firm and patient in pain and adversity and throughout all periods of panic. Such are the people of Truth; the God fearing (2:177).So crux of Goodness is, to believe in God and to be on the right side of history.Now, proceed forward to understand what grand great divine mystery works in multiplicity of religions. In fact, at the outset, man would live in the paradise of divine guidance, and pay heed to the engravings on his heart (91:7-10). Then a flash of consciousness sparked on his “self” and sense of possession was enticed in him that caused his ouster from that state of mind and due to greed and diverging benefits this unlucky creature got divided into various groups. Humanity was a single community then they got divided into groups(10:19; 2:213). Was it a result of sheer selfishness or egotism on part of man or divine mystery prevails here?“If your Lord had so willed, He would have made mankind one community, but they continue to remain divided (11:118). So whether traits of divisions or disputations are intrinsic to man or earned one, in any case are best suited to divine scheme. By it a competitive world of goodness come to fore. Abstract “ and We have sent down to you the Book in truth, confirming the books that existed already before it and protecting it….for each one of you[several communities]We have appointed a Law and a Way of Conduct [While the essence of religion is identical]. If God had so willed, He would have made all of you one community; so compete in goodness. To God shall you all return and He will tell you [the truth] about what you have been disputing (5:51).Hence the positive value or raison detre of multiplicity of religion is to compete in goodness, in this regard no community is God’s darling; Muslims are warned of their substitute if fail to deliver (9:38). Abraham on being appointed as leader of men asked the fate of his progeny, simple reply was “We do not extend promise to the unjust ones” (2:114).Noah was rebuked over the fate of his son. In Divine scheme justice prevails. Be it an individual or community, in competition law of struggle does matter. In the world of unity of Din and plurality of Sharia (42:13) only those prevail who protect and enhance the fruits of power, peace and prosperity. If all the mankind were on the same religion, how insipid the 4 Islam and Modernity in Crosshairs of History world might be. Among the communities which deserves, iscoroneted. Hence, Quran vehemently condemns the election theory of Jews: that Jews or the darling community of God; for an irreversible Election of individual or community runs against the tenor of justice in Divine scheme. So exclusivist claims of Jews and Christians are condemned in Quran. “They say none shall enter the gardens except Jews (as the Jews say) and Christians (as the Christian say) (2:111). On their exclusivist claims for monopoly on guidance Quran exhorts that guidance is God’s guidance (2:135; 2:120; 5:18). Zenith of their self- deception and haughtiness lies in the fact despite both follow or recite the Book but say guidance is exclusively their prerogative (2:113). In nutshell, so far we have tried to establish the fact that unity of Din and plurality of Sharia finds its roots in Quran (42:13). Abstract Hence when God intends to bless some community, equips it with a special kind of knowledge.That is because 5 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 that community merits the mercies of the Almighty, otherwise God does not extend His promise to the unjust ones (2:124). So, on a broader canvas, knowledge and science rules the world. Afghani utters same reality: “there was, is, and will be no ruler in the world except science. If we look at the Chaldean conquerors, like Semi-ramis, who reached the borders of Tartary and India, the true conquerors were not the Chaldeans but science and knowledge. The Egyptians who increased their realm, and Ramses 11 called Sosestris, who reached Mesopotamia according to some and India according to others- it was not the Egyptians but science that did it. The Phoenicians who, with their ships, gradually made colonies of British Isles, Spain, Portugal, and Greece – in reality it was science not Phoenicians, which so expanded their power. Alexander never came to India or conquered Indian; rather what conquered Indians was science….. Ignorance had no alternative to prostrating itself humbly before science and acknowledging its submission. In reality, sovereignty has never left the abode of science. However, this true, which is science, is continually changing capitals. Sometimes, it has moved from East to West, and other times from West to East8”. Same echoes inMaulanaMaudoodi while he says only that community is entitled to rule the world which proves itself best guardian of the garden of universe; otherwise guards are apt to be changed. Of course! God is sitting in a watch-tower (89:14). So this is mashiyya or divine mystery operational behind multiplicity of religions, I think.Any community when it becomes decadent and decrepit, after loss of moral vigor is de-seated from celestial and as a corollary territorial realms, it can prolong its existence but its exit is inevitable. Though this activity involves loss of mankind, reshuffle brings fresh blood to the veins of aging humanity and keeps the perennial moral values alive. Crux of (22:40- 41; 2:251) tells the same. ) After Din, religion and multiplicity of religions being discussed, a cursory glance of term modernity is overdue. Modernity has many implications and connotations. To Karen Armstrong, modernity is a clean departure from hypothesis towards rationality and finds its roots in European Renaissance, which is perhaps termed as cognitive revolution by Noam Chomsky. Abstract And there miraculously works divine wisdom in multiplicity of religion; for without it evaporates the milieu of‘compete in goodness’, also rather than pure divine mystery inherently people intend to divide in groups due to pettiness (10:19; 2:213), but final integration and spiritual homogeneity is the ultimate destiny of mankind; for Sooner or later the ethical ideal of monotheism (3:64) has to materialize on the realm of history. Pointing to this reality, Karen Armstrong strikes a redeeming note “the ideal of Quran of the “final unity of humanity” could not be realized so far because of the wars of Islam and Christianity. And later, European aggression in its various forms, could not allow the infinite meaning of the verse (3:64) to the countries of Islam in the shape of what is called Muslim Nationalism7”. As a corollary, the agent of human unity (Muslim community) due to European ideological incursions got divided on artificial constructs of caste, color, creed and language. g g In reality, Divine Scheme operates within the framework of multiplicity of religions or communities when Quran stays faith and goodness as the touchstone for evaluating them.“Those who believe [Muslims], the Jews, the Christians, and the Sabeans- whosoever believe in God and the Last Day and do good deeds, they shall have their reward from their Lord, shall have nothing to fear, nor shall they come to grief (2:62; cf . 5:69).Henceforth, universal goodness is recognized on theological realm, with belief in God and the Last Day as the linchpin of or necessary underpinnings. In Divine scheme only the people of excellence and virtue entertain the right to exist and prosper but active goodness (not passive) must succeed is clear from the tone tenor and tune of Quran. It keeps the battlefield hot, same goes for the community what is right on an individual level: the man can have nothing but what he strives for (53:39). In a nutshell to compete in goodness (5:51) is the major import of multiplicity of religions. How stale the world might be if without evolution of proper weltanschauung, or essential experience mankind get unified on a single platform. Different communities have to excel in virtue, and what virtue can be more valuable or precious than creative knowledge – the raison detre of man’s creation (2:31). Abstract This latter development had been more revolutionary in its impact on societies than any other event in history9”.Hence, change is the greatest sign of God, modernity is a natural response to that change, while Islam is the primordial religion on which Godhas originated mankind….this is the straight religion…and do not be [O Muslims!] among those who associate [partners with God], nor among those who split up their religion into sects, each sect rejoicing in what it has (30:30- 32). Quran calls Muhammad as a Hanif, true monotheist and his religion as the “ straight religion [al-din al-qayyim”] while on the other end of spectrum paganism and sectarianism are splits or deviations from the main stream. Islam is Din, Judaism and Christianity are religions of Islam and if “ for every community there had been a guide” is a guide, all are religions of Islamemanated from same source umm al-kitab (mother of all books), kitab al maknun(Hidden Book)and are destined to same destiny, unity of religion, the ultimate destiny of mankind. At critical juncture, despite prophets’ painfully immense struggle to get humanity on uniform platform, Islam accepted validity and role of other religions on the realm of history. So, Islam the God’s handiwork being the Din e Hanif (true religion) is in harmony to nature just as we say “magnetic needle is true to the north. At normative level, man as an artifact, shaped up by the creative hand of God, is pure, true, free, inclined to rightness, equipped with true understanding about his own position in the universe and God’s goodness, wisdom and power.That is why popular tradition says man has been created on Din e Fitrat. But oblivious of his privileged status he succumbs to carnal pleasure. In nutshell, Din and modernity are both historic phenomena, so instead of supplanting or substituting each other both should go hand in hand. Instead of parting ways Islam and Modernity in Crosshairs of History to another, in our age from agrarian/pastoral mode to the capitalist/industrial mode of production. The shift from one to another mode of production invariably brings revolutionary changes in society. It compels a new logic of social and economic life, threatens inherited styles of life, and forces transformations in the relationship of land, labor and capital. Abstract As such, it requires adaptations to new ways of being and doing, and demands drastic changes in human values and in the relations of sexes, classes, individuals, families and communities. It transforms the co-relation and arrangement of living spaces, requires change in how the workplace is organized, how new skills are gathered and distributed and how people are governed…when this process of change sets in, older values and ways of life become outdated and dysfunctional much faster than newer ones, more appropriate values and ways of life strike roots. The resulting social and cultural mutations are experienced by people both as threat and loss. For millennia humanity had experienced this unsettling process, for example when it moved from Stone Age to the age of iron, or when it discovered fire and shifted from hunting and gathering to agriculture. But never had this process been more intense and more revolutionary than it became with the rise of capitalism and industrial mode of production. This latter development had been more revolutionary in its impact on societies than any other event in history9”.Hence, change is the greatest sign of God, modernity is a natural response to that change, while Islam is the primordial religion on which Godhas originated mankind….this is the straight religion…and do not be [O Muslims!] among those who associate [partners with God], nor among those who split up their religion into sects, each sect rejoicing in what it has (30:30- 32). Quran calls Muhammad as a Hanif, true monotheist and his religion as the “ straight religion [al-din al-qayyim”] while on the other end of spectrum paganism and sectarianism are splits or deviations from the main stream. Islam is Din, Judaism and Christianity are religions of Islam and if “ for every community there had been a guide” is a guide, all are religions of Islamemanated from same source umm al-kitab (mother of all books), kitab al maknun(Hidden Book)and are destined to same destiny, unity of religion, the ultimate destiny of mankind. At critical juncture, despite prophets’ painfully immense struggle to get humanity on uniform platform, Islam accepted validity and role of other religions on the realm of history. So, Islam the God’s handiwork being the Din e Hanif (true religion) is in harmony to nature just as we say “magnetic needle is true to the north. Abstract Some perceive the wholesale rejection of religion or separation of church and state as modernity- product of secularism. Question is whether man from day first is not rational? Are we sure that rationalism of our time would be sound reality in annals of history or after millennia its major part would prove conjectural and absurd. Whether the slogan of French Revolution Freedom, liberty and fraternity was the thought product of modern man abhorrent of religion or it was religion which over millennia had infused these concepts in conscience of man through prophets and guides but religious orthodoxy distorted the same due to its vested interests. So to remove ambiguities of modernity let us get it out of its ore. A sane sage soul, Ibn e Khaldun of Modern timesDrEqbalahmad says “Modernity is a historical process. It refers to the development of societies from one mode of production 6 Islam and Modernity in Crosshairs of History to another, in our age from agrarian/pastoral mode to the capitalist/industrial mode of production. The shift from one to another mode of production invariably brings revolutionary changes in society. It compels a new logic of social and economic life, threatens inherited styles of life, and forces transformations in the relationship of land, labor and capital. As such, it requires adaptations to new ways of being and doing, and demands drastic changes in human values and in the relations of sexes, classes, individuals, families and communities. It transforms the co-relation and arrangement of living spaces, requires change in how the workplace is organized, how new skills are gathered and distributed and how people are governed…when this process of change sets in, older values and ways of life become outdated and dysfunctional much faster than newer ones, more appropriate values and ways of life strike roots. The resulting social and cultural mutations are experienced by people both as threat and loss. For millennia humanity had experienced this unsettling process, for example when it moved from Stone Age to the age of iron, or when it discovered fire and shifted from hunting and gathering to agriculture. But never had this process been more intense and more revolutionary than it became with the rise of capitalism and industrial mode of production. Abstract At normative level, man as an artifact, shaped up by the creative hand of God, is pure, true, free, inclined to rightness, equipped with true understanding about his own position in the universe and God’s goodness, wisdom and power.That is why popular tradition says man has been created on Din e Fitrat. But oblivious of his privileged status he succumbs to carnal pleasure. In nutshell, Din and modernity are both historic phenomena, so instead of supplanting or substituting each other both should go hand in hand. Instead of parting ways to 32). Quran calls Muhammad as a Hanif, true monotheist and his religion as the “ straight religion [al-din al-qayyim”] while on the other end of spectrum paganism and sectarianism are splits or deviations from the main stream. Islam is Din, Judaism and Christianity are religions of Islam and if “ for every community there had been a guide” is a guide, all are religions of Islamemanated from same source umm al-kitab (mother of all books), kitab al maknun(Hidden Book)and are destined to same destiny, unity of religion, the ultimate destiny of mankind. At critical juncture, despite prophets’ painfully immense struggle to get humanity on uniform platform, Islam accepted validity and role of other religions on the realm of history. So, Islam the God’s handiwork being the Din e Hanif (true religion) is in harmony to nature just as we say “magnetic needle is true to the north. At normative level, man as an artifact, shaped up by the creative hand of God, is pure, true, free, inclined to rightness, equipped with true understanding about his own position in the universe and God’s goodness, wisdom and power.That is why popular tradition says man has been created on Din e Fitrat. But oblivious of his privileged status he succumbs to carnal pleasure. In nutshell, Din and modernity are both historic phenomena, so instead of supplanting or substituting each other both should go hand in hand. Instead of parting ways 7 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 with modernity, religion should envelop it. If modern man is confident enough to dispose of his daily affairs, it is failure of religious elite who could not touch the pace of modern thought. Man has covered a long journey from Stone Age to our industrial era. Abstract Each era has had its own temper, but throughout history greatness, power, peace and prosperity has prostrated before tolerance, syncretism, openness and pluralism. Eqbal Ahmad rightly says where ideas do not clash, diverse influences, knowledge, viewpoints, and cultures do not converge, civilization does not thrive and greatness eludes. Same is élan of Islam, because when it collided with byzantine and Persian empires how successfully it absorbed and assimilated other cultures and religions in its folds. Now, a cursory glance on origin and development of Islam would be sufficed to explore an organic link between Islam and modernity. To analyze alchemy of Islam we would have to see what was the social milieu and status of humanity at moral plane when Islam emerged on the stage of history. Let us listen to the credible voice of a great historian who states it with a galling back. “It seemed then that the great civilization that it had taken four thousand years to construct was on the verge of disintegration, and that mankind was likely to return to that condition of barbarism where every tribe and sect was against the next, and law and order were unknown. The older tribal sanctions had lost their power. Hence the old imperial methods would no longer operate. The new sanctions created by Christianity were working division and destruction instead of unity and order. It was a time fraught with tragedy. Civilization, like a gigantic tree whose foliage had overarched the world, and whose branches had borne the golden fruits of art and science and literature, stood tottering its trunk no longer alive with the flowing sap of devotion and reverence, but rotted to the core, driven by the storms of war, and held together only by the cords of ancient customs and laws, that might snap at any moment. Was there any emotional culture that could be brought in to gather mankind once more into unity and to save civilization? This culture must be something of a new type, for the old sanctions and ceremonials were dead, and to build up others of same kind would be the work of centuries10”. Abstract In nutshell, History with a sever fatigue, at that particular juncture was assiduously crying for fresh message from God, for the bipolar world of Byzantine and Persia was enmeshed in moral morass and carnal pleasures, perhaps reached at the point of no return, standing at the threshold of devastation and utter annihilation. The rudderless gigantic ship of human civilization had hit the sand. Humanity was simply adrift. Ordinarily, in such a crisis God sends prophets. So, Muhammad (PBUH), appears at the Ordinarily, in such a crisis God sends prophets. So, Muhammad (PBUH), appears at the 8 Islam and Modernity in Crosshairs of History geographical location where three continents of Asia, Africa and Europe meet, with program of Divine guidance among the people untouched by any of the ancient cultures, for the book of wisdom reveals that most often there is an essential discontinuity between a decrepit and decayed civilization and its successor. Most often, there is no ready-made and quick succession to a decaying civilization. Rather, God renders the slate clean and makes a new beginning than tolerate a symbiosis of decadent and virile. Therefore Iqbal rightly says that the birth of Islam is the birth of inductive logic, so to him “Prophet of Islam seems to stand between ancient and the modern world. In so far as the source of his revelation is concerned he belongs to the ancient world (last in chain of prophetic succession); in so far as spirit of his revelation is concerned he belongs to the modern world11”. With intensive insight of history enhancing the same argument iqbal says “… European culture, on its intellectual side, is only a further development of some of the most important phases of the culture of Islam12”. What it inculcated in its followers and what transpired on the realm of history alchemy of Islam reveals that it struck cognitive revolutions in human thought which still continues13: Wherever whatever the worlds of color and fragrance you see, or whatever the world is entertaining the hope to be marvelous, perfect or excel, either it imbued from Muhammad (PBUH) or isgroping for (in search of) Muhammad(PBUH). Abstract It is because Quran is the greatest miracle of Holy Prophet (PBUH) and according to it God’s prophets and human messengers are recipients of some special or extra ordinary power which emanates from the ultimate source of all being and which fills the hearts of these prophets with something which is light whereby they see and know things the way others are not able to. It is Amr, Preserved Tablet or Mother of Books including Quran which opens to the sacred souls. Reality speaks to them, but in case of Muhammad (PBUH) it spoke to him in such an utmost and ultimate sense that He (PBUH) enveloped all reality and which was total in its comprehensive sweep. Summarizing this grand fact saysIbn e Khaldun. “Quran is alone among the divine books, in that our prophet received it directly in the words and phrases in which it appears. In this respect it differs from the Torah, the Gospel and other heavenly books. The prophets received them in the form of ideas during the state of revelation. After their return to a human state, they expressed those ideas in their own ordinary words. Therefore, those books do not have the attribute of ‘inimitability’. The other prophets received their books in a manner similar to that in which our prophet received ideas that he attributed to God, such as are found in many traditions. The fact that he received the Quran directly, in its literal form is attested by the statement of the Muhammad on authority of his Lord who said: ‘do not move your tongue too fast to con this revelation. We Ourself shall see to its collection and recital14”. So, if Quran is a guide, God talked directly to Muhammad in utmost 9 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 an ultimate sense, for all the previous prophets were nothing but Muhammad in making, and Din approaches to its logical end or evolutionary zenith. The entire gamut and drift of his prophetic career suggests he had to inject a moral order in flesh and blood of history that needed a viable socio-economico cum political order and so long as power remains on the side of corrupt political, mercantile and religious elites of Mecca that was not to be materialized. In short, this is the execution of al-amana trust(33:73), or fulfillment of primordial covenant. Abstract In IbneKhaldun’s view being at the equator, land of Arabian Peninsula was equipped with sound credentials to bring forth a best of religions. Already, there existed religious fermentation, a sort of Messianism or desire for new Arab prophet (35:42); and Meccans rejected Judaism and Christianity for they wanted to excel or do better than others which is evident from Quran (6:157-58). Quran points to the presence ofHanifs (men inclined to truth) among the community who had arrived at monotheism but instead of ritualistic belief in God, “Muhammad’s monotheism was, from the very beginning linked up with a humanism and a sense of social and economic justice whose intensity is no less than the intensity of the monotheistic idea, so that whoever carefully reads the early Revelations of the Prophet cannot escape the conclusion that the two must be regarded as expressions of the same experience (see 107:1-7)15”. Therefore, ethical ideal of Oneness of God requires a social moral order, an ethical egalitarian order devoid of gross socio-economic disparities. If God is one, mankind is also one. When socio-economic justice and monotheistic idea part ways, ethical ideal of Tauheed reduces to mere husk, polytheism emerges and social inequalities give rise to islands of opulence in a sea of poverty, earth is filled with corruption. Therefore, Muhammad’s Monotheism was, and proved springboard of social reforms. For this utmost thrust of Prophet’s message was to strike one God-one humanity paradigm, and Jews and Christians are addressed as the people of the book, when appeared not getting on withthis endeavor, it sets him with a problem of first theological order. His is top-notch an enterprise to succeed, while mecca’s inertia shows no healthy signs, journey of Taif multiplies that frustration. There is left no choice but to emigrate to Madina, establish there a power base to ultimately hold the control of Mecca. In Hitti’s sharp but half-baked analysis it was not a sudden flight, rather well-considered strategy. But color of his lens gets hazy when in Medenese phase he terms Adhan, substitution of Sabbat by Friday, change of Qibla from Jerusalem to Mecca etc as the Arabianization of Islam16; for monotheism and agenda of socio-economic reforms and consequent egalitarianism transcends any national ideal. On the other hand, Prophet’s letters to the neighboring kings amply demonstrate that Islam was bound to expand out of Arabian Peninsula. Abstract However, manipulation of the actual forces and materieux of history to the Prophets is as essential as to the worldly leaders. That is because Arabian conditioning was necessary if Islam was to develop as an effective religion in the world17 or in IbneKhaldun’s words role 10 Islam and Modernity in Crosshairs of History of actual power and strength (asabiyya) was necessary for the initial launch of Islam18. Therefore in Medina eighteen months after Hijra, to soothe the theological problem of Prophet’s fantastic scheme of one God-one humanity, the Muslims are recognized as a separate community (22:78), moreover median community(2:141) or in utmost sense the best community (3:11o; 3:104) along with Jews and Christianity.Yet, I think it was in face of social historic forces, otherwise “o People of the Book! Come on a platform that is common between us- that we serve not except God (3:64) is of eternal import and a signpost towards ultimate human integration, which requires necessary human evolution and mature environs of globalization. Also, the principles of humanitarianism, egalitarianism, socio-economic justice, universal goodness have nothing to do with Arab nationalism rather, universal moral values. However, to make history field of Divine activity, prophet’s resolve knows no limits, for he knew that mature spirituality lies in staying the course till success that is evident from vicissitudes ofinner spiritual history of prophet. y Prophet Muhammad blossomed in full glare of history; he constituted a moral social order by harnessing materieux of history. As before mentioned, emigration to Medina was a considered plan, ultimate goal was to conquer Mecca [Geneva of tribes, commercial hub of Peninsula, Vatican City of Arabs] by establishing power base in Medina, to many critics reconnaissance missions in Medina were nothing but to decorate a decisive battlefield of Badar, offensive in nature if we pay heed to Quran (8:5-7). Humiliated in dust, Meccans in 3/625 with an army of three thousands pounded Madina, and due to the disciplinary violation Muslims suffered a heavy blow, for exposed flank of Muslims was attacked. Yet when Muslims recovered, Meccans left the battlefield and marched home. So, according to the war norms victory stayed with Muslims.But the battle had shaken the hearts of Muslims to the core. God revived their morale after a fair criticism (3:139-142). What transpired, Uhd filled upMeccans cup of iniquity, while believers were assayed, their dross purified for spirituality grooms in adversity. Abstract Moral obliquity taints the intellect. In a pride, understanding turns upside down, a bar in front and a bar behind, so the victim can neither progress nor turn back. Injustice is basically reflexive. On the other hand God never lets the power of discernment or correct judgment of his party (believers) die, keeps moral fervor a perennial force alive. Two years after Uhd, in 5/627 Meccans with an army of ten thousands at the instigation of Jews of Khyaber again laid siege to Medina. It was an army of confederates, therefore called Ahzab: polytheists, Beduins and Jews etc hatched plan to occupy Medina. Resolve and discerning power of the believers came victorious, and the ditches dug in front of exposed parts of Medina proved ultimate graveyard of Meccans’ efforts to thwart Muhammad’s movement for establishing moral order.Jews were meted out justice. Now, prophet zeroes in on Mecca. A plan for lesser pilgrimage was something more than a mere ritual, a power display, sublime aggressive move rather than protective posture which points toward political acumen of 11 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 prophet (PBUH). It was a calculated risk to take stock of situation in Mecca.it culminated in Pact of Hudaybiya. Quran termed it as manifest victory; for a cool courage brings such reward. On one side Islam was recognized as an equal power, on the other it opened door for free promulgation of Islam throughout Arabia and thence through the world. Not the least, by striking truce prophet blocked any imminent danger of joint adventure against Islam. So after crushing snake (conspiracies of Jews) in backyard, prophet was waiting in the wings to take out the last thorn in the side of Islam. Muhammad and his companions performed minor pilgrimage next year in 7/628. However, in 8/629 Muhammad seized Mecca on high moral grounds; for Meccans were party to a conflict in which their opponents had alliance with Muslims.Meccans negotiated a peaceful surrender, now the whole of Arabian Peninsula was under the feet of Prophet. At culmination of this decisive victory, having pieces of stale soaked bread at house of Umm e Hani he said self-abnegation is my pride. So self- effacing he was that he referredevery bit of his achievement and success to God (110:1-3). Now Arabs embraced Islam en mass except city of Taif and the tribes of Hawazin presented stiff resistence. Abstract Here in battle of Hawazan first time despite larger in strength Muslims felt themselves earth-less, but succor of God came and they snatched victory from the jaws of defeat.Here disruption occurred on division of beauty, but conflict resolution bears testimony to the broad gauge magnanimity of prophet.Ansars objected: ours are more sacrifices, while new converts are getting more from booty. Prophet asked! Were you not poor, then due to me God made you rich? All replied, yes of course! Had not mutual dissentions sapped your lives? I resolved the same and you got unity? Alright replied Ansar! Prophet said you are speaking half-truth. Now I acknowledge your favors and you say yes! Was I not helpless and you provided me refuge? Was it not because of you that we became rich? Is it not right that today the whole Arabianpeninsula is under my feet because of you? In standing ovation they cried yes! Then is it not good that they should get booty and Muhammad goes back to Medina with you. We accept it O prophet of God was a reply. Prophet made Medina his permanent seat. From here in 9/630 conducted a campaign against northern Christian Arabs of Transjordania.Even a month before his death had ordered expedition to the North. Suffice to prove that Islam was bound to expand out the boundaries of Peninsula, letters to the emperors and kings of Persian and Byzantine Empire are icing on the cake. Accustomed to nationalism, orientalists are on futile effort to prove that these campaigns were no more than to free Arabs from Persian and byzantine colonialism. Their rigged intellect sees shifts in Muhammad’s stance every now and then, but fact is there we not see even an iota of change in rock solid integrity and character of prophet, Meccan verses (29:1-10) elaborate enough that Medinese and Meccan phase of Prophet’s life is highly interfaced and had organic link rather than even juxtaposition of two discrete periods. Though, revelation helped on crucial stages, yet it was razor-sharp intelligence prophet (PBUH). It was a calculated risk to take stock of situation in Mecca.it culminated in Pact of Hudaybiya. Quran termed it as manifest victory; for a cool courage brings such reward. On one side Islam was recognized as an equal power, on the other it opened door for free promulgation of Islam throughout Arabia and thence through the world. Abstract Not the least, by striking truce prophet blocked any imminent danger of joint adventure against Islam. So after crushing snake (conspiracies of Jews) in backyard, prophet was waiting in the wings to take out the last thorn in the side of Islam. Muhammad and his companions performed minor pilgrimage next year in 7/628. However, in 8/629 Muhammad seized Mecca on high moral grounds; for Meccans were party to a conflict in which their opponents had alliance with Muslims.Meccans negotiated a peaceful surrender, now the whole of Arabian Peninsula was under the feet of Prophet. At culmination of this decisive victory, having pieces of stale soaked bread at house of Umm e Hani he said self-abnegation is my pride. So self- effacing he was that he referredevery bit of his achievement and success to God (110:1-3). Now Arabs embraced Islam en mass except city of Taif and the tribes of Hawazin presented stiff resistence. Here in battle of Hawazan first time despite larger in strength Muslims felt themselves earth-less, but succor of God came and they snatched victory from the jaws of defeat.Here disruption occurred on division of beauty, but conflict resolution bears testimony to the broad gauge magnanimity of prophet.Ansars objected: ours are more sacrifices, while new converts are getting more from booty. Prophet asked! Were you not poor, then due to me God made you rich? All replied, yes of course! Had not mutual dissentions sapped your lives? I resolved the same and you got unity? Alright replied Ansar! Prophet said you are speaking half-truth. Now I acknowledge your favors and you say yes! Was I not helpless and you provided me refuge? Was it not because of you that we became rich? Is it not right that today the whole Arabianpeninsula is under my feet because of you? In standing ovation they cried yes! Then is it not good that they should get booty and Muhammad goes back to Medina with you. We accept it O prophet of God was a reply. Prophet made Medina his permanent seat. From here in 9/630 conducted a campaign against northern Christian Arabs of Transjordania.Even a month before his death had ordered expedition to the North. Suffice to prove that Islam was bound to expand out the boundaries of Peninsula, letters to the emperors and kings of Persian and Byzantine Empire are icing on the cake. Abstract Accustomed to nationalism, orientalists are on futile effort to prove that these campaigns were no more than to free Arabs from Persian and byzantine colonialism. Their rigged intellect sees shifts in Muhammad’s stance every now and then, but fact is there we not see even an iota of change in rock solid integrity and character of prophet, Meccan verses (29:1-10) elaborate enough that Medinese and Meccan phase of Prophet’s life is highly interfaced and had organic link rather than even juxtaposition of two discrete periods. Though, revelation helped on crucial stages, yet it was razor-sharp intelligence 12 Islam and Modernity in Crosshairs of History grounded in faith, painfully honest and ambitious enterprise of moral order and armed to the teeth statesmanshipof Muhammad that bequeathed to humanity both a set of ideals and a concrete way of achieving those ideals, which still constitute the best solution for human crisis.It is easy to travel with the speed of clouds; easier to weigh the earth, but understanding of human affairs is the complex task. It was intense spirituality and rigorous pragmatismof Muhammad which bent history to a definite course. Departure of prophets would prove water-shed event in history of religions. After them foundational phase gets over, interpretive phase sets in. Being revolutionary in nature, first phase,period of inception strikes hard on status quo, sclerotic modes of life depart and new values strike roots. In this 23 years’ period the complex edifice of the monopolies of religious-politico- cum mercantile elites crumbles to dust and second era the period of collection and application sets in. This era brought Persian and Byzantine empires under the pale of Islam and assimilative-deductive thought activity remained élan of this period. Wherever, whatever was necessary Islamassimilated, rejected and adjusted itself to the new currents.In this 29 years period which straddles even the major portion of third period of Islamic history there was an organic link between Sunnah,Ijma and Ijtehad, which later on reverses and Ijtehad comes to a standstill. Second period constitutes the time of four righteous caliphs. Endeavors of Omer the second caliph whom Shah Wali Allah says Mujtahid e Mutliq state, to them Ijtehad was to devise strategies which are supportive to moral life and pave the way for progress, peace and prosperity. The third era of Islam commences from Omayyad dynasty and ends at the start of Seljuk period. Abstract S h ij f M di diff d f th t f I E ithi h i Ahl-al-Ra’ye.Shafi the pupil of Malik Bin Anas, teacher of Ahmad Bin Hunmble and contemporary of Al-Shaibani held the seat of Cairo and adopted rather synthetic approach to devise Islamic Jurisprudence.Before Shafi’s formulation, there was an organic link between Sunnah, ijtehadand ijma.Sunnah in earlier generations of Islam was considered as the ideal or normative behavior of community. It was a tradition that preserved certain moral value and if preservation of thatvalue demanded ijtehad it was enhanced and that product of ijtehad also calledsunnah then a sort of concensus(informal ijma) developed on that Sunnah, in a final analysis it was a continuum phenomenon.That is because SnoukHurgronje held that the Muslims themselves added to the Sunnah of the prophet until almost all product of Muslim thought and practice came to be justified as the Sunnah of the prophet. To some, in early period Sunnah meant the practice of the Muslims themselves.This informal ijma did not rule out difference of opinion. Sunnah-ijma of Madina differed from that of Iraq. Even within each region differences existed, and in a natural way ijmawas being arrived through differences in local usage and different interpretations.Fresh thinking and interpretation was going on, but in later second and especially third century Hadith develops into an overwhelming movement and substitutes the twin principles of Ijtehad and ijma and relegates these two to the lowest position and severed the organic relationship between the two. This came under the exigencies of time and space, because Sunnah of different regions- Hijjaz, Iraq, Egypt etc became divergent on almost every issue of detail. It was purely for administrative purpose to struck uniformity in Muslim empire. Otherwise, gauge the flexibility and broadness of Sunnah from a single instance that when community was plunged in labyrinths of Predeterminism and Free Will (Jabrites and Qadrites schools), Omayyad caliph Abd-al-Mailk b. Marwan asked KhajaHasan-al-Basri is there any hadith regarding the issue. He replied in a letter that though there is no hadith of the prophet in favor of the free will and human responsibility, nevertheless this is the sunnah of the prophet. It means behavior of prophet and his companions implicitly contradict the doctrine of predeterminism. It shows Sunah of Prophet to earlier generation was rather a pointer in a direction than laid out series of rules. To it Quran refers”of course! Abstract This the most creative phase may be termed as the interpretive period of Islam, major impulse of this phase involves challenges and responses. During this period emerged different schools of philosophy, law and mysticism in Islam. At initial phase in seed of Islam there was an organically linked whole complex of fiqh, Kalam and Tassawuf; for (Tafaquhufiddin) was employed for intellectual efforts in all the three domains. Despite being a book of Human guidance on morality Quran regulates the crucial human affairs by concrete laws, Similarly despite its major thrust on faith- in- action it inculcates sort of spirituality in believers( see the mystic experience of Prophet in 17:1; 53:1-12 and 13-18;81:19-25) , also it elaborates key theological issues but the first generation had nothing to do with such problems because its major relevance was with the do’s and dont's to construct a socio-moral order. Naturally the seed of Islam had to sprout into these different disciplines which were present in a single whole in initial phase. With outward expansion of Islam Kufa, Basra in Iraq, Fustat (present Cairo) in Egypt initially cantonment areas developed into melting pot of knowledge and sciences. There developed three traditions: the tradition of Hijjaz, Iraq and Egypt (it included Africa that time). Here in Hijjaz the dominant factor was Hadith and Sunnah of Prophet therefore called Ahl-al- hadith. The major thrust of Iraqi tradition was on opinion therefore termed 13 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 Ahl-al-Ra’ye.Shafi the pupil of Malik Bin Anas, teacher of Ahmad Bin Hunmble and contemporary of Al-Shaibani held the seat of Cairo and adopted rather synthetic approach to devise Islamic Jurisprudence.Before Shafi’s formulation, there was an organic link between Sunnah, ijtehadand ijma.Sunnah in earlier generations of Islam was considered as the ideal or normative behavior of community. It was a tradition that preserved certain moral value and if preservation of thatvalue demanded ijtehad it was enhanced and that product of ijtehad also calledsunnah then a sort of concensus(informal ijma) developed on that Sunnah, in a final analysis it was a continuum phenomenon.That is because SnoukHurgronje held that the Muslims themselves added to the Sunnah of the prophet until almost all product of Muslim thought and practice came to be justified as the Sunnah of the prophet. To some, in early period Sunnah meant the practice of the Muslims themselves.This informal ijma did not rule out difference of opinion. Abstract You have indeed an ideal model in Apostle of God (33:21). This was the early thought activity of the Muslim, and ijtehad and ijma were its essential complements. However, it was Shafi who gave the status of finality to Ijma, and it became necessarily formal and total one, to strike equilibrium and uniformity in the empire. Ijma ceases to be as dynamic ongoing democratic process. Itbecame static and imposed one process.Sunnah rather than a general directive became absolutely literal and specific which relies on Hadith. In his principles of Islamic Jurisprudence, next place he assigns to Sunnah of the Companions, especially first four righteous Caliphs. In third place in his Schema comes Ijma, lastly he accepts Ijtehad. By reversal 14 Islam and Modernity in Crosshairs of History even in modern constitutional democracies no clause of the constitution gets the status of finality, if need arises it can be amended or removed by new consensus, but our consensuses of earlier generations or latter renovators are final in nature even though we know that sometimes Omer’s Ijtehad on face value seemed contradicting Quran but on broader canvass fulfilling the moral elan of Quran. Distribution of booty on conquest of Iraq is a case in point. So, “Al-Shafi’s genius provided a mechanism that gave stability to our medieval socio-religious fabric but at the cost, in the long run, of creativity and originality19”. It was in a fetus of this scheme to get Islam passive entity rather than an active force or being master of its own destiny. However, in the same third period of challenge and response of Muslim history which constitutes four centuries from the end of righteous caliphacy (661/61) to the beginning of Seljuk (1055/447) proved seed bed of philosophic, legal and mystic traditions along with history, hermeneutics, seerah, Hadith, literature, theology and other disciplines. This period evoked internal and external challenges and presented successful responses. During the wars of Ali and Muayia a fanatic group named Kharijites came to fore that marks the first schism in history of Islam. It presented a first ever extremist group which spilled blood of innocent Muslims on menial issues of dogma. Abstract However, in the same third period of challenge and response of Muslim history which constitutes four centuries from the end of righteous caliphacy (661/61) to the beginning of Seljuk (1055/447) proved seed bed of philosophic, legal and mystic traditions along with history, hermeneutics, seerah, Hadith, literature, theology and other disciplines. This period evoked internal and external challenges and presented successful responses. During the wars of Ali and Muayia a fanatic group named Kharijites came to fore that marks the first schism in history of Islam. It presented a first ever extremist group which spilled blood of innocent Muslims on menial issues of dogma. Highly venerated in orientalists formulations as first social democrats in Islam, for to them any righteous guy was entitled to be caliph even if he is slave, but why does western academic orthodoxy ignores their extremist formulations. They slaughtered countless Muslims on pretext of difference of opinion, or their definition of Islam.If their strict observations had been applied, Muslims would have been reduced to smaller community. It was high time to define Islam, Muslim. This challenge brought forth the first philosophic school of Murijites( English equivalent postponement or hope). To counter this severe tumult,Murijites said anybody who says KalmaTayyiba and count himself member of Muslim community would be considered Muslim, and so far as his conduct is concerned it comes in the domain of God to decide on the Day of Judgment. It is hoped God would treat him mercifully. On one hand it countered Kharijites’ tumult successfully; on the other it gave carte blanche to the Omayyad and later dictatorial regime. With the conquest of Persia Zoroastrianism andMagian thoughts were seeping into Muslim Ideologue, Sabr, Taqdir, Tawakkaland other terminologies lost their original meaning and vigor. Now Sabr takes on the meaning to forebear the excesses of Omayyad caliphs, Taqdeer stayed as something pre-written fate of humanity and Tawakkal means without struggle hoping best in future.Murijites developed into new school of Jabarites which believed in predeterminism. It justified injustice and tyrannies of Omayyads. It gave rise to moral perversion and apathy. Its antithesis Qadarite came to fore whose major thrust was free-will. It became torch-bearer of Amar bil- marufwanahianilmunkir and said man is free in his thought and action, so responsible for his deeds. Omayyad executed its two founders one after other. Abstract 15 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 But Damascus had no recipe tosilence the pious sermons ofHasan al-Basri the staunch supporter of Qadarites.Qadarites struck a beautiful balance between the indefinite powers of God and human free-will-the most difficult question of philosophy and religion. However, the efforts of this school were confined to the religious realm, while Kufa, Basra in Iraq and Fustat in Egypt have become melting pot of philosophy and sciences. Spain, central Asia, Iran and Indus valley were under the pale of Islam, two great empires of Byzantine and Persia have crumbled to dust, former because of its religious colonialism, the latter due to hotchpotch of internal conspiracies .Now, Zoroastrianism,Maniism, Mazdakism, Budhismalong with Judaism and Christianity had surrendered their political sovereignty, historically religio- cultural sovereignties had been the toughest area to withdraw from. Nestorian Christians had translated Greek philosophy and ideas were seeping into Muslim discourse, Qadarite’s mere religio-moral school of Hasan al- Basari proved insufficient to withstand queries of Greek dialectics, time was ripe for turning the practical religiosity of Arabian Peninsula into dialectical theology.Hijjaz soon after separation between state and religion in Omayyad period, or shift of capital first by Ali from Medina to Kufa, then by Omayyad to Damascus finally by Abbasid to Baghdad confined itself to Sunnah and Hadith, whatever law developed there was due to interest in these disciplines. Hence exigencies of time brought forth fourth school of Islamic philosophy called Mutazilla. The first rationalist school of Islam was an utter response to external challenges which weaved out fabric of Islamic theology: oneness of God, free will and attributes of God, justice, reward and punishment, good and bad etc with such a finesse that defies description, but the major challenge to any movement is to maintain equilibrium, they lost it and stayed intellect at par with revelation. Philosophy is a double edged sword its one end proves something the other can equally refute it. So their utter reliance on rationale invoked reaction on the side of fundamentalists. The fifth school of Islamic thought called Asharite emerged on the scene. Its founder Abul-Hasan al- Ashari was student of Imam Ahmad B. Humble, who himself was pupil of Shafi the student of Malik b. Anas; the first authentic compiler of Hadith groomed in simple religious tradition of Hijjaz. It was in a sense equivalent of reversal of the religious evolutionary thought. Abstract Perhaps once more, literalism, fundamentalism, decontextualized Hijjazi tradition reigns supreme on realm of theology. Free-will sacrificed on the altar of indefinite powers of God to an extent thatman became an agent of God’s plan. It flatly refused free will of man. That even resonates well in an eighteenth century Indian poet Mir. ﻧﺎﺣﻖ ﮨﻢ ﻣﺠﺒﻮﺭﻭں ﭘہ ﺗﮩﻤﺖ ﮨﮯ ﻣﺨﺘﺎﺭی ﮐﯽ g y ﻧﺎﺣﻖ ﮨﻢ ﻣﺠﺒﻮﺭﻭں ﭘہ ﺗﮩﻤﺖ ﮨﮯ ﻣﺨﺘﺎﺭی ﮐﯽ ﭼﺎﮨﮯ ﮨﻴﮟ ﺳﻮ ﺍٓپ ﮐﺮے ﮨﻴﮟ ﮨﻢ ﮐﻮ ﻋﺒﺚ ﺑﺪﻧﺎﻡ ﮐﻴﺎ Even when Asharites honed their wit with dialectics to counter Mutazila, were equally rejected by extreme right wing of orthodoxy.Asharite and ahl-hadith animosity failed to struck an agreed upon theology. However, in this tussle Tasawwuf and philosophy absorbed the best of Muslim 16 Islam and Modernity in Crosshairs of History minds.Tasuwwuf became a great appeal against orthodoxy; for it ceases to be an ever innovative force. From the time of righteous caliphs to the installment of Seljuk, along with schools of philosophy, law (fiqh) also progressed by leaps and bounds. From the beginning of second century to the end of third century many law schools flourished to administer an overwhelmingly large empire, von Kremer attributes half of Muslim triumphs to our doctors of Law, and stays Muslim Law well-coherent next to the Code Romane20. Sunni Fiqhs of Abu-Hanifa (d.150),Imam Malik (d.179), Imam Shafi (d.204), Imam Ahmad b. Humble(d.241), and Shia fiqhs of Imam Jaffar al-Sadiq(d.151) and Imam Zaid bin Ali bin Hussain Bin Ali (d.122) came to fore during the same time. They devised and harnessed different geographies of legislations. Even principally pupils more often differed with their teachers;Hanafites relied on Principle of preference, Shafi on reason or logic, Malikites on public good while equity had been the part and parcel of all schools.All schools of fiqh were sprouting naturally on the realm of Islam, and followers of one fiqah were at ease to follow the others if need arises.Soon after it we enter fourth era of Islamic thought that begins with the rise of Seljuk Dynasty and ends at the death of Imam Ghazali. This would be termed as period of standardization and consolidation of Islamic thought. In a sense tree of Islamic civilization blossomed at full zenith, fruit of each discipline was artificially ripened by Ghazalianentrepreneurship. Abstract Before him, in third period of Islamic thought, inductive school of Hanafites and deductive school of Shafites along with other fiqh schools catered well to the needs of developing and ever expanding political realm of Islam. With Ghazali dawns the school of Maqasid, instead of narrow interpretations of law ( qayas, istehsan, MasalihMursalahetc ) the higher objectives of law are formulated. In fact, by operating synthesis of the deductive and inductive approaches before them, extracting and classifying the higher objectives of law a new Usul school came to fore.Fiqah as opposed to its earlier status as in earlier generations was an all-out intellectual effort equivalent of Ijtehad perhaps turned to Roman equivalent of Jurisprudence. Abu al-Maali al-Juwayni(d. 478/1085),mentor of al-Ghazali (d. 505/1111) laid down the foundation of school of Maqasid by categorization of rulings according to divine law giver’s intentions and the effective causes of the rulings by classifying them vertically according to their degree of importance and priority identifying five different levels(p.61). In his horizontal categorization come religion, life and chastity. Al-Ghazali his pupil takes up this Herculean task to its logical conclusion. Though his link from school of Shaffi,he opted for Istehsan of Hanafite and Masalih al-Mursalah of Malik (in absence of relevant texts, an utter reliance on public interest). He says: “ ….what we mean by maslaha is preserving the objective of law (shar’) that consists in five ordered things: preserving religion (din), life(nafs), reason (aql), progeny (nasl) and property (amwal). What ensures the preservation of those five principles is maslahah, what goes against their preservation is mafsadah and preventing it is maslahah21” Ibn-e-Qayyam says“the basis of minds.Tasuwwuf became a great appeal against orthodoxy; for it ceases to be an ever innovative force. From the time of righteous caliphs to the installment of Seljuk, along with schools of philosophy, law (fiqh) also progressed by leaps and bounds. From the beginning of second century to the end of third century many law schools flourished to administer an overwhelmingly large empire, von Kremer attributes half of Muslim triumphs to our doctors of Law, and stays Muslim Law well-coherent next to the Code Romane20. Sunni Fiqhs of Abu-Hanifa (d.150),Imam Malik (d.179), Imam Shafi (d.204), Imam Ahmad b. Humble(d.241), and Shia fiqhs of Imam Jaffar al-Sadiq(d.151) and Imam Zaid bin Ali bin Hussain Bin Ali (d.122) came to fore during the same time. They devised and harnessed different geographies of legislations. Abstract What ensures the preservation of those five principles is maslahah, what goes against their preservation is mafsadah, and preventing it is maslahah21”.Ibn-e-Qayyam says“the basis of 17 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 Sharia is wisdom and welfare of the people in this world as well as the hereafter. This welfare lies in complete justice, mercy, wellbeing and wisdom. hereafter. This welfare lies in complete justice, mercy, wellbeing and wisdom. Anything that departs from justice to oppression, from mercy to harshness, from welfare to misery, and from wisdom to folly has nothing to do with sharia22”.These sources feed well to the latter scholars, some added honor to five categories. Ghazali proved springboard for latter reflections well up to the time of Shatibi (d.790/1388).Ghazali, with his spiritual itinerary struck harmony among ever feuding stakeholders of mysticism, law and kalam. “He not only reconstituted orthodox Islam, making Sufism an integral part of it, but also was a great reformer of Sufism, purifying it of un-Islamic elements and putting it at the service of orthodox religion. As such he represents a final step in a long developing history. Sufism received, through his influence, the approval of ijma, or consensus of the community. Islam received a new vigor of life and a popular appeal which won large areas in Africa, Central Asia and India. Henceforth, often a great theologian and a great Sufi was one and the same person, a development which produced in due course a series of original thinkers who reconstituted Muslim theology on a new and more integral basis, than the old formal Kalam23”.To date from Morocco to Malaya madaris of Islam are catechism of the syllabus formulated by Ghazali.Dew drops of his theological ideas diffused wellonto the thirsty theological realms of Christian West.To Hitti, works of Ghazali as Fatihat al-Ulum, tahafutalphilasifah, al-Iqtisad fi al-itiqad, IhyaUlum al-Din, al-Mustasfa min Ilm al-Usuletc marked influence on Jewish and Christian scholasticism. “Thomas Aquinas, one of the greatest theologians of Christianity, and latter Pascal were indirectly affected by the ideas of al-Ghazali, who of all Moslem thinkers came nearest to subscribing to Christian views. The scholastic shell constructed by al-Ashari and al-Ghazali has held Islam to the present day, but Christendom succeeded in breaking through its scholasticism, particularly at the time of the Protestant Revolt. Abstract Since then the West and the East have parted company, the former progressing while the latter stood still24”.Actually, in dictatorial era people find solace in mysticism. That was the case with al- Ghazali. His spiritual itinerary turned him into harmonized blend of spirituality and pragmatism. In his constructs, interests of community and Seljuk find conformity, in-depth analysis show Seljuk come prairie while community posteriori. Being Sufism in vogue, Ghazali attacked philosophy in his Tahafut al-philasifah. His chief opponent Averroes, the Spanish thinker of Islam, unconditional guardian of Aristotelian school’s doctrine of immortality of active intellect and its grand interpreter, somewhat, in defense of Greek thought rather than philosophy by writing tahafut al-tahafah punched above his weight. On the other hand Ghazali based religion on philosophical skepticism. In actuality, Quran stays ears, eyes and fuad (faculty of heart, mind cum spirit) as essential tools of knowledge (32:9). Former, eyes and ears help more in empirical sciences, latter the fuad, faculty of heart in its mature spiritual essence the locus of re elation grasps metaph sical aspects of hereafter. This welfare lies in complete justice, mercy, wellbeing and wisdom. Anything that departs from justice to oppression, from mercy to harshness, from welfare to misery, and from wisdom to folly has nothing to do with sharia22”.These sources feed well to the latter scholars, some added honor to five categories. Ghazali proved springboard for latter reflections well up to the time of Shatibi (d.790/1388).Ghazali, with his spiritual itinerary struck harmony among ever feuding stakeholders of mysticism, law and kalam. “He not only reconstituted orthodox Islam, making Sufism an integral part of it, but also was a great reformer of Sufism, purifying it of un-Islamic elements and putting it at the service of orthodox religion. As such he represents a final step in a long developing history. Sufism received, through his influence, the approval of ijma, or consensus of the community. Islam received a new vigor of life and a popular appeal which won large areas in Africa, Central Asia and India. Abstract Even principally pupils more often differed with their teachers;Hanafites relied on Principle of preference, Shafi on reason or logic, Malikites on public good while equity had been the part and parcel of all schools.All schools of fiqh were sprouting naturally on the realm of Islam, and followers of one fiqah were at ease to follow the others if need arises.Soon after it we enter fourth era of Islamic thought that begins with the rise of Seljuk Dynasty and ends at the death of Imam Ghazali. This would be termed as period of standardization and consolidation of Islamic thought. In a sense tree of Islamic civilization blossomed at full zenith, fruit of each discipline was artificially ripened by Ghazalianentrepreneurship. Before him, in third period of Islamic thought, inductive school of Hanafites and deductive school of Shafites along with other fiqh schools catered well to the needs of developing and ever expanding political realm of Islam. With Ghazali dawns the school of Maqasid, instead of narrow interpretations of law ( qayas, istehsan, MasalihMursalahetc ) the higher objectives of law are formulated. In fact, by operating synthesis of the deductive and inductive approaches before them, extracting and classifying the higher objectives of law a new Usul school came to fore.Fiqah as opposed to its earlier status as in earlier generations was an all-out intellectual effort equivalent of Ijtehad perhaps turned to Roman equivalent of Jurisprudence. Abu al-Maali al-Juwayni(d. 478/1085),mentor of al-Ghazali (d. 505/1111) laid down the foundation of school of Maqasid by categorization of rulings according to divine law giver’s intentions and the effective causes of the rulings by classifying them vertically according to their degree of importance and priority identifying five different levels(p.61). In his horizontal categorization come religion, life and chastity. Al-Ghazali his pupil takes up this Herculean task to its logical conclusion. Though his link from school of Shaffi,he opted for Istehsan of Hanafite and Masalih al-Mursalah of Malik (in absence of relevant texts, an utter reliance on public interest). He says: “ ….what we mean by maslaha is preserving the objective of law (shar’) that consists in five ordered things: preserving religion (din), life(nafs), reason (aql), progeny (nasl) and property (amwal). Abstract Henceforth, often a great theologian and a great Sufi was one and the same person, a development which produced in due course a series of original thinkers who reconstituted Muslim theology on a new and more integral basis, than the old formal Kalam23”.To date from Morocco to Malaya madaris of Islam are catechism of the syllabus formulated by Ghazali.Dew drops of his theological ideas diffused wellonto the thirsty theological realms of Christian West.To Hitti, works of Ghazali as Fatihat al-Ulum, tahafutalphilasifah, al-Iqtisad fi al-itiqad, IhyaUlum al-Din, al-Mustasfa min Ilm al-Usuletc marked influence on Jewish and Christian scholasticism. “Thomas Aquinas, one of the greatest theologians of Christianity, and latter Pascal were indirectly affected by the ideas of al-Ghazali, who of all Moslem thinkers came nearest to subscribing to Christian views. The scholastic shell constructed by al-Ashari and al-Ghazali has held Islam to the present day, but Christendom succeeded in breaking through its scholasticism, particularly at the time of the Protestant Revolt. Since then the West and the East have parted company, the former progressing while the latter stood still24”.Actually, in dictatorial era people find solace in mysticism. That was the case with al- Ghazali. His spiritual itinerary turned him into harmonized blend of spirituality and pragmatism. In his constructs, interests of community and Seljuk find conformity, in-depth analysis show Seljuk come prairie while community posteriori. Being Sufism in vogue, Ghazali attacked philosophy in his Tahafut al-philasifah. His chief opponent Averroes, the Spanish thinker of Islam, unconditional guardian of Aristotelian school’s doctrine of immortality of active intellect and its grand interpreter, somewhat, in defense of Greek thought rather than philosophy by writing tahafut al-tahafah punched above his weight. On the other hand Ghazali based religion on philosophical skepticism. In actuality, Quran stays ears, eyes and fuad (faculty of heart, mind cum spirit) as essential tools of knowledge (32:9). Former, eyes and ears help more in empirical sciences, latter the fuad, faculty of heart in its mature spiritual essence the locus of revelation, grasps metaphysical aspects of 18 Islam and Modernity in Crosshairs of History reality, guzarjaaql se aagay… dil e binabhi. The golden mean of information brought by ears, eyes and fuad points toward reality.Ghazali’s spiritual itinerary catapulted him to assert more reliance on fuad,ibn-Rushd immersed himself in scientific school of Aristotlebased on empirical sciences in its major import. In other words he stood in the camp ofMutazila. Abstract That is because East feeds on Plato and Ghazali, while West on Aristotle and Averroes, former spiritual, latter empirical in its outlook. East is more concerned with his self, God and world, and West’s attitude is this-worldly. However, except excessive reliance on predeterminism, Asharite thinkers chose constructive path and paved a way for modern forms of idealism, though major thrust of their movement remained to defend orthodoxy with a weapon of Greek dialectic, which would have proved futile if Ghazali had not endorsed it. History bears witness that major bulk of humanity tends to shake balance of thought. Same happened with Ghazali’s constructs, he was construed as opponent of philosophy or streaks of his thought did so, with his departure Muslims banned philosophy on themselves, even if al-Ghazali in his profound reflections terms the universe as “outspread book (al-kitab al-manshur)” which is theological as well as physical mirror of the “written book (al-kitab- al-mastur)”; for Quran itself vociferously points towards the signs spread in cosmos. Hats downto the resilienceof orthodoxy, which takes of its choice and leaves other under its hat from these genies of Islam! If someone claims that Deoband and its clones are followers of Shah Wali Allah he is simply ignorant of Shah’s thought. Same is the case of Ibn e Tamiyyah’s followers. Ghazali leaves a wide scope for generations to come, to continually exhaust their faculties of ears, eyes and intuition (fuad) in exploring harmonies between revealed book and unfurled revelation (universe) to resolve their theological as well as practical problems; for the revealed text neither stifles nor directs the mind, it liberates it at the heart of the Universe: the world speaks by itself, autonomously , and it is human intelligence’s task to understand its language, vocabulary, semantics, rules, grammar and grand symmetry (67:3- 4) in creation and order. In nutshell, text (kitab-al-manshur) and context (kitab-al-satur) rather than contradicting or supplanting envelop, augment and reinforce each other.But whether it is intellectual lethargy of his followers or his misconstrued constructs, or his apparent attacks on philosophy, Ghazali is also maligned in some academic circles who say “but for Al-ashari and al- Ghzali the Arabs might have been a nation of Galileos, Keplers and Newtons. Abstract By their denunciations of science and philosophy, by their exhortations that besides theology and law no other knowledge was worth acquiring, they did more to stop the progress of the Muslims world than other Muslim scholiasts. And up to this day their example is held forth as a reason for ignorance and stagnation25”. Anyway, fourth period of Islamic thought might be termed as period of consolidation and standardization, product of Ghazalian entrepreneurship Henceforth, after Ghazali, ushers in the fifth period of Islamic thought 19 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 termed as period of stagnation and blind imitation. To some extent it still continues with some extra exceptional aberrations such as those of al- Razi(d.606/1209), al-Bayddawi (d.685/1286), Ib as-Subki(d.771/1369), at- Tufi(716/1316) and Ibn e Taymiyyah (d.728/1327), al-sutiand in near past Sarhandi, Shah Wali Allah etc.All these proved harmless ozone’s filtered rays for the glacier of our thought or gradually got absorbed in the same.For al- Asharite’spredetermnism got its zenith in al-Ghazali and al-Razi’s constructs when they vied with each other in producing ever fresh arguments to prove that man can be said “to act only” metaphysically , not really, since the only real “actor” is God? Today, any real theorist of Islam needs not to frighten of fishes in the pool because crocodiles have devoured them. However, two centuries after Ghazali a Spanish scholar Shatbi (d.790/1388) emerges to scene with a theory and methodology that transcend legal school and he integrate those tools of inductive and deductive logic devised by earlier schools. He integrated into his approach , all the works and methodologies produced in the meanwhile by scholars from the other schools- Shafi, Hanafi,Hanbali, Zahiri (with IbnHazm, died 455/1064 ) and the various Shii schools. His ultra- synthetic approach enhanced the epistemological frame-work of school of objectives with a new dimension: Maqasid al-Mukallaf stemming from the accountable [man] along with earlier specified as maqasid al-shari determined by Divine Lawgiver. Further human interests are classified into primary and secondary (maqasid-al-asliyyah ,maqasid al-tabiyyah)He struck equilibrium and collapsed extremes into middle. His insight of history compels him to say that though an action may be deemed permissible, recommended, lawful, objectionable or even lawful in itself, its status can be changed according to context in which it is considered and judged. Abstract For example, caliph Omer’s decisionto suspend the punishments for poor thieves in times of famine, was based on justice rather than literal implementation. If it be, it would have run against the higher objective of justice, since the poor thieves have been twice victimized. Similarly, while attacking BanuNadhir, cutting tree was to strengthen the seizure of the very tribe apparently violation of (2:205) but context changed its very status.Furthermore, to al-shatibi, Quran of Mecca is of universal import while of Medina is rendering, illustration and concrete implementation of those principles in that historical context.To great minds of Islam text determines major principles and sometimes states number of details, while sunnah’s function is to detail, clarify and illustrate the same. It liberates Quran from the specific contextual interpretation of Medina period. Those renderings cannot close off the Quran and the Divine Lawgivers’ higher, universal objectives into a particular, historically dated, and quite specific interpretation.On these grounds, Omer refused to divide the lands of Iraq among soldiers as booty against the literal interpretation of Quran and Sunnah in favor of tenor of Quran and an Ideal model of the prophet (PBUH); for both converge on social justice and fair play.Omer buttresses his stand by (59:9-10).In sum total, Shatbi toe the framework of Omer the second Caliph an termed as period of stagnation and blind imitation. To some extent it still continues with some extra exceptional aberrations such as those of al- Razi(d.606/1209), al-Bayddawi (d.685/1286), Ib as-Subki(d.771/1369), at- Tufi(716/1316) and Ibn e Taymiyyah (d.728/1327), al-sutiand in near past Sarhandi, Shah Wali Allah etc.All these proved harmless ozone’s filtered rays for the glacier of our thought or gradually got absorbed in the same.For al- Asharite’spredetermnism got its zenith in al-Ghazali and al-Razi’s constructs when they vied with each other in producing ever fresh arguments to prove that man can be said “to act only” metaphysically , not really, since the only real “actor” is God? Today, any real theorist of Islam needs not to frighten of fishes in the pool because crocodiles have devoured them. However, two centuries after Ghazali a Spanish scholar Shatbi (d.790/1388) emerges to scene with a theory and methodology that transcend legal school and he integrate those tools of inductive and deductive logic devised by earlier schools. Abstract He integrated into his approach , all the works and methodologies produced in the meanwhile by scholars from the other schools- Shafi, Hanafi,Hanbali, Zahiri (with IbnHazm, died 455/1064 ) and the various Shii schools. His ultra- synthetic approach enhanced the epistemological frame-work of school of objectives with a new dimension: Maqasid al-Mukallaf stemming from the accountable [man] along with earlier specified as maqasid al-shari determined by Divine Lawgiver. Further human interests are classified into primary and secondary (maqasid-al-asliyyah ,maqasid al-tabiyyah)He struck equilibrium and collapsed extremes into middle. His insight of history compels him to say that though an action may be deemed permissible, recommended, lawful, objectionable or even lawful in itself, its status can be changed according to context in which it is considered and judged. For example, caliph Omer’s decisionto suspend the punishments for poor thieves in times of famine, was based on justice rather than literal implementation. If it be, it would have run against the higher objective of justice, since the poor thieves have been twice victimized. Similarly, while attacking BanuNadhir, cutting tree was to strengthen the seizure of the very tribe apparently violation of (2:205) but context changed its very status.Furthermore, to al-shatibi, Quran of Mecca is of universal import while of Medina is rendering, illustration and concrete implementation of those principles in that historical context.To great minds of Islam text determines major principles and sometimes states number of details, while sunnah’s function is to detail, clarify and illustrate the same. It liberates Quran from the specific contextual interpretation of Medina period. Abstract Those renderings cannot close off the Quran and the Divine Lawgivers’ higher, universal objectives into a particular, historically dated, and quite specific interpretation.On these grounds, Omer refused to divide the lands of Iraq among soldiers as booty against the literal interpretation of Quran and Sunnah in favor of tenor of Quran and an Ideal model of the prophet (PBUH); for both converge on social justice and fair play.Omer buttresses his stand by (59:9-10).In sum total, Shatbi toe the framework of Omer the second Caliph an 20 Islam and Modernity in Crosshairs of History absolute Mujaddid and the first critical and independent mind in Islam who, at the last moments of the Prophet, had the moral courage to utter these remarkable words: ‘The Book of God is sufficient for us’26.However, in fifth period of Islamic thought of blind imitation and stagnation moral apathy not only slipped Muslim’s grip on realm of power, spring of their fresh thought stopped gushing out, especially from 18th century on, once master of their own destiny were serving the history of others when industrialized imperial Europe with fresh vigor locked horns with empires of Islam. During the heydays at outset, Muslims were psychologically invincible, at the helm, politically masters of situation, and Byzantine a spent force, so the dramatic expansion of Islamic dominance occurred at the expanse of Christendom. For seven hundred years Muslims ruled Spain, if Safavid had not been in Persia, Ottomans had been brought all the Europe under the pale of Islam. Safavids’ power at the backyard rather than rival forces rendered Ottoman’s occupation of France unfulfilled task. Hence, it is only Islam with which West has fourteen centuries fluctuating history of territorial, religious and cultural boundaries. Crusades and wars of Islam obliterated sense of decency and unbiased appraisal of each on both sides. Even Quran and Prophet(PBUH) were not spared, though Muslim by faith are obliged to venerate Biblical chain of Prophets, so better sense prevailed there, yet Judaism and Christianity was misjudged and misrepresented by medieval Muslim thinkers, for they saw Jews and Christians from the prism of Medenese phase. It obscured their vision of any possibility of mutual accommodation. Abstract Revivalists like Alice in wonderland have continually to wander in wonders; for the idealist knew only to sacrifice without having pearl of their choice. Be it the revivalist movement of 1857 freedom war or before it the puritanical movement of Sayyed Ahmed Brailvi, Ikhwan of Egypt or Jamaat e Islami of Pakistan. To many observers, it was holy alliance of orthodoxy, feudal elite and military which forced secession of Bangla Dash to get rid of progressive majority. The alliance still continues. Doctrine of Necessity still looms large over Muslim polities; for it was al-Ghazali and his successor IbnJamma who said that public tolerance of bad ruler was preferable to anarchy and civil war, on the other side theological authorities of Islam like al-Mawardi, al-Baghdadi and al-Baqilani held that an unjust and unrighteous ruler should not claim obedience against just contender. But Ghazalian constructs reign supreme on Muslim’s mindset and Muslims are hostage to the tripartite alliance of dictators, clergy and Sufis. The same three elements have taken wind out of Islam’s sails. To be back on terra firma, Muslims need to grapple with these categories and grasp the nettle firmly. For this, Muslims have to recourse to Quran with an independent mind; infer laws of history according to dictates of time rather than blindly following medieval constructs; for our problems and constraints are different in our own milieu. Though Quran claims it is easy to comprehend, yet it is as deep as life itself, for it took twenty three years to be revealed.The simplest complex, Quran is as straightforward and coherent as life itself. In Churchill’s phrase life is riddle wrapped in mystery, inside an enigma. So,to take Quran as lifeless dicta would be equivalent of “crushing its gossamer wings to powder”. In this transitional period of Islamic thought idealists, the stupid friends of Islam entertain proclivity to depreciate materieux of history (which even the Prophet of Islam was bound to harness), consequently are unable to bend history to a specific definitive course. Future of Islam lies with the modernists who can make history field of Divine activity by working with the complicated twists and enmeshing folds of the materials of history.Pragmatists or non-idealists know only the exterior of life on cost of higher values. Quran vehemently exhorts that man instead of social animal is a moral being. Abstract On the other side, rivalry of Islam was too deep seated in psyche of Christian Europe that at the end of First World War while conquering Palestine commander of allied forces Lord Allenby saidthat today the crusade is over.In a terse and nude analysis, centuries old fluctuating history of sea-saw struggle between Islam and Christianity took a new turn with Napoleon Bonaparte’s invasion of Egypt in 1798 and British colonization of Mughal India in 18th and 19th centuries and break-up of Ottoman empire proved a last nail in the coffin of Islam’s political cum military power. Henceforth, moral epistemology of imperial Europe (white man’s burden, superman, survival of the fittest, now clash of civilization) divested even last speck of confidence, a loss greater than political one for the later thrives on the former. Russia in our times is on resurge (take her stand on Syria, Ukraine), it’s politico-economic cum military demise at Asia’s heart (Afghanistan) failed to diminish her huge ego.We lost our wings in cage, and are unable to grow even after freedom. Self-cognizant individuals and communities pile stock of straws while in cage, and weave out nests on freedom spree. However, Western long hegemony caused intense soul search in Muslim world, it is norm of history when a civilization hits the rock bottom, and at that point of fundamental crisis it evokes three responses in its adherents. First the revivalists who seek solace in pristine Islam, second the traditionalist who unconditionally surrender to the tradition and are rather apathetic to future 21 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 believe in uniform decline of community. The third one is modernist, harmonized blend of reason and tradition, the class of enlightened and progressive intellectuals. Be it the Jinnah of Pakistan or Jamal Abd al-Nasir of Egypt, Ahmad Sukarno of Indonesia or HabibBourguiba of Tunisia are a few and familiar who did marvelous job to get rid their communities of colonial yokes. Extra-exceptional among them is Mustafa Kemal who gave coupe de grace to imperial Britain in Cyprus, ignominy of which haunted Churchill till last breath of his life. However, soon after de-colonization mushrooming of Marshal Law regimes in Muslim world from Morocco to Indonesia swept the tide of such a people and brought forth holy lot of traditionalists easily dove- tailed with dictators. Abstract Philosophy in our curricula has to cease as a disciplina non grata ; a perennial intellectual need has to be allowed to flourish 22 Islam and Modernity in Crosshairs of History for its own sake and for the sake of other disciplines, since it inculcate a much needed analytical critical spirit and generates new ideas that become important intellectual tools for other sciences, not least for religion and theology. At this crucial juncture, Islam needs ta’miri school of Farabi and Ibn al-Arabi (not to reinvent them but like them bring forth new ideas) rather than Takfiri school of Ibn e Taimiyyah and Ghazali, however political emancipation is first step toward that destiny, declining moments would bring forth new Ghazalis and Ibn e Taimiyyahs. Societies devoid of socio- economic justice can be Muslims but never be truly monotheistic at all. This work is licensed under a Creative Commons Attribution 4.0 International License. References 1. Netche claimed so. 1. Netche claimed so. 2. Keddie, Nikki, Islamic Response to Western Imperialism, University of California Press Berkeley, Loss Angeles,p.107, California library Reprint Series 1983 3. Iqbal, Reconstruction of Religious Thoughts In Islam, published by JavaidIqbal son of Late Dr Mohammad Iqbal printed by sh Mohammad Ashraf, at the Ashraf Press, 7 Aibak Road Lahore,p.8, Reprinted February 1960 4. See Karen Armstrong’s Battle of God. Conclusion of this work portrays so. First published in Great Britain by HarperCollinsPublishers 2000 5. Al-TurqulHukmiyyafissiyasatish-shariyyya, p.14; cf Jalundhari, Rashid, Islamic Sharia and its Application with Special Reference to Pakistan, MaktabaJadeed Printing Press Lahore,p.5, First Edition 2003 6. See Kitabulalimwal-mutaalim, p. 5, 6. cf Jalundhari, Rashid, Islamic Sharia and its Application with Special Reference to Pakistan, MaktabaJadeed Printing Press Lahore,p.5, First Edition 2003 7. Malik, Fateh Muhammad, Article: Iqbal’s Vision of a Composite Muslim- Christian-Jewish Nationalism, Published in Iqbal Review journal of Iqbal Academy, Pakistan April 2003. Also see The Guardian, London, and Thursday, June 20, 2002. 8. Keddie, Nikki, Islamic Response to Western Imperialism, p.103; California library Reprint Series 1983 9. Article, Ahmad Eqbal, Roots of the Religious Right, or see Between Past and Future (Selected Essays on South Asia by Eqbal Ahmad, Oxford University Press, p. 281-282. 10. Iqbal, Muhammad, Reconstruction of Religious Thoughts in Islam, p.146-47. 23 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 Al-Duhaa, Jan-June 2021, Volume: 02, Issue: 01 11. Iqbal, Muhammad, Reconstruction of Religious Thoughts in Islam, p.126 12. Iqbal, Muhammad, Reconstruction of Religious Thoughts in Islam,p.7 13. Iqbal, Muhammad, Javaid Nama. Shaikh Muhammad Basheer and Sons, Jalaludin Hospital building, Urdu Bazar Lahore. 14. The MuqaddimahIbnKhaldun: translated and introduced by Franz Rosenthal abridged and edited by N.J Dawood, Bollingen series Princeton University Press Princeton and Oxford, p.75.First Princeton Classic Edition with a new introduction by Bruce B. Lawrence, 2005. 15. FazlurRehman, Islam, Anchor Books Doubleday and Company, Inc, Edition 1968, p.2 15. FazlurRehman, Islam, Anchor Books Doubleday and Company, Inc, Edition 1968, p.2 16. See Hitti, Philip K; History of Arabs from the Earliest Times to Present; fifth edition. London Machmillan and co Ltd 1953 p.118 16. See Hitti, Philip K; History of Arabs from the Earliest Times to Present; fifth edition. London Machmillan and co Ltd 1953 p.118 17. See Hujjat Allah al-Baligha,Cairo 1322 AH,1, 93ff 17. See Hujjat Allah al-Baligha,Cairo 1322 AH,1, 93ff 17. See Hujjat Allah al-Baligha,Cairo 1322 AH,1, 93ff 18. IbnKhaldun, Muqaddima, Engl… 18. IbnKhaldun, Muqaddima, Engl… 18. IbnKhaldun, Muqaddima, Engl… 19. FazlurRahman, Islamic Methodology in History, Central Institute of Islamic Research Karachi 1965, p.24 19. FazlurRahman, Islamic Methodology in History, Central Institute of Islamic Research Karachi 1965, p.24 20. Iqbal, Muhammad, Reconstruction of Religious Thoughts in Islam,p.168 21. Ibid. 1:286-287. 21. Ibid. 1:286-287. 22. IbnQayyim al-Jawziyyah, I’lam al-muwaqqi’in(1955), vol .3,p.14 23. FazlurRahman, Islam, p.169 23. FazlurRahman, Islam, p.169 24. See Hitti, Philip K; History of Arabs , p.432 24. See Hitti, Philip K; History of Arabs , p.432 25. Syed Ameer Ali, The Spirit of Islam, Sang e Meel Publications, 2005,p.486-87. 26. Iqbal, Muhammad, Reconstruction of Religious Thought in Islam, p.162. 24
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Review of: "The Correlation between Poverty and Crime in Pakistan"
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Qeios ID: 7GYNM8 · https://doi.org/10.32388/7GYNM8 Qeios, CC-BY 4.0 · Review, January 9, 2024 Review of: "The Correlation between Poverty and Crime in Pakistan" Lihong Yang1 1 University of Southern California Lihong Yang1 1 University of Southern California Potential competing interests: No potential competing interests to declare. Potential competing interests: No potential competing interests to declare. The paper offers a valuable contribution to the exploration of the poverty-crime nexus in Pakistan. Strengthening empirical support, enhancing conceptual clarity, and suggesting future research directions would further solidify the paper's significance and appeal to a wider audience. It would benefit from incorporating more empirical evidence to support its claims. Including case studies, statistical data, or real-world examples would enhance the credibility of the findings. Qeios ID: 7GYNM8 · https://doi.org/10.32388/7GYNM8 1/1
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Influence of Coexisting Chronic Kidney Disease and Left Ventricular Hypertrophy on Changes in FGF23 and the Renin-Angiotensin-Aldosterone-System
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Kohei Okamoto Kohei Okamoto  Kobe University Graduate School of Medicine Hideki Fujii  (  fhideki@med.kobe-u.ac.jp ) Kobe University Graduate School of Medicine Shunsuke Goto  Kobe University Graduate School of Medicine Kentaro Watanabe  Kobe University Graduate School of Medicine Keiji Kono  Kobe University Graduate School of Medicine Shinichi Nishi  Kobe University Graduate School of Medicine Kobe University Graduate School of Medicine Hideki Fujii  (  fhideki@med.kobe-u.ac.jp ) Kobe University Graduate School of Medicine Shunsuke Goto  Kobe University Graduate School of Medicine Kentaro Watanabe  Kobe University Graduate School of Medicine Keiji Kono  Kobe University Graduate School of Medicine Shinichi Nishi  Kobe University Graduate School of Medicine Kobe University Graduate School of Medicine Influence of Coexisting Chronic Kidney Disease and Left Ventricular Hypertrophy on Changes in FGF23 and the Renin-Angiotensin-Aldosterone- System Influence of Coexisting Chronic Kidney Disease and Left Ventricular Hypertrophy on Changes in FGF23 and the Renin-Angiotensin-Aldosterone- System Influence of Coexisting Chronic Kidney Disease and Left Ventricular Hypertrophy on Changes in FGF23 and the Renin-Angiotensin-Aldosterone- System Kohei Okamoto Influence of Coexisting Chronic Kidney Disease and Left Ventricular Hypertrophy on Changes in FGF23 and the Renin-Angiotensin-Aldosterone- System Kohei Okamoto Research Article nic kidney disease, fibroblast growth factor 23, left ventricular hypertrophy, renin-angiotensin-aldosterone system cense:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full Lic Loading [MathJax]/jax/output/CommonHTML/jax.js Page 1/15 Abstract Serum fibroblast growth factor 23 (FGF23) levels and the renin-angiotensin-aldosterone system (RAAS) are elevated in chronic kidney disease (CKD) patients, and their association with left ventricular hypertrophy (LVH) has been reported. However, whether the FGF23 elevation is the cause or result of LVH remains unclear. At 10 weeks, male C57BL/6J mice were divided into four groups: Sham, CKD (5/6 nephrectomy), LVH (transaortic constriction), and CKD/LVH group. At 16 weeks, the mice were sacrificed, and blood and urine, cardiac expressions of FGF23 and RAAS-related factors, and cardiac histological analyses were performed. Heart weight, serum FGF23 levels, and cardiac expression of FGF23 and RAAS-related factors, except for angiotensin-converting enzyme 2, more increased in the CKD/LVH group compared to the other groups. A significant correlation between LVH and cardiac expressions of FGF23 and RAAS-related factors was observed. Furthermore, there was a significantly close correlation of the cardiac expression of FGF23 with LVH and RAAS-related factors. The coexisting CKD and LVH increased serum and cardiac FGF23 and RAAS-related factors, and there was a significant correlation between them. A close correlation of cardiac, but not serum FGF23, with LVH and RAAS suggested that local FGF23 may be associated with LVH and the RAAS activation. Introduction Fibroblast growth factor 23 (FGF23) is a crucial phosphaturic hormone, and its serum level increases as kidney function declines [1]. FGF23 is mainly produced in the bone and interacts with the FGF23 receptor (FGFR) in the presence of klotho in the kidney proximal tubule. In this way, phosphorus diuresis is promoted in the kidney. However, FGF23 also has many other actions [2]. One of them is a potential action on left ventricular hypertrophy (LVH) progression [3, 4]. Although previous experimental studies have reported that considerably high levels of serum FGF23 could induce LVH via FGFR4-mediated calcineurin- NFAT pathway activation even in the absence of αKlotho [4], clinical studies have shown a significant correlation between serum FGF23 levels and LVH even in patients with a mild or moderate elevation of serum FGF23 levels [3]. In addition, it has also been reported that the FGF23 gene deletion mice underwent transverse aortic constriction (TAC) reveal LVH [5]. Although patients with tumor-induced osteomalacia have remarkably high serum FGF23 levels and FGF23- related hypophosphatemia, serum FGF23 levels are not associated with LVH [6]. Thus, whether LVH progresses by an increase in FGF23 or whether the occurrence of LVH increases FGF23 are still unclear, and we do not yet have a consensus on which is the primary cause. Recent studies have demonstrated a close correlation between FGF23 and the renin-angiotensin-aldosterone system (RAAS) [7]. The RAAS is an important control system for blood pressure and causes LVH [8]. Therefore, we focused on the RAAS as a common factor for an increase in FGF23 and the exacerbation of LVH. This study aimed to verify FGF23 and RAAS changes and their correlation using a chronic kidney disease (CKD) model, a model for pressure overload-induced cardiac hypertrophy, and a combined model. This study aimed to verify FGF23 and RAAS changes and their correlation using a chronic kidney disease (CKD) cardiac hypertrophy, and a combined model. Animal characteristics and biochemical measurements The characteristics and biochemical data at 16 weeks in the sham, CKD, LVH, and CKD/LVH groups are shown in Table 1. BP was significantly elevated in the CKD and CKD/LVH groups. Serum Cr levels, phosphate levels, and uAlb excretion significantly increased in the CKD and CKD/LVH groups compared to the sham group. Serum calcium levels were comparable among the four groups. Although uAlb excretion was comparable between the sham and LVH groups, and serum Cr levels were slightly elevated in the LVH group compared to the sham group. Serum iPTH levels were elevated in the CKD and LVH groups compared to the sham group, although there was no statistically significant difference; however, they were significantly higher in the CKD/LVH group. Serum 25D levels were similar among all the study groups, and serum 1,25D levels decreased by CKD induction. Urinary 8-OHdG levels were significantly higher in the CKD and CKD/LVH groups than in the sham group. As shown in Fig. 1, although serum iFGF23 levels were significantly elevated in the CKD, LVH, and CKD/LVH groups compared to the sham group, there was no statistically significant difference among the three groups, except for the sham group. In contrast, serum aldosterone levels increased by CKD and LVH induction, and they were additionally increased by coexisting CKD and LVH. Loading [MathJax]/jax/output/CommonHTML/jax.js Page 2/15 Table 1 Animal characteristics at 16 weeks   Sham (N = 6) CKD (N = 6) LVH (N = 6) CKD/LVH (N = 6) Body weight (g) 29. Animal characteristics and biochemical measurements 5 ± 0.7 25.1 ± 0.8# 28.7 ± 0.6* 25.7 ± 1.0#, ♰ SBP (mmHg) 86.2 ± 0.6 97.6 ± 2.0# 87.2 ± 1.8 107.2 ± 4.0#, ♰ HR (/min) 516.3 ± 12.3 493.1 ± 12.2 490.7 ± 15.4 472.8 ± 14.7 Cr (mg/dL) 0.14 ± 0.02 0.44 ± 0.03# 0.23 ± 0.02* 0.47 ± 0.09#, ♰ Ca(mg/dL) 9.87 ± 0.30 9.83 ± 0.80 10.03 ± 0.76 9.02 ± 0.81 P (mg/dL) 8.1 ± 0.6 9.7 ± 0.4# 8.8 ± 0.3 10.3 ± 0.3# iPTH (pg/mL) 29.0 ± 11.6 77.6 ± 16.2 50.6 ± 7.0 111.6 ± 39.1# 25(OH)D (pg/mL) 61.7 ± 2.3 61.8 ± 3.5 59.7 ± 3.2 63.3 ± 4.0 1, 25(OH)2D (pg/mL) 227.0 ± 18.9 184.8 ± 10.4 222.2 ± 24.6 178.0 ± 9.9 uAGT (ng/mgCr) 3.32 ± 0.85 3.36 ± 0.94 7.13 ± 2.55#, * 9.62 ± 2.10#, * uAlb (µg/mgCr) 144.6 ± 9.5 1156.3 ± 221.2# 156.1 ± 5.7* 1356.8 ± 145.6#, ♰ u8-OHdG (ng/mgCr) 53.9 ± 1.8 99.4 ± 16.1# 82.8 ± 8.3 101.3 ± 9.2# #: vs Sham, P < 0.05, *: vs CKD, P < 0.05, ♰: vs LVH, P < 0.05. CKD, chronic kidney disease; LVH, left ventricular hypertrophy; SBP, systolic blood pressure; HR, heart rate; Cr, creatinine; Ca, calcium; P, phosphate; iPTH, intact parathyroid hormone; uAGT, urinary angiotensinogen; uAlb, urinary albumin; u8-OHdG, urinary 8-hydroxy-2’-deoxyguanosine. Cardiac Parameters After Induction Of Ckd And Lvh Cardiac parameters evaluated using echocardiography and heart weight are shown in Table 2. As for the LV dimension, LV end-diastolic diameter and LV end- ystolic diameter were smaller in the CKD/LVH group compared to other groups. Both anterior and posterior wall of the LV remarkably thickened in the LVH nd CKD/LVH group. Systolic function revealed as ejection fraction (EF) was significantly reduced in the LVH and CKD/LVH groups compared to the sham and CKD groups. The relative heart weight (heart weight/body weight) was significantly higher in the order of the CKD/LVH, LVH, CKD, and sham groups. Cardiac parameters evaluated using echocardiography and heart weight are shown in Table 2. As for the LV dimension, LV end-diastolic diameter and LV end- systolic diameter were smaller in the CKD/LVH group compared to other groups. Both anterior and posterior wall of the LV remarkably thickened in the LVH and CKD/LVH group. Systolic function revealed as ejection fraction (EF) was significantly reduced in the LVH and CKD/LVH groups compared to the sham and CKD groups. The relative heart weight (heart weight/body weight) was significantly higher in the order of the CKD/LVH, LVH, CKD, and sham groups. Table 2 Animal characteristics at 16 weeks   Sham (N = 6) CKD (N = 6) LVH (N = 6) CKD/LVH (N = 6) LVDd (mm) 3.77 ± 0.76 3.45 ± 0.76 3.47 ± 0.29 3.15 ± 0.08 LVDs (mm) 2.78 ± 0.06 2.67 ± 0.06 2.75 ± 0.22 2.55 ± 0.07#, * LVAW (mm) 0.75 ± 0.02 0.80 ± 0.04 1.23 ± 0.04#, * 1.30 ± 0.04#, * LVPW (mm) 0.71 ± 0.03 0.75 ± 0.04 1.18 ± 0.05#, * 1.20 ± 0.04#, * FS (%) 25.8 ± 0.4 23.0 ± 0.2# 20.9 ± 0.3#, * 19.3 ± 0.8#, * EF (%) 57.7 ± 0.6 52.9 ± 0.4 49.1 ± 0.5#, * 46.0 ± 1.7#, * Heart weight (mg) 131.3 ± 3.3 116.2 ± 2.3 181.6 ± 5.2#, * 178.2 ± 8.5#, * Relative heart weight (mg/g) 4.45 ± 0.07 4.64 ± 0.08 6.33 ± 0.16#, * 6.96 ± 0.36#, * #: vs Sham, P < 0.05, *: vs CKD, P < 0.05, ♰: vs LVH, P < 0.05. CKD, chronic kidney disease; LVH, left ventricular hypertrophy; LVDd, left ventricular diastolic diameter; LVDs, left ventricular systolic diameter; LVAW, left ventricular anterior wall; LVPW, left ventricular posterior wall; FS, fractional shortening; EF, ejection fraction. Correlation between FGF23, RAAS-related factors and cardiac hypertrophy and fibrosis Serum FGF23 levels, cardiac FGF23 expressions (mRNA and immunohistochemical staining), and RAAS-related factors were related to cardiac hypertrophy parameters (width and size of cardiomyocyte) and mRNA expressions of cardiac hypertrophy and fibrosis-related factors. However, the correlation of serum FGF23 levels with them was weaker than that of cardiac FGF23 expressions and RAAS-related factors (Table 3). Although cardiac FGF23 expressions were significantly correlated with all the RAAS-related factors, serum FGF23 was significantly correlated with only cardiac mRNA expression of AT1R and AGT and the immunohistochemical expressions of angiotensin II and ACE (Table 4). Thus, the correlation of cardiac FGF23 expression with RAAS-related factors was closer than that of serum FGF23 with RAAS-related factors. Table 3 Correlation of each FGF23 and RAAS data with cardiac parameters   HW rHW Width Area Fibrosis LVAW LVPW FS EF cANP cBNP cα- MHC cβ- MHC cC iFGF23 .212 .517** .617** .518** .698** .449* .394 − .639** − .645** .329 .299 − .181 .583** .4 cFGF23 .536** .708** .641** .717** .807** .726** .707** − .816** − .805** .399 .436* − .512* .846** .7 imFGF23 .505* .766** .649** .815** .890** .728** .709** − .820** − .814** .403 .538** − .569** .838** .6 Ald .251 .641** .666** .495* .616** .564** .566** − .629** − .625** .241 .239 − .241 .527** .5 cAT1R .624** .765** .679** .830** .768** .702** .644** − .858** − .863** .369 .427* − .651** .742** .5 cAGT .537** .674** .730** .678** .662** .779** .700** − .692** − .691** .490* .352 − .703** .742** .4 cACE .823** .784** .600** .683** .628** .735** .705** − .717** − .714** .441* .448* − .773** .738** .4 cACE2 − .690** − .778** − .613** − .701** − .622** − .752** − .702** .714** .704** − .528** − .311 .803** − .699** −  uAGT .485* .572** .528** .557** .557** .744** .694** − .624** − .612** .116 .213 − .539** .683** .5 imAng II .448* .656** .651** .562** .637** .665** .613** − .672** − .676** .301 .283 − .561** .714** .3 imACE .700** .836** .779** .853** .824** .837** .821** − .884** − .881** .462* .561** − .695** .834** .6 imACE2 − .690** − .776** − .664** − .586** − .643** − .834** − .794** .706** .705** − .489* − .476* .719** − .786** −  *: P < 0.05, **: P < 0.01. ardiac expressions of FGF23, RAAS-related factors, and cardiac hypertrophy-related factors induced by CKD and L Cardiac FGF23 mRNA expression significantly increased in the CKD and LVH groups and further increased in the CKD/LVH group. Similarly, cardiac angiotensinogen (AGT) mRNA expression significantly increased in the LVH group and further increased in the CKD/LVH group. The cardiac AT1R and ACE mRNA expression significantly increased in the CKD/LVH group. In contrast, cardiac ACE2 mRNA expression significantly decreased in the LVH group and further decreased in the CKD/LVH group (Fig. 4). In addition, the immunohistochemical analysis also showed that expression of FGF23 and ACE significantly increased in the CKD/LVH group. Corresponding with RT-PCR results, angiotensin II significantly increased in the LVH and CKD/LVH groups, and the expression of ACE2 significantly decreased in the LVH and CKD/LVH groups (Fig. 5). As for bone FGF23 mRNA expression, it slightly increased in the LVH group compared to the sham group and remarkably increased by CKD induction (Fig. 6). As for bone FGF23 mRNA expression, it slightly increased in the LVH group compared to the sham group and re mRNA expression of factors related to cardiac hypertrophy and fibrosis increased by CKD and LVH induction The cardiac β-MHC, ANP, BNP, Col1a2, and Col3a1 mRNA expressions significantly increased, whereas cardiac α-MHC mRNA expression significantly decreased in the CKD/LVH group. In the LVH group, cardiac β-MHC mRNA expression also significantly increased, and cardiac α-MHC mRNA expression significantly decreased compared to the sham group (Fig. 7). Cardiac Parameters After Induction Of Ckd And Lvh CKD, chronic kidney disease; LVH, left ventricular hypertrophy; LVDd, left ventricular diastolic diameter; LVDs, left ventricular systolic diameter; LVAW, left ventricular anterior wall; LVPW, left ventricular posterior wall; FS, fractional shortening; EF, ejection fraction. Loading [MathJax]/jax/output/CommonHTML/jax.js Loading [MathJax]/jax/output/CommonHTML/jax.js Page 3/15 As shown in Fig. 2, cardiomyocyte width and area increased by LVH and CKD induction. Coexisting LVH and CKD are related to the further development of cardiac hypertrophy. Cardiac fibrosis was also significantly deteriorated in the CKD and CKD/LVH groups (Fig. 3). Cardiac expressions of FGF23, RAAS-related factors, and cardiac hypertrophy-related factors induced by CKD and LVH Discussion We demonstrated that 1) CKD deteriorated LVH and cardiac fibrosis induced by TAC; 2) serum FG23 and aldosterone levels increased in the CKD and LVH groups, and further elevation of these levels was observed in the CKD/LVH group; 3) similarly, intracardiac FGF23, hypertrophy-related factors, fibrosis-related factors, and RAAS-related factors increased in the LVH and CKD groups and dramatically increased in the CKD/LVH group; 4) among the RAAS-related factors, only ACE2 decreased by LVH, CKD, and CKD/LVH induction; 5) heart weight was significantly correlated with serum FGF23 and aldosterone levels and intracardiac expression of FGF23 and the RAAS-related factors. It is well known that pressure overload and CKD induce LVH. The development of LVH in these conditions involves various pathophysiological mechanisms. In pressure overload-induced LVH, cardiomyocyte hypertrophy and interstitial fibrosis reportedly occur through the activated mitogen-activated protein kinase (MAPK) pathway [14], increase in oxidative stress [15], and the activation of RAAS [16]. The results of our study also showed an increase in urinary excretion of 8-OHdG, an elevation of serum aldosterone levels, and an increase in the expression of AT1R, AGT, and ACE in the heart. In CKD, not only these factors but also CKD-related factors are involved in the progression of LVH. Recent compelling evidences suggest that CKD and mineral bone disorder (CKD-MBD) is strongly linked to the progression of cardiovascular disease in these populations [17]. Among the CKD-related factors, phosphate, calcium, FGF23, PTH, Klotho, and 1, 25 (OH)2 D were thought to play key roles. Many experimental and clinical studies have reported the correlation between FGF23 and LVH [18, 19]. It has been reported that the elevation of FGF23 directly causes myocardial hypertrophy even if klotho does not coexist with FGFR [4, 20]. Among FGFRs, FGFR4 is thought to have an important role in the progression of LVH through the activation of the calcineurin-NFAT pathway [4, 20]. However, the previous experimental study using cardiomyocyte-specific calcineurin A-transgenic mice showed that LVH induced an increase in myocardial and serum FGF23 [21]. In other words, this might mean that activation of the calcineurin-NFAT pathway could increase FGF23. Therefore, we performed an experimental study using a surgical model of CKD, LVH, and CKD/LVH to ascertain changes in RAAS and CKD-MBD parameters, including FGF23. Although serum and cardiac FGF23 increased in CKD as previously reported, these also increased even in LVH without CKD. In addition, further elevation of FGF23 was observed in CKD/LVH. Discussion However, as serum FGF23 levels in the CKD/LVH group were approximately 2-fold higher compared to those in the sham group, they did not seem to be enough to bind to FGFR without klotho. On the other hand, serum aldosterone levels in the CKD group rose approximately 5-fold as high as those in the sham group, and intracardiac mRNA expression of RAAS-related factors also increased in the CKD/LVH group. In our previous study using hypertensive model rats, an increase in serum FGF23 levels was also trivial. In contrast, an increase in serum aldosterone levels was much greater in the hypertensive group than in the sham group [22]. Furthermore, serum FGF23 levels were not correlate with LVH in pediatric CKD patients with preserved kidney function and FGF23-related hypophosphatemic diseases [6, 23]. From these findings, it is speculated that the influence of RAAS on LVH is greater than that of FGF23. To date, various studies have reported the interaction between RAAS and FGF23 [24–26]. An in vitro study using cardiomyocytes demonstrated that Ang II and aldosterone significantly upregulated FGF23 mRNA expression, suggesting that activation of the RAAS leads to the induction of FGF23 in cardiac myocytes [27]. In contrast, FGF23 also increased intracellular angiotensin II expression in cardiomyocytes [7]. Both RAAS and FGF23 are thought to be closely related to the pathophysiological mechanisms of the progression of LVH and cardiac fibrosis. Our previous study showed a significant relationship between serum FGF23 and aldosterone levels, and both were significantly correlated with heart weight [22]. Even in the present study, both RAAS-related factors and FGF23 increased in cardiac tissues in the CKD and LVH groups. The increase was manifest, particularly in the CKD/LVH group. Serum FGF23 levels, intracardiac FGF23 expression, serum aldosterone levels, and intracardiac RAAS-related factors expressions were significantly correlated with heart weight. Particularly, a significantly close correlation between intracardiac RAAS-related factors and FGF23 was observed. There is surely a close link between intracardiac RAAS and FGF23, and this interaction could play a key role in the progression of LVH. We assessed the influence of LVH on CKD-MBD parameters. The increase in FGF23 decreases 1, 25 (OH)2 D by reducing the expression of 1α-hydroxylase [28] and led to increased renin activity [29]. Correlation between FGF23, RAAS-related factors and cardiac hypertrophy and fibrosis HF, heart weight; rHF, relative heart weight; LVAW, left ventricular anterior wall; LVPW, left ventricular posterior wall; FS, fractional shortening; EF, ejection fractio atrial natriuresis peptide; cBNP, cardiac brain natriuresis peptide; cα-MHC, cardiac α-myosin heavy chain; cβ-MHC, cardiac β-myosin heavy chain; cCol1a2, car I α2; cCol3a1, cardiac collagen type III α1; iFGF23, intact fibroblast growth factor 23; cFGF23, cardiac fibroblast growth factor 23; imFGF23, immunohistochem of cardiac fibroblast growth factor 23; Ald, aldosterone; cAT1R, cardiac angiotensin II type 1 receptor; cAGT, cardiac angiotensinogen; cACE, cardiac angiotens enzyme; cACE2, cardiac angiotensin-converting enzyme 2; uAGT, urinary angiotensinogen; imAng II, immunohistochemical expression of cardiac angiotensin immunohistochemical expression of cardiac angiotensin-converting enzyme; imACE2, immunohistochemical expression of cardiac angiotensin-converting en Table 3 d RAAS d HF, heart weight; rHF, relative heart weight; LVAW, left ventricular anterior wall; LVPW, left ventricular posterior wall; FS, fractional shortening; EF, ejection fractio atrial natriuresis peptide; cBNP, cardiac brain natriuresis peptide; cα-MHC, cardiac α-myosin heavy chain; cβ-MHC, cardiac β-myosin heavy chain; cCol1a2, car I α2; cCol3a1, cardiac collagen type III α1; iFGF23, intact fibroblast growth factor 23; cFGF23, cardiac fibroblast growth factor 23; imFGF23, immunohistochem of cardiac fibroblast growth factor 23; Ald, aldosterone; cAT1R, cardiac angiotensin II type 1 receptor; cAGT, cardiac angiotensinogen; cACE, cardiac angiotens enzyme; cACE2, cardiac angiotensin-converting enzyme 2; uAGT, urinary angiotensinogen; imAng II, immunohistochemical expression of cardiac angiotensin immunohistochemical expression of cardiac angiotensin-converting enzyme; imACE2, immunohistochemical expression of cardiac angiotensin-converting en HF, heart weight; rHF, relative heart weight; LVAW, left ventricular anterior wall; LVPW, left ventricular posterior wall; FS, fractional shortening; EF, ejection fractio atrial natriuresis peptide; cBNP, cardiac brain natriuresis peptide; cα-MHC, cardiac α-myosin heavy chain; cβ-MHC, cardiac β-myosin heavy chain; cCol1a2, car I α2; cCol3a1, cardiac collagen type III α1; iFGF23, intact fibroblast growth factor 23; cFGF23, cardiac fibroblast growth factor 23; imFGF23, immunohistochem of cardiac fibroblast growth factor 23; Ald, aldosterone; cAT1R, cardiac angiotensin II type 1 receptor; cAGT, cardiac angiotensinogen; cACE, cardiac angiotens enzyme; cACE2, cardiac angiotensin-converting enzyme 2; uAGT, urinary angiotensinogen; imAng II, immunohistochemical expression of cardiac angiotensin immunohistochemical expression of cardiac angiotensin-converting enzyme; imACE2, immunohistochemical expression of cardiac angiotensin-converting en HF, heart weight; rHF, relative heart weight; LVAW, left ventricular anterior wall; LVPW, left ventricular posterior wall; FS, fractional shortening; EF, ejection fractio atrial natriuresis peptide; cBNP, cardiac brain natriuresis peptide; cα-MHC, cardiac α-myosin heavy chain; cβ-MHC, cardiac β-myosin heavy chain; cCol1a2, car I α2; cCol3a1, cardiac collagen type III α1; iFGF23, intact fibroblast growth factor 23; cFGF23, cardiac fibroblast growth factor 23; imFGF23, immunohistochem of cardiac fibroblast growth factor 23; Ald, aldosterone; cAT1R, cardiac angiotensin II type 1 receptor; cAGT, cardiac angiotensinogen; cACE, cardiac angiotens enzyme; cACE2, cardiac angiotensin-converting enzyme 2; uAGT, urinary angiotensinogen; imAng II, immunohistochemical expression of cardiac angiotensin immunohistochemical expression of cardiac angiotensin-converting enzyme; imACE2, immunohistochemical expression of cardiac angiotensin-converting en Loading [MathJax]/jax/output/CommonHTML/jax.js Page 4/15 Page 4/15 Table 4 Correlation of each FGF23 data with RAAS-related factors   Ald cAT1R cAGT cACE cACE2 uAGT imAng II imACE imACE2 iFGF23 .388 .665** .556** .285 − .269 .164 .511* .518** − .297 cFGF23 .567** .780** .722** .663** − .689** .626** .713** .785** − .666** imFGF23 .554** .820** .762** .664** − .623** .554** .729** .862** − .705** Ald, aldosterone; cAT1R, cardiac angiotensin II type 1 receptor; cAGT, cardiac angiotensinogen; cACE, cardiac angiotensin-converting enzyme; cACE2, cardiac angiotensin-converting enzyme 2; uAGT, urinary angiotensinogen; imAng II, immunohistochemical expression of cardiac angiotensin II; imACE, immunohistochemical expression of cardiac angiotensin-converting enzyme; imACE2, immunohistochemical expression of cardiac angiotensin-converting enzyme 2; iFGF23, intact fibroblast growth factor 23; cFGF23, cardiac fibroblast growth factor 23; imFGF23, immunohistochemical expression of cardiac fibroblast growth factor 23. Correlation between FGF23, RAAS-related factors and cardiac hypertrophy and fibrosis Ald, aldosterone; cAT1R, cardiac angiotensin II type 1 receptor; cAGT, cardiac angiotensinogen; cACE, cardiac angiotensin-converting enzyme; cACE2, cardiac angiotensin-converting enzyme 2; uAGT, urinary angiotensinogen; imAng II, immunohistochemical expression of cardiac angiotensin II; imACE, immunohistochemical expression of cardiac angiotensin-converting enzyme; imACE2, immunohistochemical expression of cardiac angiotensin-converting enzyme 2; iFGF23, intact fibroblast growth factor 23; cFGF23, cardiac fibroblast growth factor 23; imFGF23, immunohistochemical expression of cardiac fibroblast growth factor 23. Blood Pressure Measurements Systolic blood pressure (BP) was measured using tail-cuff plethysmography (Model MK-2000; Muromachi Kikai Co. Ltd., Japan). The mice rested for 15min to reduce stress-induced BP elevation. The mean value was determined using multiple readings (at least 10). The BP evaluation was performed at the end of the study period. Blood And Urine Measurement Collected blood was centrifuged for 15 min at 3,000 rpm and stored at -80℃ until analysis. Serum creatinine (Cr) levels were measured using a Fuji Dri-chem 3500 (FUJIFILM Japan, Tokyo, Japan). Serum phosphate, intact parathyroid hormone (iPTH), intact fibroblast growth factor 23 (iFGF23), and aldosterone levels were measured using ELISA (phosphate: FUJIFILM Japan, Tokyo, Japan; iPTH: Cloud-Clone Corp., Houston, Texas, USA; iFGF23: Kainos Laboratories, Inc., Tokyo, Japan; Aldosterone: Abcam, Cambridge, UK). Urinary excretion of albumin (uAlb), urinary Cr levels, urinary angiotensinogen (uAGT) levels, and 8- hydroxydeoxyguanosine (u8-OHdG) were also determined using ELISA (uAlb: FUJIFILM Wako Shibayagi Corporation, Gunma, Japan; u-Cr: FUJIFILM Wako Pure Chemicals Corporation, Osaka, Japan; uAGT: MyBioSource, San Diego, CA, USA; u8-OHdG: Japan Institute for Control of Aging, Shizuoka, Japan). Serum 25-hydroxyvitamin D (25D) and 1,25-dihydroxyvitamin D (1,25D) levels were measured using 25D125I radioimmunoassay kit (DIAsource ImmunoAssays S.A., Nivelles, Belgium) and a TFB 1,25D radioimmunoassay kit (Immunodiagnostic Systems Ltd., Boldon, UK), respectively. Echocardiographic Measurements The mice were mildly anesthetized with 1.0 % isoflurane. Echocardiography was performed using a commercially available echocardiographic system (F37; Hitachi Aloka Medical, Ltd, Tokyo, Japan). The wall thickness and ejection fraction (EF) were measured using a two-dimensional short-axis view of the left ventricle (LV) at the papillary muscle level. These studies were performed at 16 weeks. Animals Male C57BL/6J mice were obtained from SLC Japan Inc., Shizuoka, Japan. The mice were housed with food and water available ad libitum in light- and temperature-controlled environments. The mice were randomly divided into four groups at 10 weeks of age: sham-operated mice (sham, n = 6), induced-CKD mice (CKD, n = 6), induced-LVH mice (LVH, n = 6), and induced-CKD and induced-LVH mice (CKD/LVH, n = 6). At 10 weeks, TAC was performed for mice in the LVH and CKD/LVH groups. Briefly, the mice were anesthetized with isoflurane, and medetomidine, midazolam, and butorphanol were intraperitoneally administered. They were intubated with 22 G vascular catheters and then artificially ventilated (Small Animal Ventilator MK-V100, Muromachi Kikai). The thorax was opened, the aorta was exteriorized and a ligature was placed around the aorta as previously described [9]. The needle used for ligation was 27 G. At 11 weeks, a left 2/3 nephrectomy was performed, and one week later, a right nephrectomy was also performed for mice in the CKD and CKD/LVH groups. For mice in the sham group, sham operations were performed at 10, 11, and 12 weeks of age. Echocardiographic parameters were measured and 24-hour urine samples were collected from each mouse using a metabolic cage before the mice were sacrificed. At 16 weeks, these mice were sacrificed under anesthesia. Blood samples for serum measurements were collected from the right ventricle, and the hearts and bones were removed for RNA extraction and histomorphological analysis. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Experiment Facility Ethics Committee, Graduate School of Medicine, Kobe University (Permit Number: P160707-R2). All surgery was performed under anesthesia with 1.5 % isoflurane with intraperitoneal medetomidine, midazolam, and butorphanol premedication (0.15 mg/kg medetomidine, 2 mg/kg midazolam, and 2.5 mg/kg butorphanol. Efforts were made to minimize suffering by following the ARRIVE guidelines for reporting experiments involving animals [10, 11]. A randomized protocol was used in the experiments with a total of 24 mice. Discussion In addition, 1, 25 (OH)2 D is also considered a crucial factor in the progression of LVH because cardiac-specific vitamin D receptor knock-out mice showed marked cardiac hypertrophy [30], and a vitamin D receptor activator could prevent the progression of LVH independently of RAAS [31]. The present study also showed that serum 1, 25 (OH)2 D decreased in the CKD and CKD/LVH groups despite similar serum 25 (OH) D levels. ACE2 is an enzyme that can convert angiotensin I to angiotensin (1–9) and angiotensin II to angiotensin (1–7) [32, 33]. Angiotensin (1–9) and angiotensin (1–7) can lower BP by their vasodilative effect. Thus, they could prevent the LVH progression. ACE2-deficient mice developed cardiac dysfunction and LVH, Loading [MathJax]/jax/output/CommonHTML/jax.js Page 5/15 Page 5/15 enhanced oxidative stress, and inflammatory cytokine expression [34–36]. Interestingly, intracardiac ACE2 expression dramatically decreased in the LVH and CKD + LVH groups, and ACE is supposed to be an important factor in the progression of LVH. It has been reported that FGF23 decreases ACE2 expression [37] and that plasma ACE2 activity is impaired in CKD patients [38]. Therefore, this mechanism might greatly contribute to the progression of LVH in CKD. The most important limitation of this study is that we could not clarify the first occurrence regarding the relationship between RAAS and FGF23. From the degree of change in FGF23, FGF23 could not be a contributor to the progression of LVH. However, there is a possibility that FGF23 may induce LVH in a paracrine or autocrine manner, or both. FGF23 and RAAS enhance the expression of cardiac hypertrophy- and cardiac fibrosis-related factors via various pathways, such as the MAPK pathway, the TGF-β-Smad pathway, the NFκB pathway, and the NFAT-CnB pathway [14, 21, 39]. Unfortunately, our study could not adequately evaluate these pathways. As for intracardiac calcineurin B expression, there was no significant difference among the study groups. Moreover, we did not perform an interventional study with the FGFR blocker, ACE inhibitor, angiotensin receptor blocker, or aldosterone blocker. To resolve this issue, we need to plan a further study. In conclusion, coexisting CKD and LVH further increased serum aldosterone and FGF23 levels and cardiac expression of FGF23 and RAAS-related factors. There was a significantly close correlation of not serum but cardiac FGF23 with LVH and RAAS, suggesting that local FGF23 may be associated with the activation of the RAAS and LVH. Histological And Immunohistochemical Analyses Histological And Immunohistochemical Analyses The hearts were removed, weighed, and fixed in 10 % formaldehyde. The paraffin block was made by immersing the samples in ethanol, xylene, and paraffin in the embedding device at room temperature. The paraffin blocks were cut into 2-µm sections and stained with hematoxylin-eosin for routine histology and Loading [MathJax]/jax/output/CommonHTML/jax.js Page 6/15 Page 6/15 Page 6/15 Sirius red for morphometric studies. FGF23, angiotensin II, angiotensin converting enzyme (ACE), and angiotensin converting enzyme 2 (ACE2) expression in the heart were assessed with anti- FGF23 monoclonal antibodies (R&D Systems, Minneapolis, MN, USA), anti-angiotensin II monoclonal antibodies (Novus Biologicals, LLC, Littleton, Colorado, USA), anti-ACE monoclonal antibodies (Abcam, Cambridge, UK), and anti-ACE2 monoclonal antibodies (R&D Systems, Minneapolis, MN, USA). In brief, heart slices were preincubated with blocking agents and then incubated with the primary antibodies mentioned above for 60 min at room temperature (ACE, FGF23) and overnight at 4 ℃ (ACE2, angiotensin II). Biotinylated Anti-rat and Anti-goat IgG (Vector Laboratories, Burlingame, CA, USA) and an avidin: biotinylated enzyme complex (VECTASTAIN Elite ABC Reagent; Vector Laboratories, Inc., Burlingame, CA, USA) for FGF23 and ACE2, and a universal immunoperoxidase polymer (Histofine Simple Stain mouse MAX PO, anti-rat and anti-rabbit; Nichirei, Tokyo, Japan) for ACE and angiotensin II were used for immunostaining. The percentage of positive areas of Sirius red staining, relative immune-positive area, and cardiomyocyte width and area in the LV sections were calculated as mean values in 20 randomly selected microscopic fields using ImageJ (ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA). As for assessing the immune-positive area, the relative value was defined as that in each group or that in the sham group. All evaluations were performed in a blinded manner. RNA E t ti A d R l ti P l Ch i R ti RNA Extraction And Real-time Polymerase Chain Reaction As previously reported [12, 13], total RNA was extracted from the mouse heart and bone samples with an ISOGEN kit (Wako Pure Chemicals Industries, Ltd, Osaka, Japan) according to the manufacturer’s instructions. ReverTra Ace™ qPCR RT Kit (TOYOBO Co., Ltd., Osaka, Japan) was used to create cDNA using an oligo-dT primer as recommended. The synthesized cDNA was stored at -80℃ until analysis with quantitative PCR. Conflict of interest statement All authors declared no competing interests. Statistical analysis Collected data were analyzed using the computer software application IBM SPSS statistics version 25.0 (IBM Corp., Armonk, NY, USA) for all statistical analyses. Values are presented as means ± SEM. The Mann–Whitney U test was used to analyze the significance of the differences between the two groups. The Kruskal-Wallis test was used to assess the differences between the four groups. Pearson’s and Spearman’s correlation coefficients were used to analyze relationships between the variables. A P value of < 0.05 was considered statistically significant. Author contributions K.O. designed and performed experiments and wrote the manuscript. H.F., S.G., K.W., K.K., S.N. contributed to discussions. H.F. is the guarantor of this work and takes responsibility for the integrity of the data and the accuracy of the data analysis. Histological And Immunohistochemical Analyses mRNA expression was examined with real- time PCR using Applied Biosystems 7500 Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) with the SYBR Green Assay with Thunderbird SYBR qPCR Mix (TOYOBO Co., Ltd., Osaka, Japan) following the manufacturer’s protocol. The analysis was performed with the relative quantification method of the Applied Biosystems 7500 Real-Time PCR Software. The relative amount the mRNA samples was normalized to GAPDH mRNA. For PCR analysis, we used the following primers: α-myosin heavy chain (α-MHC), (5′-ATGTTAAGGCCAAGGTCGTG-3′, 5′-CACCTGGTCCTCCTTTATGG-3′), β-myosin heavy chain (β- MHC), (5′-AGCATTCTCCTGCTGTTTCC-3′, 5′-GAGCCTTGGATTCTCAAACG-3′), atrial natriuresis peptide (ANP), (5′-AGGCAGTCGATTCTGCTTGA-3′, 5′- CGTGATAGATGAAGGCAGGAAG-3′), brain natriuresis peptide (BNP), (5′-TAGCCAGTCTCCAGAGCAATTC-3′, 5′-TTGGTCCTTCAAGAGCTGTCTC-3′), collagen type I α2 (Col1a2), (5′-GCAGGTTCACCTACTCTGTCCT-3′, 5′-CTTGCCCCATTCATTTGTCT-3′), collagen type III α1 (Col3a1), (5′-TCCCCTGGAATCTGTGAATC-3′, 5′- TGAGTCGAATTGGGGAGAAT-3′), connective tissue growth factor (CTGF), (5′-CACAGAGTGGAGCGCCTGTTC-3′, 5′-GATGCACTTTTTGCCCTTCTTAA-3′), FGF23, (5′-ACAAGGACACCTAAACCGAACAC-3′, 5′-AGCTACTGACTGGTCCTATCACA-3′), AGT, (5′-TCTCTTTACCCCTGCCCTCT-3′, 5′-GAAACCTCTCATCGTTCCTTG-3′), AT1R, (5 ( -ℂA ⊤GTℂAℂCGATG ∀G - 3′ , 5′ - TGCAGGTGAC ⊤TGGℂAC - 3′ ), ACE, (5′ - ℂCTAGAG ∀∀TCGℂ⊤C ⊤G - 3′ , 5′ - CG ∀GATAℂAℂAGT -TGGGCAAACTCTATGCTG-3′, 5′-TTCATTGGCTCCGTTTCTTA-3′), Renin, (5′-CCTCTACCTTGCTTGTGGGATT-3′, 5′-CTGGCTGAGGAAACCTTTGACT-3′), GAPDH, (3′-GCAAAGTGGAGATTGTTGCCA-5′, 3′-AATTTGCCGTGAGTGGAGTCA-5′). 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Takashi, Y. et al. Patients with FGF23-related hypophosphatemic rickets/osteomalacia do not present with left ventricular hypertrophy. Endocr Res. 42, 132–137 (2017). Loading [MathJax]/jax/output/CommonHTML/jax.js Loading [MathJax]/jax/output/CommonHTML/jax.js Page 7/15 Page 7/15 7. Mhatre, K. N. et al. Crosstalk between FGF23- and angiotensin II- mediated Ca2+ signaling in pathological cardiac hypertrophy. Cell Mol Life Sci. 75, 4403– 4416 (2018). 8. De Stefano, G. M. M. F. et al. Aldosterone is associated with left ventricular hypertrophy in hemodialysis patients. Ther Adv Cardiovasc Dis. 10, 304–313 (2016). 9. Stein, A. B. et al. The PTIP-associated histone methyltransferase complex prevents stress-induced maladaptive cardiac remodeling. Plos One. 22, e0127839; 10.1371/journal.pone.0127839. eCollection (2015) 10. Kilkenny, C., Browne, W. J., Cuthill, I.C., Emerson, M., Altman, D. G. Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. PLoS Biol. 8, e1000412 ; 10.1371/journal.pbio.1000412 (2010). 11. McGrath, J. C., Drummond, G. B., McLachlan, E. M., Kilkenny, C., Wainwright, C. L. Guideline for reporting experiments involving animals: the ARRIVE guidelines. Br J Pharmacol. 160, 1573–1576 (2010). ti-oxidative effect of AST-120 on kidney injury after myocardial infarction. Br J Pharmacol. 173, 1302-1313 (2016) 2. Fujii, H. et al. Anti-oxidative effect of AST-120 on kidney injury after myocardial infarction. Br J Pharmacol. 173 12. Fujii, H. et al. Anti-oxidative effect of AST-120 on kidney injury after myocardial infarction. Br J Pharmacol. 173, 1302-1313 (2016). 13. References Enhanced susceptibility to biomechanical stress in ACE2 null mice is prevented by loss of the p47 (phox) NADPH oxidase subunit. Cardiovasc Res. 91, 151–161 (2011). 36. Oudit, G. Y. et al. Angiotensin II-mediated oxidative stress and inflammation mediate the age-dependent cardiomyopathy in ACE2 null mice. Cardiovasc Res. 75, 29–39 (2007). 7. Dai, B. et al. A comparative transcriptome analysis identifying FGF23 regulated genes in the kidney of a mouse 44161; 10.1371/journal.pone.0044161 (2012). 37. Dai, B. et al. A comparative transcriptome analysis identifying FGF23 regulated genes in the kidney of a mouse CKD model. PLoS One. 7, e 44161; 10.1371/journal.pone.0044161 (2012). 8. Anguiano, L. et al. Circulating angiotensin-converting enzyme 2 activity in patients with chronic kidney disease disease. Nephrol Dial Transplant. 30, 1176–1185 (2015). irculating angiotensin-converting enzyme 2 activity in patients with chronic kidney disease without previous histo al Transplant. 30, 1176–1185 (2015). 38. Anguiano, L. et al. Circulating angiotensin-converting enzyme 2 activity in patients with chronic kidney disease without previous history of cardiovascular disease. Nephrol Dial Transplant. 30, 1176–1185 (2015). 9. Lang, F. et al. Phosphate Homeostasis, Inflammation and the Regulation of FGF-23. Kidney Blood Press Res. 43 39. Lang, F. et al. Phosphate Homeostasis, Inflammation and the Regulation of FGF-23. Kidney Blood Press Res. 43, 1742–1748 (2018). References Fujii, H., Hamada, Y., Fukagawa, M. Bone Formation in Spontaneously Diabetic Torii - newly established model of non-obese Type 2 Diabetes rats. Bone. 42, 372-379 (2008). 14. Lei, B. et al. Transient activation of p38 MAP kinase and up-regulation of Pim-1 kinase in cardiac hypertrophy despite no activation of AMPK. J Mol Cell Cardiol. 45, 404–410 (2008). 15. Zhan, M. et al. Pathological cardiac hypertrophy alters intracellular targeting of phosphodiesterase type 5 from nitric oxide synthase-3 to natriuretic peptide signaling. Circulation. 126, 942–951 (2012). 16. Rockman, H. A. et al. ANG II receptor blockade prevents ventricular hypertrophy and ANF gene expression with pressure overload in mice. Am J Physiol. 266: H2468–2475 (1994). 7. Fujii, H. & Joki, N. Mineral metabolism and cardiovascular disease in CKD. Clin Exp Nephrol. 21, 53–63 (2017). 18. Gutierrez, OM. et al. Fibroblast growth factor 23 and left ventricular hypertrophy in chronic kidney disease. Circulation. 119, 2545–2552(2009). 18. Gutierrez, OM. et al. Fibroblast growth factor 23 and left ventricular hypertrophy in chronic kidney disease. C 19. Mirza, M. A. I., Larsson, A., Melhus, H., Lind, L., Larsson, T. E. Serum intact FGF23 associate with left ventricular mass, hypertrophy and geometry in an elderly population. Atherosclerosis. 207, 546–551 (2009). 0. Grabner, A. et al. Activation of cardiac fibroblast growth factor receptor 4 causes left ventricular hypertrophy. Ce . et al. Cardiac hypertrophy elevates serum levels of fibroblast growth factor 23. Kidney Int. 94, 60-71 (2018). 21. Matsui, I. et al. Cardiac hypertrophy elevates serum levels of fibroblast growth factor 23. Kidney Int. 94, 60-71 (2018). 22. Fujii, H. et al. Changes in serum and intracardiac fibroblast growth factor 23 during the progression of left ventricular hypertrophy in hypertensive model rats. Clin Exp Nephrol. 23, 589-596 (2019). neider MF, et al. FGF23 and left ventricular hypertrophy in children with CKD. Clin J Am Soc Nephrol. 13, 45-52 (20 3. Mitsnefes, M. M. Betoko A, Schneider MF, et al. FGF23 and left ventricular hypertrophy in children with CKD. Cli 24. Böckmann, I. et al. FGF23-mediated activation of local RAAS promotes cardiac hypertrophy and fibrosis. Int J Mol Sci. 20, 4634; 10.3390/ijms20184634 (2019). 25. Pi, M. et al. Cardiovascular interactions between fibroblast growth factor-23 and angiotensin II. Sci Rep. 8, 12398; 10.1038/s41598-018-30098-1 (2018). 26 Zhang B et al Up-regulation of FGF23 release by aldosterone Biochem Biophys Res Commun 470 384–390 (2016) 5. Pi, M. et al. Table 2.  Animal characteristics at 16 weeks Table 2. Animal characteristics at 16 weeks Sham (N = 6) CKD (N = 6) LVH (N = 6) CKD/LVH (N = 6) LVDd (mm) 3.77 ± 0.76 3.45 ± 0.76 3.47 ± 0.29 3.15 ± 0.08 LVDs (mm) 2.78 ± 0.06 2.67 ± 0.06 2.75 ± 0.22 2.55 ± 0.07#, * LVAW (mm) 0.75 ± 0.02 0.80 ± 0.04 1.23 ± 0.04#, * 1.30 ± 0.04#, * LVPW (mm) 0.71 ± 0.03 0.75 ± 0.04 1.18 ± 0.05#, * 1.20 ± 0.04#, * FS (%) 25.8 ± 0.4 23.0 ± 0.2# 20.9 ± 0.3#, * 19.3 ± 0.8#, * EF (%) 57.7 ± 0.6 52.9 ± 0.4 49.1 ± 0.5#, * 46.0 ± 1.7#, * Heart weight (mg) 131.3 ± 3.3 116.2 ± 2.3 181.6 ± 5.2#, * 178.2 ± 8.5#, * Relative heart weight (mg/g) 4.45 ± 0.07 4.64 ± 0.08 6.33 ± 0.16#, * 6.96 ± 0.36#, * #: vs Sham, P < 0.05, *: vs CKD, P < 0.05, ♰: vs LVH, P < 0.05. : vs Sham, P 0.05, : vs CKD, P 0.05,  : vs LVH, P 0.05. CKD, chronic kidney disease; LVH, left ventricular hypertrophy; LVDd, left ventricular diastolic diameter; LVDs, left ventricular systolic diameter; LVAW, left ventricular anterior wall; LVPW, left ventricular posterior wall; FS, fractional shortening; EF, ejection fraction. , , , , , CKD, chronic kidney disease; LVH, left ventricular hypertrophy; LVDd, left ventricular diastolic diameter; LVDs, left ventricular systolic diameter; LVAW, left ventricular anterior wall; LVPW, left ventricular posterior wall; FS, fractional shortening; EF, ejection fraction. Table 3. , HF, heart weight; rHF, relative heart weight; LVAW, left ventricular anterior wall; LVPW, left ventricular posterior wall; FS, fractional shortening; EF, ejection fraction; cANP, cardiac atrial natriuresis peptide; cBNP, cardiac brain natriuresis peptide; cα-MHC, cardiac α-myosin heavy chain; cβ-MHC, cardiac β-myosin heavy chain; cCol1a2, cardiac collagen type I α2; cCol3a1, cardiac collagen type III α1; iFGF23, intact Loading [MathJax]/jax/output/CommonHTML/jax.js #: vs Sham, P < 0.05, *: vs CKD, P < 0.05, ♰: vs LVH, P < 0.05. *: P < 0.05, **: P < 0.01. #: vs Sham, P < 0.05, *: vs CKD, P < 0.05, ♰: vs LVH, P < 0.05. Tables Loading [MathJax]/jax/output/CommonHTML/jax.js Loading [MathJax]/jax/output/CommonHTML/jax.js Page 8/15 Page 8/15 Page 8/15 Table 1. Animal characteristics at 16 weeks b e . a c a acte st cs at 6 ee s Sham (N = 6) CKD (N = 6) LVH (N = 6) CKD/LVH (N = 6) Body weight (g) 29. 5± 0.7 25.1 ± 0.8# 28.7 ± 0.6* 25.7 ± 1.0#, ♰ SBP (mmHg) 86.2 ± 0.6 97.6 ± 2.0# 87.2 ± 1.8 107.2 ± 4.0#, ♰ HR (/min) 516.3 ± 12.3 493.1 ± 12.2 490.7 ± 15.4 472.8 ± 14.7 Cr (mg/dL) 0.14 ± 0.02 0.44 ± 0.03# 0.23 ± 0.02* 0.47 ± 0.09#, ♰ Ca(mg/dL) 9.87 ± 0.30 9.83 ± 0.80 10.03 ± 0.76 9.02 ± 0.81 P (mg/dL) 8.1 ± 0.6 9.7 ± 0.4# 8.8 ± 0.3 10.3 ± 0.3# iPTH (pg/mL) 29.0 ± 11.6 77.6 ± 16.2 50.6 ± 7.0 111.6 ± 39.1# 25(OH)D (pg/mL) 61.7 ± 2.3 61.8 ± 3.5 59.7 ± 3.2 63.3 ± 4.0 1, 25(OH)2D (pg/mL) 227.0 ± 18.9 184.8 ± 10.4 222.2 ± 24.6 178.0 ± 9.9 uAGT (ng/mgCr) 3.32 ± 0.85 3.36 ± 0.94 7.13 ± 2.55#, * 9.62 ± 2.10#, * uAlb (μg/mgCr) 144.6 ± 9.5 1156.3 ± 221.2# 156.1 ± 5.7* 1356.8 ± 145.6#, ♰ u8-OHdG (ng/mgCr) 53.9 ± 1.8 99.4 ± 16.1# 82.8 ± 8.3 101.3 ± 9.2# CKD, chronic kidney disease; LVH, left ventricular hypertrophy; SBP, systolic blood pressure; HR, heart rate; Cr, creatinine; Ca, calcium; P, phosphate; iPTH, intact parathyroid hormone; uAGT, urinary angiotensinogen; uAlb, urinary albumin; u8-OHdG, urinary  8-hydroxy-2’- deoxyguanosine. Table 2. Animal characteristics at 16 weeks 0.05, *: vs CKD, P < 0.05, ♰: vs LVH, P < 0.05. Table 2.  Animal characteristics at 16 weeks fibroblast growth factor 23; cFGF23, cardiac fibroblast growth factor 23; imFGF23, immunohistochemical expression of cardiac fibroblast growth factor 23; Ald, aldosterone; cAT1R, cardiac angiotensin II type 1 receptor; cAGT, cardiac angiotensinogen; cACE, cardiac angiotensin- converting enzyme; cACE2, cardiac angiotensin-converting enzyme 2; uAGT, urinary angiotensinogen; imAng II, immunohistochemical expression of cardiac angiotensin II; imACE, immunohistochemical expression of cardiac angiotensin-converting enzyme; imACE2, immunohistochemical expression of cardiac angiotensin-converting enzyme 2. Table 4. Correlation of each FGF23 data with RAAS-related factors   Ald cAT1R cAGT cACE cACE2 uAGT imAng II imACE imACE2 iFGF23 .388 .665** .556** .285 -.269 .164 .511* .518** -.297 cFGF23 .567** .780** .722** .663** -.689** .626** .713** .785** -.666** imFGF23 .554** .820** .762** .664** -.623** .554** .729** .862** -.705** Table 4. Correlation of each FGF23 data with RAAS-related factors Ald, aldosterone; cAT1R, cardiac angiotensin II type 1 receptor; cAGT, cardiac angiotensinogen; cACE, cardiac angiotensin-converting enzyme; cACE2, cardiac angiotensin-converting enzyme 2; uAGT, urinary angiotensinogen; imAng II, immunohistochemical expression of cardiac angiotensin II; imACE, immunohistochemical expression of cardiac angiotensin-converting enzyme; imACE2, immunohistochemical expression of cardiac angiotensin-converting enzyme 2; iFGF23, intact fibroblast growth factor 23; cFGF23, cardiac fibroblast growth factor 23; imFGF23, immunohistochemical expression of cardiac fibroblast growth factor 23. Ald, aldosterone; cAT1R, cardiac angiotensin II type 1 receptor; cAGT, cardiac angiotensinogen; cACE, cardiac angiotensin-converting enzyme; cACE2, cardiac angiotensin-converting enzyme 2; uAGT, urinary angiotensinogen; imAng II, immunohistochemical expression of cardiac angiotensin II; imACE, immunohistochemical expression of cardiac angiotensin-converting enzyme; imACE2, immunohistochemical expression of cardiac angiotensin-converting enzyme 2; iFGF23, intact fibroblast growth factor 23; cFGF23, cardiac fibroblast growth factor 23; imFGF23, immunohistochemical expression of cardiac fibroblast growth factor 23. Ald, aldosterone; cAT1R, cardiac angiotensin II type 1 receptor; cAGT, cardiac angiotensinogen; cACE, cardiac angiotensin-converting enzyme; cACE2, cardiac angiotensin-converting enzyme 2; uAGT, urinary angiotensinogen; imAng II, immunohistochemical expression of cardiac angiotensin II; imACE, immunohistochemical expression of cardiac angiotensin-converting enzyme; imACE2, immunohistochemical expression of cardiac angiotensin-converting enzyme 2; iFGF23, intact fibroblast growth factor 23; cFGF23, cardiac fibroblast growth factor 23; imFGF23, immunohistochemical expression of cardiac fibroblast growth factor 23. Table 2.  Animal characteristics at 16 weeks Correlation of each FGF23 and RAAS data with cardiac parameters HW rHW Width Area Fibrosis LVAW LVPW FS EF cANP cBNP cα- MHC cβ- MHC cCol1a2 cCol3a1 3 .212 .517** .617** .518** .698** .449* .394 -.639** -.645** .329 .299 -.181 .583** .493* .528** 3 .536** .708** .641** .717** .807** .726** .707** -.816** -.805** .399 .436* -.512* .846** .709** .445* 23 .505* .766** .649** .815** .890** .728** .709** -.820** -.814** .403 .538** -.569** .838** .683** .487* .251 .641** .666** .495* .616** .564** .566** -.629** -.625** .241 .239 -.241 .527** .500* .423* R .624** .765** .679** .830** .768** .702** .644** -.858** -.863** .369 .427* -.651** .742** .555** .443* .537** .674** .730** .678** .662** .779** .700** -.692** -.691** .490* .352 -.703** .742** .497* .473* .823** .784** .600** .683** .628** .735** .705** -.717** -.714** .441* .448* -.773** .738** .457* .335 2 -.690** -.778** -.613** -.701** -.622** -.752** -.702** .714** .704** -.528** -.311 .803** -.699** -.400 -.226 .485* .572** .528** .557** .557** .744** .694** -.624** -.612** .116 .213 -.539** .683** .510* .312 II .448* .656** .651** .562** .637** .665** .613** -.672** -.676** .301 .283 -.561** .714** .389 .277 E .700** .836** .779** .853** .824** .837** .821** -.884** -.881** .462* .561** -.695** .834** .645** .444* 2 -.690** -.776** -.664** -.586** -.643** -.834** -.794** .706** .705** -.489* -.476* .719** -.786** -.437* -.423* : P < 0.05, : P < 0.01. HF, heart weight; rHF, relative heart weight; LVAW, left ventricular anterior wall; LVPW, left ventricular posterior wall; FS, fractional shortening; EF, ejection fraction; cANP, cardiac atrial natriuresis peptide; cBNP, cardiac brain natriuresis peptide; cα-MHC, cardiac α-myosin heavy chain; cβ-MHC, cardiac β-myosin heavy chain; cCol1a2, cardiac collagen type I α2; cCol3a1, cardiac collagen type III α1; iFGF23, intact Loading [MathJax]/jax/output/CommonHTML/jax.js Page 9/15 fibroblast growth factor 23; cFGF23, cardiac fibroblast growth factor 23; imFGF23, immunohistochemical expression of cardiac fibroblast growth factor 23; Ald, aldosterone; cAT1R, cardiac angiotensin II type 1 receptor; cAGT, cardiac angiotensinogen; cACE, cardiac angiotensin- converting enzyme; cACE2, cardiac angiotensin-converting enzyme 2; uAGT, urinary angiotensinogen; imAng II, immunohistochemical expression of cardiac angiotensin II; imACE, immunohistochemical expression of cardiac angiotensin-converting enzyme; imACE2, immunohistochemical expression of cardiac angiotensin-converting enzyme 2. Figures g Figure 1 Serum FGF23 and aldosterone levels among the study groups. Black bar: serum FGF23 levels White bar: serum aldosterone levels #: vs Sham group, P<0.05; *: vs CKD group, P<0.05. Figure 1 Serum FGF23 and aldosterone levels among the study groups. Black bar: serum FGF23 levels White bar: serum aldosterone levels #: vs Sham group, P<0.05; *: vs CKD group, P<0.05. Loading [MathJax]/jax/output/CommonHTML/jax.js Page 10/15 Page 10/15 Figure 2 Histological measurement of cardiomyocytes. Representative photomicrographs of cardiomyocytes with hematoxylin-eosin (magnification, ×400; Bar = 20 μm) Left upper panel, sham group; right upper panel, CKD group; left lower panel, LVH group; right lower panel, CKD/LVH group (CKD, chronic kidney disease; LVH, left ventricular hypertrophy). a. Width of cardiomyocytes b. Area of cardiomyocytes Values are expressed as means ± SEM. #: vs Sham, P < 0.05; *: vs CKD, P < 0.05; ♰: vs LVH, P < 0.05. Figure 2 Histological measurement of cardiomyocytes. Representative photomicrographs of cardiomyocytes with hema μm) Left upper panel, sham group; right upper panel, CKD group; left lower panel, LVH group; right lower panel, LVH, left ventricular hypertrophy). a. Width of cardiomyocytes b. Area of cardiomyocytes Values are expressed CKD, P < 0.05; ♰: vs LVH, P < 0.05. Figure 2 Histological measurement of cardiomyocytes. Representative photomicrographs of cardiomyocytes with hematoxylin-eosin (magnification, ×400; Bar = 20 ) f l h i h l C l f l l i h l l C (C h i kid di Figure 2 Histological measurement of cardiomyocytes. Representative photomicrographs of cardiomyocytes with hematoxylin-eosin (magnification, ×400; Bar = 20 μm) Left upper panel, sham group; right upper panel, CKD group; left lower panel, LVH group; right lower panel, CKD/LVH group (CKD, chronic kidney disease; LVH, left ventricular hypertrophy). a. Width of cardiomyocytes b. Area of cardiomyocytes Values are expressed as means ± SEM. #: vs Sham, P < 0.05; *: vs CKD, P < 0.05; ♰: vs LVH, P < 0.05. Loading [MathJax]/jax/output/CommonHTML/jax.js Page 11/15 Page 11/15 gure 3 aluation of cardiac fibrosis. a. Representative photomicrographs of cardiomyocytes with Sirius red (magnification, ×400; Bar = 20 μm) Left upper panel, am group; right upper panel, CKD group; left lower panel, LVH group; right lower panel, CKD/LVH group (CKD, chronic kidney disease; LVH, left ventricular pertrophy). b. Fibrosis area of the heart Values are expressed as means ± SEM. Figures #: vs Sham, P < 0.05; *: vs CKD, P < 0.05; ♰: vs LVH, P < 0.05. Figure 3 Evaluation of cardiac fibrosis. a. Representative photomicrographs of cardiomyocytes with Sirius red (magnification, ×400; Bar = 20 μm) Left upper panel, sham group; right upper panel, CKD group; left lower panel, LVH group; right lower panel, CKD/LVH group (CKD, chronic kidney disease; LVH, left ventricular hypertrophy). b. Fibrosis area of the heart Values are expressed as means ± SEM. #: vs Sham, P < 0.05; *: vs CKD, P < 0.05; ♰: vs LVH, P < 0.05. Loading [MathJax]/jax/output/CommonHTML/jax.js Page 12/15 Page 12/15 Page 12/15 Figure 4 mRNA expressions of FGF23 and RAAS-related factors in the heart. a. Fibroblast growth factor 23 (FGF23) b. Angiotensin II type 1 receptor (AT1R) c. Angiotensinogen (Ang) d. Renin e. Angiotensin-converting enzyme (ACE) f. Angiotensin-converting enzyme 2 (ACE2) Values are expressed as means ± SEM. #: vs Sham, P < 0.05; *: vs CKD, P < 0.05; ♰: vs LVH, P < 0.05. Evaluation of the expressions of FGF23 and RAAS-related factors by immunohistochemical staining. A. Representative photomicrographs of cardiomyocytes with immunohistochemical staining for FGF23 and each RAAS-related factors B. Semiquantitative evaluation of FGF23 and each RAAS-related factors in the heart a. Fibroblast growth factor 23 (FGF23) b. Angiotensin II (Ang II) c. Angiotensin-converting enzyme (ACE) d. Angiotensin-converting enzyme 2 (ACE2) Values are expressed as means ± SEM. #: vs Sham, P < 0.05; *: vs CKD, P < 0.05; ♰: vs LVH, P < 0.05. mRNA expressions of cardiac hypertrophy and fibrosis-related factors in heart. a. α-myosin heavy chain (α-MHC) b. β-myosin heavy chain (β-MHC) c. collagen type I α2 (Collagen 1a2) d. collagen type III α1 (Collagen 3a1) Values are expressed as means ± SEM. #: vs Sham, P < 0.05; *: vs CKD, P < 0.05; ♰: vs LVH, P < 0.05. Figure 4 mRNA expressions of FGF23 and RAAS-related factors in the heart. a. Fibroblast growth factor 23 (FGF23) b. Angiotensin II type 1 receptor (AT1R) c. Angiotensinogen (Ang) d. Renin e. Angiotensin-converting enzyme (ACE) f. Angiotensin-converting enzyme 2 (ACE2) Values are expressed as means ± SEM. # vs Sham, P < 0.05; *: vs CKD, P < 0.05; ♰: vs LVH, P < 0.05. Page 13/15 Figure 5 Loading [MathJax]/jax/output/CommonHTML/jax.js Loading [MathJax]/jax/output/CommonHTML/jax.js Page 13/15 Evaluation of the expressions of FGF23 and RAAS-related factors by immunohistochemical staining. A. Representative photomicrographs of cardiomyocytes with immunohistochemical staining for FGF23 and each RAAS-related factors B. Semiquantitative evaluation of FGF23 and each RAAS-related factors in the heart a. Fibroblast growth factor 23 (FGF23) b. Angiotensin II (Ang II) c. Angiotensin-converting enzyme (ACE) d. Angiotensin-converting enzyme 2 (ACE2) Values are expressed as means ± SEM. #: vs Sham, P < 0.05; *: vs CKD, P < 0.05; ♰: vs LVH, P < 0.05. Figure 6 RNA i f FGF23 i b V l d ± SEM # Sh P 0 05 ♰ LVH P 0 05 Figure 6 mRNA expression of FGF23 in bone. Values are expressed as means ± SEM. #: vs Sham, P < 0.05; ♰: vs LVH, P < 0.05. Figure 6 RNA expression of FGF23 in bone. Values are expressed as means ± SEM. #: vs Sham, P < 0.05; ♰: vs LVH, P < 0.0 Figure 7 Loading [MathJax]/jax/output/CommonHTML/jax.js Page 14/15 Figure 7 Loading [MathJax]/jax/output/CommonHTML/jax.js Figure 7 Page 14/15 mRNA expressions of cardiac hypertrophy and fibrosis-related factors in heart. a. α-myosin heavy chain (α-MHC) b. β-myosin heavy chain (β-MHC) c. collagen type I α2 (Collagen 1a2) d. collagen type III α1 (Collagen 3a1) Values are expressed as means ± SEM. #: vs Sham, P < 0.05; *: vs CKD, P < 0.05; ♰: vs LVH, P < 0.05. Loading [MathJax]/jax/output/CommonHTML/jax.js Page 15/15
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Role of chloride in the oxidative decarboxylation of amino acids by chloramine-T
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Role of chloride in the oxidative decarboxylation of amino acids by chloramine-T B. Thimme Gowda*, S. Darlin Quine and P. S. Krishna Kumar Department of Post-Graduate Studies and Research in Chemistry, Mangalore University, Mangalagangotri-574 J 99, India. E-mail : gowdabt@yahoo.com Manufcrrpt rec e1ved 15 November 1999 aLcepted 28 Ap11l 2000 Studies and Research in Chemistry, Mangalore University, Mangalagangotri-574 J 99, oo.com Manufcrrpt rec e1ved 15 November 1999. aLcepted 28 Ap11l 2000 Kinetics of' oxidative decarboxylation of amino acids by chloramine-T in the presence of chloride have been studied in aqueous perchloric acid over a wide range. The rate-[H+] plots show nearly bell-shaped profiles in the presence of added chloride. The rate-dependence in [CAT] changes from second order to first order as [H+] and [Cr-] are varied. The reactions generally 1ohow fractional order kinetics in [AA] and invcr~e dependence in (WJ except in the acid range 0.05~.20 mol dm-3• Mechanism~ consistent with the observed result~ arc dbcu&\ed. The rate-limiting steps have been identified and constants of these steps calculated. Activation parameters corresponding to these steps have also been computed. Validity of Taft equation has been tested. The study cstabli..,IJCs the significant role of chloride in chloramine-T oxidations in acid medium. The chloride effect is more pronounced at high acid concentrations. As the fH+J was increased beyond 0.05 mol dm-\ the rate dependence in {CAT] was changed from second order to first order. This condition was a function ot [H+ I and [CJ-]. At low [CI-J, the change of order was ob'>erved at a Mechani~m of oxidative decarboxylation of amino acids is of considerable interest 1-4. It is one of the important biochemical processes and hence a potential area for inten- sive investigation. Although some work on the effect of chloride on the kinetics of oxidation of some amino acids by chloramine-T has been reported, there are some incon- sistencies and the work is incomplete2• The effect as func- tion of [H+] and [CJ-] has not been systematically investigated. Hence it was thought worthwhile to complete the work aimed at deducing a comprehensive mechanism for the oxidation of amino acids in the presence of chlonde and to establish the role of chloride in positive chlorine oxidations. '• 7,; • ~ .! 30 .. 0 0 zo ,. 10 [HCIOJ hnot CII...-1J r---------------~·~·----~~----~·~0 ____ __, •. 10 zo •• .0 •• tQ• ('HCU4] (Mol tlm·'J Fig. 1. (1) Plor~ of kob, 1v~ [HCI04] and (u) plot\ ol A'.,~, v' {HC1041. rcmp 303 K. Role of chloride in the oxidative decarboxylation of amino acids by chloramine-T 10 {CAnu = 50[AA]u = 20{CI j = 111 I= I 0 mol dm 1. l ti l hi h [H+J h1l t h1 h [CJ ] th11> ditiO 1> J. Indian Chern. Soc., Vol. 77, September 2000, pp. 413-420 J. Indian Chern. Soc., Vol. 77, September 2000, pp. 413-420 Results and Discussion The kinetic data on the oxidations of alanine (Ala), valine (Val), leucine (Leu), serine (Ser), glutamine (Gin) and glutamic acid (Giu) by chloramme-T (CAT) a~ func- tions of [H+] and [CJ-] are shown in Tables 1-6 and Figs. 1-4. The rates-fH+] plot!> showed nearly bell-shaped profile!> even in the presence of added chloride (Fig. 1). The rate dependence in [CAT] was changed from !>econd order to tirst order as [H+] and fCJ-] were vaned. The kinetic~ we1e therefore determined as functions ot [H+J and {CI-]. The rates were alo;o measured at different tempera- tures as functions ot LAA] and the corresponding activation parameters computed a~ described under discu~sion. The kinetics at very low [H+] (<0.05 mol dm-3) were compli- cated and the rate showed second order kinetJcs m [CAT], fractional order in [AA] and inver!>e dependence in [H+J. Fig. 1. (1) Plor~ of kob, 1v~ [HCI04] and (u) plot\ ol A'.,~, v' {HC1041. rcmp 303 K. 10 {CAnu = 50[AA]u = 20{CI j = 111 I= I 0 mol dm 1. relatively high [H+J, wh1le at h1gh [CJ-] th11> conditiOn wa1> achieved even at a lower [H+J (Table 6). The change ot rate-dependence may be due to the change m reactrve tm m of the oxidant. The rates showed posJtrve dependence m [H+] in the narrow range of 0 05-0.20 mol dm-1 The rate exhibited shght dependence 1n fAA). The reaction\ beyond 413 eudo-first and second order rate constants for the oxidative decarboxylation of amino acids (AA) by chloramme- At 10~ [Cl-](mol dm-3), (I) 10' k:,·<s-1);. (2) 10 k•b• (dm3 mol-1 s Ala Val ---------- ---------- 5.0 20.0 1.0 5.0 20.0 1.0 (1) (2) (l) (2) (l) (2) (!) (2) (1) (2) (1) (2) 9.7 16.5 60 60 14.7 - 4.5 - 8.3 26.3 - 6.9 - 12.9 93.9 - 18 . .1 - - 26.6 8.3 18.4 53.3 7.4 10.7 36.7 60 - 133.3 - 25.6 - 40.0 99.9 - 30.6 75.9 - 133.3 - 19.9 - 39.7 - 111.1 - 11.5 28.4 - 70.4 - 6.3 11.1 - 44.0 - 3.0 6.4 - 15.9 - 2.2 - 7.7 6.5 - 10.9 - 2.8 - 8.5 5.4 4.3 2.8 7.0 - 3.1 - 5.4 - 3.3 - 4.5 3.0 - 2.3 - 3.3 AJ. = 3.33, I= 1.0 mol dm-3• 60.1 - 133.3 - 67.6 - Ill.! Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T Ar~Q,NCl +II+ ta~t J..s ~ 2 HOCI + 2 Ar \O>NII HOCI + S A1S02NH + H+ ~ ~ Produtl\ Ar'iOoNHo Patlz 3 K6 ----=- lntermcdt.tlL (YJ - ArSO~NIICI + CI- ra,l /..7 Produu~ ~low Path 4 Ks Y' /..9 Y' H20 ~ HOCI A SO Nih 11 (I F1R 2 F1R 2 Plot' ot A,b, v' [AA] (A) 101[CAT], = '\11 [HC104l = 111 I = I 0 mol dm 1 10' fCI 1 (mol dm 1) = 1 0 (1) ~ 0 (11) and (B) 101 (CAT], = 2[HCI04] = 111 1 = 1 0 mol dm \ lO'[Cl ] (mol dm 1) = ~ 0 (1) 20 0 (n) F1R 2 Plot' ot A,b, v' [AA] (A) 101[CAT], = '\11 [HC104l = 111 I = I 0 mol dm 1 10' fCI 1 (mol dm 1) = 1 0 (1) ~ 0 (11) and (B) 101 (CAT], = 2[HCI04] = 111 1 = 1 0 mol dm \ lO'[Cl ] (mol dm 1) = ~ 0 (1) 20 0 (n) t/(H+] ldM11 MOl 1) ,, 20 ,, 101 [cl"l C'"OI 4..rJ) •• 8 15• !I 10 IS 2.0 1/[M'J{dm'-1') - . .. 101 [el"l C'"ot dm 1 ) ---.. 1<1~ 3 (J) Ploh of ~ •b' v' 1/[H+] and (II} plot' of A b' \~ [Cl ] (1) 101 [CA fl.,= ~0 [AA] = 20 [CI 1 = 1 0 (mol dm 1) 1nd (11) 101 [CATI = ~0 IAA] = l 0 (mol dm-1) 10' [HCl04] (mol dm 1) = 'i 0 (A) 'iOO (B) t/(H+] ldM11 MOl 1) ,, 20 ,, !I 10 IS 2.0 1/[M'J{dm'-1') - 101 [cl"l C'"OI 4..rJ) •• 8 15• . .. 101 [el"l C'"ot dm 1 ) ---.. 101 [cl"l C'"OI 4..rJ) •• 8 15• . .. 101 [el"l C'"ot dm 1 ) ---.. Path 4 Path 4 Ks Y' 1<1~ 3 (J) Ploh of ~ •b' v' 1/[H+] and (II} plot' of A b' \~ [Cl ] (1) 101 [CA fl.,= ~0 [AA] = 20 [CI 1 = 1 0 (mol dm 1) 1nd (11) 101 [CATI = ~0 IAA] = l 0 (mol dm-1) 10' [HCl04] (mol dm 1) = 'i 0 (A) 'iOO (B) /..9 Y'+H20 ~ HOCI + ArSOoNih+ 11+ +(I I-IOCI+S • z .. - .6$+ UK'"' mot-') -·· ... ""140 .. ,., " .. . .. ~ 10 I -~ ·- •• r .. Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T --. , .. • •• i ! - ,rJ £AA] C molfiM 11 , • c; . 6 ~ ••• L• iL "• a .. ~ . .. • • • 101[AA] hool dlllf"1) ---.... . !! • ... . .. ... 101(AA] (mol'~~"'') - F1R 2 Plot' ot A,b, v' [AA] (A) 101[CAT], = '\11 [HC104l = 111 I = I 0 mol dm 1 10' fCI 1 (mol dm 1) = 1 0 (1) ~ 0 (11) and (B) 101 (CAT], = 2[HCI04] = 111 1 = 1 0 mol dm \ lO'[Cl ] (mol dm 1) = ~ 0 (1) 20 0 (n) --. , .. • •• i ! - ,rJ £AA] C molfiM 11 , • • L• "• . .. • • • 101[AA] hool dlllf"1) ---.... Mecluumms of OJI.tdatwn The ob.,el\ ed kmet1c..o, tOJ the oxidation ot ammo ac..Jd., m the p1 e'ienc..e ot c..hlo11de 111 the entne acid 1 • .mge may be expl.uned b) the wmptehen s1ve mecham!.m 'hown 111 Sc..heme I c; . 6 ~ • iL a .. ~ . !! • ... . .. ... 101(AA] (mol'~~"'') - Path 1 ~ ~ + H+ last ~ lntu mcdt.tlL (X) ~-- td't /..1 -- Produll~ 'low X+ ArS02NHCI Path 2 K-! Results and Discussion - 43.9 - 85.7 20.2 35.8 69.2 17.4 - 1.9 11.9 - 3.1 7.3 5.0 3.5 4.4 5.1 3.6 23.8 9.5 6.0 3.2 ( 1) 8.3 16.7 3.5 4.9 6.8 11.3 7.0 4.6 Leu 5.0 (2) 44.4 46.3 42.9 34.1 11.6 6.4 20.0 (1) 10.4 20.5 23.0 18.6 14.7 9.4 5.8 6 7 74 48 41 29 Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T Pseudo-first order rate constants for the oxidative decarboxylation of amino acids by chloramine-Tin the presence of chloride at high I HCIO~l as [CJ] varied at 303 K* 102 [Cil mol dm-3 2.0 1.0 3.2 2.0 3.0 5.0 10.0 20.0 5.0 6.0 7.3 9.1 10.9 Ala 5.0 2.9 4.2 4.5 4.7 5.0 5.4 2.0 2.8 8.5 11.9 *[CAn,,= 50[AAJ., = 1.0 mol dm-3 Val 5.0 3.1 3.5 4.1 4.2 5.1 5.2 104 k'obs(s-1) at 10 [HCI04] (mol dm-3) 2.0 3.4 6.6 9.0 11.3 16.6 18.6 Leu 5.0 5.6 6.3 7.0 7.1 7.7 9.4 2.0 2.7 3.8 4.6 5.2 6.0 7.7 Ser 5.0 2.8 2.9 3.0 3.1 3.4 4.8 2.0 2.9 5.3 6.8 9.0 11.3 Gin 5.0 2.1 2.3 2.4 2.7 3.0 2.0 3.4 5.3 7.1 9.2 12.8 Glu 5.0 4.6 4.9 5.3 5.6 6.1 Table 4. Pseudo-second order rate constants (kub•) for the oxidative decarboxylation of amino acids by chloramine-Tin the presence of chloride in aqueous medium at low [HCI04) = 0.03 mol dm-3 104 k •b• (dm3 mol-1 s-1) at 102 [Cil (mol dm-3) Table 3. Pseudo-first order rate constants for the oxidative decarboxylation of amino acids by chloramine-Tin the presence of chloride at high I HCIO~l as [CJ] varied at 303 K* Table 4. Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T [H+J [H+f fCAT] or kob• = K2k3[S] + Khs£H20]2 + K6k7[Cn' asfS] = K1[SH+l (4) [H+]2 [CAT] [H+] (3) k' k"rcn or kobs = k[SJ + -- + -- (5) [H+]2 [CAT] where, k = K2 k3, k' = Iq k5[H20]2 and k" = K6k7 (5) where, k = K2 k3, k' = Iq k5[H20]2 and k" = K6k7 The plots of kobs vs [AA] (Fig. 2) and k0 h, vs !CI-1 (Fig. 3) are linear in accordance with the rate law (5}. Slight decrease of the pseudo-second order rate constants. in this acid range, with increase in [CAT] is also in conformity with the rate law. The values of k for all the amino acids at different temperatures were calculated from the plots of Table 3. Pseudo-first order rate constants for the oxidative decarboxylation of amino acids by chloramine-Tin the presence of chloride at high I HCIO~l as [CJ] varied at 303 K* 102 [Cil mol dm-3 2.0 1.0 3.2 2.0 3.0 5.0 10.0 20.0 5.0 6.0 7.3 9.1 10.9 Ala 5.0 2.9 4.2 4.5 4.7 5.0 5.4 2.0 2.8 8.5 11.9 *[CAn,,= 50[AAJ., = 1.0 mol dm-3 Val 5.0 3.1 3.5 4.1 4.2 5.1 5.2 104 k'obs(s-1) at 10 [HCI04] (mol dm-3) 2.0 3.4 6.6 9.0 11.3 16.6 18.6 Leu 5.0 5.6 6.3 7.0 7.1 7.7 9.4 2.0 2.7 3.8 4.6 5.2 6.0 7.7 Ser 5.0 2.8 2.9 3.0 3.1 3.4 4.8 2.0 2.9 5.3 6.8 9.0 11.3 Gin 5.0 2.1 2.3 2.4 2.7 3.0 2.0 3.4 5.3 7.1 9.2 12.8 Glu 5.0 4.6 4.9 5.3 5.6 6.1 Table 4. Pseudo-second order rate constants (kub•) for the oxidative decarboxylation of amino acids by chloramine-Tin the presence of chloride in aqueous medium at low [HCI04) = 0.03 mol dm-3 103[CAT] 102[AA),, mol dm-3 mol dm-3 1.0 Effect of varying [CAT]., : 0.3 2.0 12.8 0.5 1.0 2.0 2.0 12.3 11.1 Ala 5.0 20.0 41.2 40.3 40.2 69.1 68.8 70.1 104 k.•b• (dm3 mol-1 s-1) at 102 [Cil (mol dm-3) Val 1.0 5.0 20.0 11.4 45.3 44.8 11.5 43.5 85.7 83.4 85 7 1.0 7.1 6.8 6 0 Leu 5.0 11.6 11 6 20.0 35.7 38.7 41 9 Ser 1.0 20 0 3.5 4.1 4 4 82.7 58.3 40 7 Gin 5.0 17.8 15.9 14 2 Table 3. Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T [H+J [H+f fCAT] or kob• = K2k3[S] + Khs£H20]2 + K6k7[Cn' asfS] = K1[SH+l (4) [H+]2 [CAT] [H+] k' k"rcn or kobs = k[SJ + -- + -- (5) [H+]2 [CAT] where, k = K2 k3, k' = Iq k5[H20]2 and k" = K6k7 The plots of kobs vs [AA] (Fig. 2) and k0 h, vs !CI-1 (Fig. 3) are linear in accordance with the rate law (5}. Slight decrease of the pseudo-second order rate constants. in this acid range, with increase in [CAT] is also in conformity with the rate law. The values of k for all the amino acids at K1 K2 k3[SH+] Ka ks[HzOf K6 k7[Cil fH+] + K1 K2[SH+] + [H+]2 + li+K6[Cil)ICAT] (2) different temperatures were calculated from the plots of Table 3. Pseudo-first order rate constants for the oxidative decarboxylation of amino acids by chloramine-Tin the presence of chloride at high I HCIO~l as [CJ] varied at 303 K* 104 k'obs(s-1) at 10 [HCI04] (mol dm-3) At low [H+], if K1, K 2 and K6 are smaller, then the rate law (2) becomes At low [H+], if K1, K 2 and K6 are smaller, then the rate law (2) becomes Table 2. Pseudo-second order rate constants for the oxidative decarboxylation of amino acids by chloramine-Tin the presence of chloride at low [HCI04] as [Cil varied at 303 K* to2 rcn mol dm-3 1.0 2.0 3.0 5.0 10.0 20.0 Ala 13.6 20.8 27.2 39.7 57.3 111.1 10 k,.,b, (dm3 mol-l s- 1) Val Leu 6.3 9.5 43.9 85.7 10.1 10.4 11.6 12.9 48.7 Ser 10.7 14.5 20.9 Gin 7.8 9.3 10.5 26.9 14.0 44.4 22.0 54.8 38.0 *103 [CAT]o =50 [AA]., = 33.3 [HC104] = 3.33. l = 1.0 mol dm-3 If we take -_I_ d[CAT] "'d ji/[CAT]j "'kob• [CAT]2 dt dt then the rate law (I) takes the form, kob• = Glu 11.9 19.7 25.6 30.6 46.4 K1 K2 k3[SH+] Ka ks[HzOf K6 k7[Cil fH+] + K1 K2[SH+] + [H+]2 + li+K6[Cil)ICAT] (2) Table 3. Pseudo-first order rate constants for the oxidative decarboxylation o [CJ] varie Table 2. Pseudo-second order rate constants for the oxidative decarboxylation of amino acids by chloramine-Tin the presence of chloride at low [HCI04] as [Cil varied at 303 K* K1 Kz k3[SH+] K~ ks[HzOf K6 k7fCn kobs = + + (3) . Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T •• . z .. o eo 110 10 0 as• .,., .. , ... o, .. ,, 6s,•CJK ... II'IIDI ') l·1~ 4 Plot' of Mr \' tJ.\"' .. .. •• • . • •• . ]. •• Path 5 (ArS02NHCit + CI- !..JO - Y" ~low Y" + S -r.w- Proc..luct~ Pwho Kr '>II S + H+ ..... ta't (A1'iO '\II,Cil+ + S "II PlOd lil-t\ -\low <,t.hlnu. I Path 5 Path 5 Path 5 (ArS02NHCit + CI- !..JO - Y" ~low Y" + S -r.w- Proc..luct~ Pwho Kr '>II S + H+ ..... ta't (A1'iO '\II,Cil+ + S "II PlOd lil-t\ -\low <,t.hlnu. I Pwho '>II l·1~ 4 Plot' of Mr \' tJ.\"' The wmbmed 1ate law tm p.tthw,ty' 1-1 IM" hcen deduc..ed, fHCI04 ] > 0 20 mol dm-1 .tl<.o <,howed tno;t mue1 kmet1cs m [CAT], but exh1b1ted h1ghe1 frJ.ct1onal 01de1 m fAA) and mve1 se f1 ac..tJOnal dependence m [H+l The 1 e.tLIJOn'i unde1 the'>e c..ond1t10n~ m.ty go th10ugh competitive p.tthways 4 .tnd 5 m Sc..heme I d[CAT] K1 K2k3[CAT]2 [SH+] K~/..~[CATI~I~H>f - ---= + --'---''---~~ dt [H+] +Kt K' [Sl-1+1 JH+I' K6 k7[CATJLCI I + ( 4 J. Indian Chern. Soc., Vol. 77, September 2000 J. Indian Chern. Soc., Vol. 77, September 2000 J. Indian Chern. Soc., Vol. 77, September 2000 Table 2. Pseudo-second order rate constants for the oxidative decarboxylation of amino acids by chloramine-Tin the presence of chloride at low [HCI04] as [Cil varied at 303 K* to2 rcn mol dm-3 1.0 2.0 3.0 5.0 10.0 20.0 Ala 13.6 20.8 27.2 39.7 57.3 111.1 10 k,.,b, (dm3 mol-l s- 1) Val Leu 6.3 9.5 43.9 85.7 10.1 10.4 11.6 12.9 48.7 Ser 10.7 14.5 20.9 Gin 7.8 9.3 10.5 26.9 14.0 44.4 22.0 54.8 38.0 *103 [CAT]o =50 [AA]., = 33.3 [HC104] = 3.33. l = 1.0 mol dm-3 If we take -_I_ d[CAT] "'d ji/[CAT]j "'kob• [CAT]2 dt dt then the rate law (I) takes the form, kob• = Glu 11.9 19.7 25.6 30.6 46.4 At low [H+], if K1, K 2 and K6 are smaller, then the rate law (2) becomes K1 Kz k3[SH+] K~ ks[HzOf K6 k7fCn kobs = + + (3) . Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T 9 = !..9[HzOJ I+Ks[H+] [Cn (7) I I I I or----- +- k' ob, - Ks!..' 9 [H+] [Cn k' 9 (8) ac1d range, - d[CAT] = 1..10 [CATIICT] dt 01 kob~ = t..Jo[CI-] + Kik 01 kob, = k10[CI-] + kJJ[S Two sets ot constants k10 an plots ot k0 b vs lAA] and k0 d[CAT] Ks k9[ArS02NHCI] [H+] [Cq [H20] - _d_t_ = I + Ks[H+j [Cn (6) , Ksk'9 [H+) [Cn , or k ob' = , where!.. 9 = !..9[HzOJ I+Ks[H+] [Cn (7) I I I I or----- +- k' ob, - Ks!..' 9 [H+] [Cn k' 9 (8) ac1d range, - d[CAT] = 1..10 [CATIICT] + KJ!..ll [CAT)[SH+] (9) dt [H+] 01 kob~ = t..Jo[CI-] + Kikll [SH+J[H+l ( 10) 01 kob, = k10[CI-] + kJJ[S] (II) Two sets ot constants k10 and k11 we1e cakulated hom the plots ot k0 b, vs lAA] and k0 b, vs [CI 1 (Fig'> 2 ~) Fmthe1 d[CAT] Ks k9[ArS02NHCI] [H+] [Cq [H20] - _d_t_ = I + Ks[H+j [Cn (6) - d[CAT] = 1..10 [CATIICT] + KJ!..ll [CAT)[SH+] (9) dt [H+] 01 kob~ = t..Jo[CI-] + Kikll [SH+J[H+l ( 10) 01 kob, = k10[CI-] + kJJ[S] (II) (9) , Ksk'9 [H+) [Cn , or k ob' = , where!.. 9 = !..9[HzOJ I+Ks[H+] [Cn (7) I I I I ( 10) (II) The plot'> of 1/k'oo.. v~ 1/[CJ-) and 1/k'rb. vs 1/[W] are hnear consJ'>tent w1th 1ate law (8) (F1g 3) The obse1 ved plots ot k0 b, vs lAA] and k0 b, vs [CI 1 (Fig> 2 ~) Fmthe1 values of k10 and k11 «t ddte1ent tempelJ.tme' we1e c.,aku lated by varymg [AA] at each tempe1atme The WI- The plot'> of 1/k'oo.. v~ 1/[CJ-) and 1/k'rb. vs 1/[W] are hnear consJ'>tent w1th 1ate law (8) (F1g 3) The obse1 ved The plot'> of 1/k'oo.. v~ 1/[CJ-) and 1/k'rb. Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T vs 1/[W] are hnear consJ'>tent w1th 1ate law (8) (F1g 3) The obse1 ved plots ot k0 b, vs lAA] and k0 b, vs [CI 1 (Fig 2 ~) Fmthe1 values of k10 and k11 «t ddte1ent tempelJ.tme' we1e c.,aku lated by varymg [AA] at each tempe1atme The WI- Table 5 Pwudo fn\1 oulu 1 11~ LOII\I,mt\ fm the oxtdallve decmboxylatwn of ammo actd~ by chlmamme T m the p1e~ence of chlo11de .ttlugh [HCI04] = 0 ~ mol dm 1 [CAT]u [AA]., 101 102 mol dm 1 mol dm 1 Aid 10 Effel.l of vm ymg I CAT )u ~ 0 20 () 010 O'iO I 0 20 10 20 20 20 20 20 29 16 29 16 2 9 17 28 18 2 9 16 n 'i4 'i4 n 'i1 Effel.t of vmymg [AA], I 0 I 0 I 0 I 0 10 0~ 10 20 10 'iO 21 18 16 24 2'i 4'i 29 16 54 11 41 6'i 16 66 89 10 16 16 1 I 11 12 21 28 11 36 4~ Val ~0 11 16 18 1 ~ 12 2 I 29 18 42 'i9 200 'i9 'i8 'i6 'i1 46 31 40 56 61 7'\ 10 ~ ~ n 5 I 5~ 'i6 32 42 5 I 52 60 Leu 'iO 200 8 0 9 6 6 9 9 'i 7 I 9 4 66 95 68 94 40 42 48 63 70 94 96 II 7 12 6 16 8 St.t 10 'iO 2'i 41 24 3'i 26 10 29 24 2 7 16 20 2 I 2 5 26 30 10 3 7 l'i 48 20 0 41 4'i 48 47 2'i 12 48 'i9 74 Gin ~0 2~ 26 28 2 I 22 I 9 21 27 12 19 Glu ~0 77 6~ 'i 'i 'iO ~ I 27 n 70 I dhll 6 l<.mLtiL d 11.1 lut lhL o\ld.tll\o.. Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T dt.L.ttboxylatwn of ammo actd~ by chlmamme T m the pre~ence of chloude at 101 K I dhll 6 l<.mLtiL d 11.1 lut lhL o\ld.tll\o.. dt.L.ttboxylatwn of ammo actd~ by chlmamme T m the pre~ence of chloude at 101 K At 102 [Cl ] (mol dm 1) Otde!'l [H+] ob~ervcd mol dm ' 111 I 0 [CAT] 0 01-005 2 0 OO'i-020 020-1 0 I 0 [AA] 0 01-0 O'i 0 2 00~-020 020-1 0 01 [H+J 001-00'i -07 Al.t 'iO 200 20 20 10 I 0 I 0 04 04 0 O'i 07 --1 I -095 I 0 20 I 0 02 02 --10 Y.tl 50 20 10 02 04 --10 200 20 10 10 04 0 04 -06 I 0 20 I 0 +ve 01 --12 Leu 50 200 20 I 0 I 0 07 02 04 --1 I 20 I 0 0 06 -0~ Se1 I 0 'iO 2 0 1--2 10 I 0 I 0 04 01 03 01 -09 --10 Gin (.(u 20 0 'i () ~ () 1-2 10 06 O'i -O'i 20 10 10 O'i 01 01 -1 I 20 10 10 02 0~ O'i -1 I I dhll 6 l<.mLtiL d 11.1 lut lhL o\ld.tll\o.. dt.L.ttboxylatwn of ammo actd~ by chlmamme T m the pre~ence of chloude at 101 K At 102 [Cl ] (mol dm 1) 'l [H+] cd mol dm ' I 0 Al.t 'iO 200 I 0 Y.tl 50 200 I 0 Leu 50 200 Se1 I 0 'iO Gin (.(u 20 0 'i () ~ () I dhll 6 l<.mLtiL d 11.1 lut lhL o\ld.tll\o.. Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T dt.L.ttboxylatwn of ammo actd~ by chlmamme T m the pre~ence of chloude at 101 K At 102 [Cl ] (mol dm 1) Otde!'l [H+] ob~ervcd mol dm ' 111 I 0 [CAT] 0 01-005 2 0 OO'i-020 020-1 0 I 0 [AA] 0 01-0 O'i 0 2 00~-020 Al.t 'iO 200 20 20 10 I 0 I 0 04 04 0 I 0 20 I 0 02 Y.tl 50 20 10 02 200 20 10 10 04 0 I 0 20 I 0 +ve Leu 50 200 20 I 0 I 0 07 02 20 I 0 0 Se1 I 0 'iO 2 0 1--2 10 I 0 I 0 04 01 Gin (.(u 20 0 'i () ~ () 1-2 10 06 20 10 10 O'i 01 20 10 10 02 0~ Table 5 Pwudo fn\1 oulu 1 11~ LOII\I,mt\ fm the oxtdallve decmboxylatwn of ammo actd~ by chlmamme T m the p1e~ence of chlo11de .ttlugh [HCI04] = 0 ~ mol dm 1 [CAT]u [AA]., 101 102 mol dm 1 mol dm 1 Aid 10 Effel.l of vm ymg I CAT )u ~ 0 20 () 010 O'iO I 0 20 10 20 20 20 20 20 29 16 29 16 2 9 17 28 18 2 9 16 n 'i4 'i4 n 'i1 Effel.t of vmymg [AA], I 0 I 0 I 0 I 0 10 0~ 10 20 10 'iO 21 18 16 24 2'i 4'i 29 16 54 11 41 6'i 16 66 89 10 16 16 1 I 11 12 21 28 11 36 4~ Val ~0 11 16 18 1 ~ 12 2 I 29 18 42 'i9 200 'i9 'i8 'i6 'i1 46 31 40 56 61 7'\ 10 ~ ~ n 5 I 5~ 'i6 32 42 5 I 52 60 Leu 'iO 200 8 0 9 6 6 9 9 'i 7 I 9 4 66 95 68 94 40 42 48 63 70 94 96 II 7 12 6 16 8 St.t 10 'iO 2'i 41 24 3'i 26 10 29 24 2 7 16 20 2 I 2 5 26 30 10 3 7 l'i 48 20 0 41 4'i 48 47 2'i 12 48 'i9 74 Gin ~0 2~ 26 28 2 I 22 I 9 21 27 12 19 Glu ~0 77 6~ 'i 'i 'iO ~ I 27 n 70 I dhll 6 l<.mLtiL d 11.1 lut lhL o\ld.tll\o.. Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T Pseudo-second order rate constants (kub•) for the oxidative decarboxylation of amino acids by chloramine-Tin the presence of chloride in aqueous medium at low [HCI04) = 0.03 mol dm-3 103[CAT] 102[AA),, mol dm-3 mol dm-3 1.0 Effect of varying [CAT]., : 0.3 2.0 12.8 0.5 1.0 2.0 3.0 2.0 2.0 2.0 2.0 12.3 11.1 10.7 10.3 Effect of varying [AA1, : 1.0 0.5 1.0 1.0 1.0 1.0 416 1.0 10.1 2.0 11.1 3.0 .• 12.7 5.0 18.3 Ala 5.0 20.0 41.2 40.3 40.2 40.7 40.0 26.0 33.3 40.0 51.1 59.4 69.1 68.8 70.1 70.0 72.3 28.3 49.2 70.4 103.6 113.9 104 k.•b• (dm3 mol-1 s-1) at 102 [Cil (mol dm-3) Val 1.0 5.0 20.0 11.4 45.3 44.8 11.5 43.5 9.4 42.3 8.3 40.4 8.3 33.3 10.2 39.2 11.5 43.8 12.1 46.0 14.2 85.7 83.4 85.7 87.1 90.2 55.6 72.4 85.6 99.9 114.3 1.0 7.1 6.8 6.0 5.9 5.6 3.2 3.7 6.0 7.4 13.7 Leu 5.0 11.6 11.6 9.8 9.4 7.5 11.6 15.3 23.3 20.0 35.7 38.7 41.9 42.1 42.4 41.9 40.9 41.9 42.1 41.5 Ser 1.0 20 0 3.5 4.1 4.4 3.9 3.2 3.8 4.4 7.4 11.5 82.7 58.3 40.7 32.7 21.4 18.7 26.9 40.7 47.6 74.9 Gin 5.0 17.8 15.9 14.2 12.8 12.8 11.9 10.7 14.4 17.1 25.4 Table 4. Pseudo-second order rate constants (kub•) for the oxidative decarboxylation of amino acids by chloramine-Tin the presence of chloride in aqueous medium at low [HCI04) = 0.03 mol dm-3 104 k.•b• (dm3 mol-1 s-1) at 102 [Cil (mol dm-3) 416 kmet1cs m th1s ac1d 1ange can be explumed by the compct1 tive pathway!. 5 and 6 111 the c.,omp1ehen!.IVe mech«m"m Under the!.e condition' the oxidant mo&tly exl'>l'> "' (ArS02NH2CI)+ k0b, vs fAAl (F1g 2 and Table 7) The conespondmg dC- tlvatiOn parameters to1 all the ammo ac1ds have also been computed (Table 7) The 1eact10ns m the ac1d 1ange 001-005 mol dm-' may mo&tly go through ~tep'i o;hown m path 4 ot Scheme I The related lute law I'i g1ven by The combmed r«te law" (9-11) deduc.,ed bao;ed on p<.~th.., 5 and 6 (Scheme I) .1cwunt tm the ob<,el ved •esult'> m thl'> ac1d range, d[CAT] Ks k9[ArS02NHCI] [H+] [Cq [H20] - _d_t_ = I + Ks[H+j [Cn (6) , Ksk'9 [H+) [Cn , or k ob' = , where!.. Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T The following typical equations are found to be valid (tigure not shown), 1< log k = -0.23 o - 2.60 log k 11 = -0.19 cr·- 1.95 log k = -0.19 Es- 2.70 log k 11 = -0.42 E, - 1.58 ( 12) ( 13) (14) ( 15) The free energies of activation in the low and high [HCI04} and [CI-1 ranges are almost constant (Tables 7, 8) signalling operation of similar mechanisms in the respec- tive ranges. Further. the plots of I).H"' vs I).S"' are linear (Fig. 4). A typical detailed mechanism of oxidation of amino acids by chloramine-T corresponding to the formation of nitrites and aldehydes as products is shown in Scheme 2 1 -~. Conclusions : This study establishes the signiticant role of chloride on the rates and mechanism of CAT oxidations. The chloride effect is more pronounced at high [H+J. The rate-dependence in [CAT! changes from second order to first order as the [H+] and [CJ-1 are increased. This change of order in [CAT] could be achieved at a lower rH+] (0.05- 0.20 mol dm-3), if the [CI-] is about 0.20 mol dm-3. or at low [CJ-]. if the [HCIO.d > 0.20 mol dm-3. The changes in kinetics of oxidations are due to the changes in reactive form of the oxidant species in the presence of chloride Leu Ser 1.0 s.o 1:0 1.1 2.0 1.1 2.3 2.3 3.3 2.1 3.4 6.4 34 46.8 46 8 47.4 5.4 5.6 5.5 54.7 -40.2 6.9 -57.3 -102.2 -84.0 72.1 71.2 72.4 H R 0 ..I•, I I ,,. Slow H-!>li£.C-C~ o.:..c1 ---- 1 1~· -ArSO:.NH~H"cr H H \ Cl d. HzN S02Ar HG ,......R N-C H/ lH Cl H,O j HfN+ S01Ar NH:+RCHO Path I Experimental Scheme 2 Gin Glu 5.0 50 1.7 I I 23 3.3 2.1 5.2 2.6 46 2 4.U 5.5 4R 55.5 44 I -51.9 -91.1 71.2 72 .l 1-w N•C -R Path II The purity of chloramine-T (f'Juka. A.G.) was checked by iodometric estimation of the amount of active l"hlorine Tnblc7. Values of kat different temperatures and the corresponding activation parameters 103 k (dm6 mor2 s-1) ot 102 [CI-](Jnol dm-·1) Temp Ala Val K 1.0 5.0 1.0 5.0 29J 1.2 5.4 0.63 2.3 29& 303 14 7.5 1.3 2.3 308 4.8 9.6 2.0 2.5 Activotion parameters calculated fromu E. Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T 74.4 30.6 57.7 58 2 (kJ mol-l) log A 10.2 3.2 9.55 7.3 t>II' 70.9 29.5 66.6 17.4 {kJ mol-1) f'SI -3.5 -130.9 -220.7 -180 7 (J mor' K-1l t>d' 72.0 69.1 73.5 72.2 (kJ rnol- 1) "See text and cqn {6). responding activation parameters have also been computed (Table 8). Leu Ser 1.0 s.o 1:0 1.1 2.0 1.1 2.3 2.3 3.3 2.1 3.4 6.4 34 46.8 46 8 47.4 5.4 5.6 5.5 54.7 -40.2 6.9 -57.3 -102.2 -84.0 72.1 71.2 72.4 Gin Glu 5.0 50 1.7 I I 23 3.3 2.1 5.2 2.6 46 2 4.U 5.5 4R 55.5 44 I -51.9 -91.1 71.2 72 .l responding activation parameters have also been computed (Table 8). responding activation parameters have also been computed (Table 8). Applicability of Taft equation5 for the oxidation of amino acids by chloramine-T in the presence of chloride ion has also been tested. The following typical equations are found to be valid (tigure not shown), 1< log k = -0.23 o - 2.60 log k 11 = -0.19 cr·- 1.95 log k = -0.19 Es- 2.70 log k 11 = -0.42 E, - 1.58 ( 12) ( 13) (14) ( 15) ( 15) The free energies of activation in the low and high [HCI04} and [CI-1 ranges are almost constant (Tables 7, 8) signalling operation of similar mechanisms in the respec- tive ranges. Further. the plots of I).H"' vs I).S"' are linear (Fig. 4). A typical detailed mechanism of oxidation of amino acids by chloramine-T corresponding to the formation of nitrites and aldehydes as products is shown in Scheme 2 1 -~. Conclusions : This study establishes the signiticant role of chloride on the rates and mechanism of CAT oxidations. The chloride effect is more pronounced at high [H+J. The rate-dependence in [CAT! changes from second order to first order as the [H+] and [CJ-1 are increased. This change of order in [CAT] could be achieved at a lower rH+] (0.05- 0.20 mol dm-3), if the [CI-] is about 0.20 mol dm-3. or at low [CJ-]. if the [HCIO.d > 0.20 mol dm-3. The changes in kinetics of oxidations are due to the changes in reactive form of the oxidant species in the presence of chloride under varying [H+j. Path II Scheme 2 Gowda et at Role ot c..hloude 10 the oxidative dec,trboxylatmn ot ammo acid' by c..hloi.tmme T dt.L.ttboxylatwn of ammo actd~ by chlmamme T m the pre~ence of chloude at 101 K At 102 [Cl ] (mol dm 1) Otde!'l [H+] ob~ervcd mol dm ' 111 I 0 [CAT] 0 01-005 2 0 OO'i-020 020-1 0 I 0 [AA] 0 01-0 O'i 0 2 00~-020 020-1 0 01 [H+J 001-00'i -07 0 O'i-0 20 Al.t 'iO 200 20 20 10 I 0 I 0 04 04 0 O'i 07 --1 I -095 I 0 20 I 0 02 02 --10 Y.tl 50 20 10 02 04 --10 020--10 -01 -O'i -07 -07 -O'i 200 20 10 10 04 0 04 -06 -07 I 0 20 I 0 +ve 01 --12 1 I -07 Leu 50 200 20 I 0 I 0 07 02 04 --1 I 08 20 I 0 0 06 -0~ -07 -07 Se1 I 0 'iO 2 0 1--2 10 I 0 I 0 04 01 03 01 -09 --10 I I 0 -06 Gin (.(u 20 0 'i () ~ () 1-2 10 06 O'i -O'i 20 10 10 O'i 01 01 -1 I O'i ...() 6 -I 2 20 10 10 02 0~ O'i -1 I O'i -O'i 417 I dhll 6 l<.mLtiL d 11.1 lut lhL o\ld.tll\o.. dt.L.ttboxylatwn of ammo actd~ by chlmamme T m the pre~ence of chloude at 101 K At 102 [Cl ] (mol dm 1) 417 J. Indian Chern. Soc., Vol. 77, September 2000 J. Indian Chern. Soc., Vol. 77, September 2000 Tnblc7. Values of kat different temperatures and the corresponding activation parameters 103 k (dm6 mor2 s-1) ot 102 [CI-](Jnol dm-·1) Temp Ala Val K 1.0 5.0 1.0 5.0 29J 1.2 5.4 0.63 2.3 29& 303 14 7.5 1.3 2.3 308 4.8 9.6 2.0 2.5 Activotion parameters calculated fromu E. 74.4 30.6 57.7 58 2 (kJ mol-l) log A 10.2 3.2 9.55 7.3 t>II' 70.9 29.5 66.6 17.4 {kJ mol-1) f'SI -3.5 -130.9 -220.7 -180 7 (J mor' K-1l t>d' 72.0 69.1 73.5 72.2 (kJ rnol- 1) "See text and cqn {6). responding activation parameters have also been computed (Table 8). Applicability of Taft equation5 for the oxidation of amino acids by chloramine-T in the presence of chloride ion has also been tested. References I. M. M. Campbell and G. Johnson, Clzem. Rev .. I 97ll. 78. 65. I. M. M. Campbell and G. Johnson, Clzem. Rev .. I 97ll. 78. 65. Kinetic measurements : The kinetic runs were made in glass-stoppered pyrex boiling tubes under pseudo order conditions with fAA] » (CAT} (5-50 times). The reac- tions were initiated by the rapid addition of known amounts of oxidant solution (0.0003-0.003 mol dm-3), pre- equilibrated at a desired temperature, to mixtures contain- ing the required amounts of amino acid (0.005-0.05 mol dm-3), perchloric acid (0.0005-0. I 0 mol dm-3), sodium chloride (0.01-0.20 mol dm-3) and water, in the boiling tube thermostated at the same temperature. Progress of the reactions was monitored for at least two half-lives by the iodometric determination of unreacted oxidant at regular intervals of time. The pseudo-second ordt:r and pseudo-first order rate constants were computed by the graphical methods and the values were reproducible within 3% error. 2. B. T. Gowda and D. S. Mahadevappa, f. Chem. Soc .. Pl'l'km 7i'am. 2, 1983, 323. 2. B. T. Gowda and D. S. Mahadevappa, f. Chem. Soc .. Pl'l'km 7i'am. 2, 1983, 323. 3. G. Gopalakrishnan and J. L. Hogg, ./. Org. Clrem .. 19!'!5, 50, 1206. 3. G. Gopalakrishnan and J. L. Hogg, ./. Org. Clrem .. 19!'!5, 50, 1206. 4. B. T. Gowda and R. V. Rao, Oxidn. Commuu .. 1987. 10. 31; 1988, 11, 149; Indian J. Clzem .. Sect. A, 1986, 25. 908; 1988. 27. 39; J Indian Chem. Soc., 1987,64, 467; 1988, 65, 339; B. T Gowda and B.S. Sherigara, Oxidn. Commwr .. 1986, 9, 165; /ndwn .1. Chem. Sect. A, 1986,25, 960; 1987.26. 960; J. Indian Cflem. Sm . 1987. 64, 158; K. K. Bane•:ii, B. Jayaram and D. S. Mahadevapp:1 . ./. Sc t. Ind. Res., 1987,46, 65; M. C. Agrawal and S. K. Upadhyay . .1. Sn Ind. Re.v., 1990,49, 13; B. T. Gowda and P. J. M. Rao. Bull. Cflem. Soc. Jpn., 1989, 62, 3303; J. Indian Clu•m. Sm .• 1990. 67,426: 1992, 69, 642, 825; B. T. Gowda and P. Ramachandra . .1. Indian Clrem. Soc., 1990, 67, 632; B. T. Gowda and K. Gectha, Oxufn Commun., 1996, 19, 75; A. C. Hegde and B. T. Gowda, Oxulrr. Commun., 1996. 19, 293; S. D. Quine and B. T. Gowda. O.Ardn. Commun., 1998, 21, 106. 5. R. W. Taft, Jr. RCH(NH2)COOH + 2 (ArS02NCJ)-Na+ ~ RCN + 2 ArS02NH2 + C02 + 2 Na+ + 2 CJ- ( 16) where, R = CH3 (Ala), (CH3)2CH (Val), (CH1h CHCH2 (Leu), HOH2C (Ser), NH2COCH2CH2 (Gin), HOOCCH2CH2 (Giu) and Ar = p-CH3C6H-J. 6. A. I. Vogel, "Quantitative Organic Analysis", Longman. London, 1958. Experimental The purity of chloramine-T (f'Juka. A.G.) was checked by iodometric estimation of the amount of active l"hlorine present in it and its aqueous stock solution (0.05 mol dm-') 418 Alo~ 0 50 - --- l" k, k" 14 24 18 4~ 28 100 66 60 161 ter; co~lcu1o~ted !rom' 649 439 117 8 89 59 181 625 1101 71 4 816 85 9 --47 7 87 8 8-10 85 8 (I:!) Table 8 Values of 101 k10 and 10 k11 at d1tferent temp At JO"(U l Vdl 200 I 0 50 200 --- --- k,. l" l, kll kto k" A.,. 035 39 110 099 20 I 6 05 13 123 210 50 36 80 I 3 19 209 300 69 52 152 I 9 859 83 2 85 8 490 491 97 5 6~ 2 , 119 124 111 62 60 14 7 81 85 I 8062 90) 4~ 5 469 999 67 6 -196 154 21 6 -118 8 -136 8 446 -77) - 910 760 1~6 2 884 884 864 91 I J. Indian Chern. Soc., Vol. 77, September 2000 spray reagent. The observed stiochiometries may be repre- sented by spray reagent. The observed stiochiometries may be repre- sented by spray reagent. The observed stiochiometries may be repre- sented by was prepared, standardised and preserved in dark coloured bottles. Chromatographically pure amino acids alanine. valine, leucine, serine, glutamine and glutamic acid (Sisco) were further assayed6. Aqueous stock solutions of the com- pounds (0. I 0 mol dm-3) were used. Sodium chloride was used as a source of chloride ion. Ionic strength of the medium was kept at 0.3 mol dm-3 by employing con- centrated aqueous solutions of sodium nitrate. All other reagents employed were of accepted grades of purity. RCH(NH2)COOH + 2 (ArS02NCJ)-Na+ ~ RCN + 2 ArS02NH2 + C02 + 2 Na+ + 2 CJ- ( 16) References in "Steric Effects m Organic Chemistry". ed M Newman, Wiley, New York. 1956; N. B. Chapman and J Shmtcr. "Correlation Analysis in Chenustry", Plenum, New York. 197!!. Stoichiometry and product analysis : The stoichio- metries of CAT-amino acid oxidations were determined at varying [H+J and [CI-J by equilibrating varying ratios of [CAT] to [AA] at 303 K. The corresponding nitriles were the major products at low [H+J and aldehydes at high [H+]. The nitriles, aldehydes, C02 and NH3 were identified by standard tests7. Toluene-p-sulfonamide, the reduced product of the oxidant was detected by paper chromato- graphy with benzyl alcohol saturated with water as the sol- vent and 0.5% vanillin in I% HCI solution in ether as the 6. A. I. Vogel, "Quantitative Organic Analysis", Longman. London, 1958. 7. S. Soloway and A. Lipschitz, Anal. Clrern .• 1952. 24. 898: "Vogel's Text Book of Quantitative Inorganic Analysis". eds. J. Ba~sett. R C. Denney, G. H. Jeffery and J. Mendham. Longrnans. London, 1978; "Vogel's Text Book of Quantitative lnorg~nic Analysi~". eds. B.S. Furniss, A. J. Hannaford. V. Rogers. P. W. G. Smith and A. R. Tatachell, Longmans, London, 1969. 420
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Investigation of the influence of high glucose on molecular and genetic responses: an in vitro study using a human intestine model
Genes & nutrition
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Tugce Boztepe and Sukru Gulec* Tugce Boztepe and Sukru Gulec* © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Dietary glucose consumption has increased worldwide. Long-term high glucose intake contributes to the development of obesity and type 2 diabetes mellitus (T2DM). Obese people tend to eat glucose-containing foods, which can lead to an addiction to glucose, increased glucose levels in the blood and intestine lumen, and exposure of intestinal enterocytes to high dietary glucose. Recent studies have documented a role for enterocytes in glucose sensing. However, the molecular and genetic relationship between high glucose levels and intestinal enterocytes has not been determined. We aimed to identify relevant target genes and molecular pathways regulated by high glucose in a well-established in vitro epithelial cell culture model of the human intestinal system (Caco-2 cells). Methods: Cells were grown in a medium containing 5.5 and 25 mM glucose in a bicameral culture system for 21 days to mimic the human intestine. Transepithelial electrical resistance was used to control monolayer formation and polarization of the cells. Total RNA was isolated, and genome-wide mRNA expression profiles were determined. Molecular pathways were analyzed using the DAVID bioinformatics program. Gene expression levels were confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Results: Microarray gene expression data demonstrated that 679 genes (297 upregulated, 382 downregulated) were affected by high glucose treatment. Bioinformatics analysis indicated that intracellular protein export (p = 0.0069) and ubiquitin-mediated proteolysis (p = 0.024) pathways were induced, whereas glycolysis/gluconeogenesis (p < 0.0001), pentose phosphate (p = 0.0043), and fructose-mannose metabolism (p = 0.013) pathways were downregulated, in response to high glucose. Microarray analysis of gene expression showed that high glucose significantly induced mRNA expression levels of thioredoxin-interacting protein (TXNIP, p = 0.0001) and lipocalin 15 (LCN15, p = 0.0016) and reduced those of ATP-binding cassette, sub-family A member 1 (ABCA1, p = 0.0004), and iroquois homeobox 3 (IRX3, p = 0.0001). Conclusions: To our knowledge, this is the first investigation of high glucose-regulated molecular responses in an intestinal enterocyte model. Our findings identify new target genes that may be important in the intestinal glucose absorption and metabolism during high glucose consumption. Keywords: Obesity, Intestine, High glucose consumption, Caco-2, ABCA1, IRX3, TXNIP, LCN15 Investigation of the influence of high glucose on molecular and genetic responses: an in vitro study using a human intestine model Tugce Boztepe and Sukru Gulec* * Correspondence: sukrugulec@iyte.edu.tr * Correspondence: sukrugulec@iyte.edu.tr Correspondence: sukrugulec@iyte.edu.tr Molecular Nutrition and Human Physiology Laboratory, Food Engineering Department, İzmir Institute of Technology, Room: 215, Urla, 35430 İzmir, Turkey p g @ y Molecular Nutrition and Human Physiology Laboratory, Food Engineering Department, İzmir Institute of Technology, Room: 215, Urla, 35430 İzmir, Turkey Correspondence: sukrugulec@iyte.edu.tr Molecular Nutrition and Human Physiology Laboratory, Food Engineering Department, İzmir Institute of Technology, Room: 215, Urla, 35430 İzmir, Turkey Boztepe and Gulec Genes & Nutrition (2018) 13:11 https://doi.org/10.1186/s12263-018-0602-x Effect of high glucose on cellular tight junctions and measurement of glucose efflux The monolayer structure formation and tight junction permeability of Caco-2 cells are commonly evaluated by measurement of transepithelial electrical resistance (TEER) along with OCLN and ZO1 gene mRNA levels [14, 15]. We grew Caco-2 cells in bicameral cell culture chambers to mimic the human intestine and measured TEER and OCLN and ZO1 mRNA expression levels to evaluate the intestinal barrier function of our experi- mental model. The TEER levels of control and high glucose-treated 21-day postconfluent Caco-2 cell groups were not significantly different (Table 1) and neither were OCLN and ZO1 mRNA levels significantly affected by high glucose treatment (Table 1). These results dem- onstrate that high glucose did not interfere with barrier formation by monolayer Caco-2 cells. The function of enterocytes involves absorption of dietary nutrients (nutrient uptake) and their transport into the blood circulation (nutrient efflux). We tested cellular glucose efflux under control and high glucose treatment conditions. Glucose efflux was significantly increased in the glucose-treated group relative to the controls at the 90-min (p = 0.0031; 1.4-fold), 120-min (p = 0.01; 1.4-fold), and 240-min (p = 0.0049; 1.9-fold) time points (Table 2), suggesting that Caco-2 cells mimic the human intestine in terms of glucose efflux in our experimental model. © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Boztepe and Gulec Genes & Nutrition (2018) 13:11 Page 2 of 8 Background may be important in the control of intestinal glucose absorption during high glucose consumption. g Consumption of high dietary glucose is a risk factor for the development of obesity and type 2 diabetes mellitus (T2DM) in humans [1, 2]. The total number of deaths attributable to high blood glucose in 2012 was estimated as 3.7 million, 1.5 million of which were due to diabetes [3]. High glucose intake leads to postprandial glycemia, insulinemia, and lipidemia in healthy overweight people [4]. Moreover, longer exposure to dietary high glucose is associated with the development of T2DM [5]. These findings indicate that control of dietary glucose intake is an important strategy to combat the development of obesity and T2DM. Recent studies have focused on the mechanisms controlling blood glucose turnover during obesity and T2DM. For example, sodium-dependent glu- cose transporter 1 (SGLT1) is a protein that has been targeted to control reabsorption of glucose from the kid- ney [6]. Moreover, hepatic glucose production from glycogen has been studied as a therapeutic target mo- lecular pathway in the development of T2DM [7]. Glucose-dependent molecular mechanisms contributing to obesity and T2DM have been extensively studied in the adipose tissue, liver, pancreas, and brain. The small intestine may also be an important tissue to investigate glucose metabolism during obesity and T2DM, as it can contribute to systemic glucose production in fasted and diabetic states [8]. Moreover, the gene expression and morphology of the small intestine are altered during obesity [9]. These adaptive changes of the small intestine may be uncontrolled during high nutrient consumption and metabolic disease states. The small intestine mainly consists of enterocytes, which directly interact with diet- ary nutrients and transfer them into the circulatory sys- tem [10]. The rate of interaction between nutrients and enterocytes varies depending on the eating habits of individuals. Quantitative reverse transcription polymerase chain reaction analysis of genes identified by microarray analysis Microarray technology was used to identify genes that were differentially expressed in experimental samples. When we compared the control and high glucose-treated groups, we identified a total of 297 and 382 genes that were signifi- cantly up- and downregulated, respectively (≥1.5-fold change; p < 0.05) in response to high glucose treatment. The list of high glucose-regulated genes is available under GEO accession number GSE97977 ( https://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE97977; Additional file 2: Table S2 and Additional file 3: Table S3). These genes were used to identify high glucose-regulated molecular pathways by DAVID bioinformatics program using KEGG Pathways database. The gene IDs were converted to DA- VID gene IDs that were associated with particular pathway annotation terms. The enrichment of molecular pathways was performed by Benjamini-Hochberg method, and p ≤0. 05 was used as a cutoff value in DAVID annotation model. Intracellular protein export (p = 0.0069) and ubiquitin- mediated proteolysis (p = 0.024) pathways were identified as significantly induced, whereas glycolysis-gluconeogenesis (p < 0.0001), pentose phosphate (p = 0.0043), and fructose- mannose metabolism (p = 0.013) pathways were signifi- cantly downregulated by high glucose treatment in Caco-2 cell monolayers after 21 days (Table 4). The detailed y Genome-wide mRNA array technology provides valuable information about the genomic regulation of cells; how- ever, technical procedures can affect microarray results [16]. Therefore, we performed RT-qPCR experiments to confirm the regulation of individual genes from the identified molecular pathways. In the intracellular protein export pathway, mRNA levels of SRP14 (1.4-fold; p = 0.002) and SRP54 (1.8-fold; p = 0.0003) and in the ubiquitin-mediated proteolysis pathway, mRNA levels of UBE2L6 (2-fold; p = 0.0007), UBE2Q2 (1.6-fold; p = 0.01), and SKP1 (1.5-fold; p = 0.03) were significantly increased in the high glucose-treated group compared with the con- trols (Table 5). However, RT-qPCR analysis of the SRP72 gene in the protein export pathway did not indicate simi- lar regulation to that determined by microarray analysis. We also selected common genes from molecular pathways that were downregulated by high glucose treatment and found that mRNA levels of ALDOA (1.5-fold; p = 0.006), ALDH2 (2.8-fold; p = 0.01), PFKP (2.9-fold; p = 0.0002), PGAM1 (1.7-fold; p = 0.03), PGD (2.1-fold; p = 0.002), and PFKFB3 (4.3-fold; p = 0.003) were significantly reduced in pathways downregulated by high glucose treatment (Table 5). High glucose alters mRNA expression levels of glucose- regulated genes Values are expressed as the mean ± SD; n = 3 independent experiments with three technical replicates Boztepe and Gulec Genes & Nutrition (2018) 13:11 Page 3 of 8 Page 3 of 8 Table 2 Cellular glucose efflux Time (min) Ctrl +Glc p value Significance 0 105.5 ± 4.50 114.5 ± 2.5 0.2225 NS 30 104.5 ± 1.50 121.0 ± 7.0 0.1477 NS 60 103.5 ± 5.50 128.0 ± 2.0 0.0526 NS 90 96.5 ± 2.50 135.5 ± 2.5 0.0031 Yes 120 105.5 ± 4.00 142.5 ± 3.5 0.0195 Yes 240 90.00 ± 5.0 169.5 ± 2.5 0.0049 Yes The glucose efflux was measured from the basolateral surface of the two experimental groups at 0-, 30-, 60-, 120-, 180-, and 240-min time points. Results were given as milligrams per deciliter. Statistical significance was determined by Student’s t test. Values are expressed as the mean ± SD; n = 3 independent experiments with three technical replicates Table 4 Functional analysis of high glucose-regulated up- or downregulated genes Upregulated pathways p value Number of genes Ubiquitin-mediated proteolysis 2.4 × 10−2 7 Protein export 6.9 × 10−3 3 Downregulated pathways Glycolysis/gluconeogenesis 3.0 × 10−5 10 Pentose phosphate 4.3 × 10−3 5 Fructose and mannose metabolism 1.3 × 10−2 5 Microarray data showed a total of 297 upregulated and 382 downregulated genes in response to high glucose treatment. The high glucose-regulated genes were subjected in DAVID bioinformatics analyze program to describe the functional molecular pathways along with the number of genes in each pathway Table 4 Functional analysis of high glucose-regulated up- or downregulated genes The glucose efflux was measured from the basolateral surface of the two experimental groups at 0-, 30-, 60-, 120-, 180-, and 240-min time points. Results were given as milligrams per deciliter. Statistical significance was determined by Student’s t test. Values are expressed as the mean ± SD; n = 3 independent experiments with three technical replicates Microarray data showed a total of 297 upregulated and 382 downregulated genes in response to high glucose treatment. The high glucose-regulated genes were subjected in DAVID bioinformatics analyze program to describe the functional molecular pathways along with the number of genes in each pathway were significantly lower in the high glucose-treated group relative to the controls (Table 3). High glucose alters mRNA expression levels of glucose- regulated genes information for high glucose-regulated pathways can be found in Additional file 4: Figure S2, Additional file 5: Fig- ure S2, Additional file 6: Figure S3, Additional file 7: Figure S4, and Additional file 8: Figure S5. Quantitative reverse transcription polymerase chain reaction analysis of genes identified by microarray analysis Table 3 mRNA expression levels of glucose-regulated genes Genes Ctrl +Glc p value Significance GLUT2 1.004 ± 0.035 0.5465 ± 0.055 0.0001 Yes GLUT5 1.014 ± 0.064 0.6490 ± 0.044 0.0004 Yes SGLT1 1.023 ± 0.083 0.7186 ± 0.040 0.0053 Yes Total RNA was isolated from the control and the high glucose-treated (+Glc) groups. RT-qPCR was performed to analyze relative expression levels of genes from the two experimental groups. Statistical significance was determined by Student’s t test. Values are expressed as the mean ± SD; n = 3 independent experiments with three technical replicates High glucose alters mRNA expression levels of glucose- regulated genes Before genome-wide array analysis was performed, we tested whether high glucose affected the regulation of glucose-related genes in Caco-2 cells. Thus, we analyzed mRNA expression of GLUT2, GLUT5, and SGLT1. mRNA expression levels of GLUT2 (1.8-fold; p = 0.0001), GLUT5 (1.5-fold; p = 0.0004), and SGLT1 (1.4-fold; p = 0.0053) Recent studies suggest that there may be a physiological interaction between enterocytes and glucose that contrib- utes to intestinal dietary glucose sensing [9, 11, 12]. In humans, high dietary glucose consumption elevates glucose levels in the lumen of the intestine and in the blood; hence, enterocytes are exposed to high levels of glucose in both the diet and the blood. This situation can be continuous in obese people, due to the consumption of foods containing high levels of glucose [13]. It is unknown how enterocytes respond to high glucose levels in either the diet or the blood. Hence, investigations to improve the understanding of the adaptive physiological changes of enterocytes in response to high glucose consumption are warranted. In this study, we examined the genome-wide molecu- lar responses of enterocytes to high glucose. Our re- sults demonstrate that chronic high glucose exposure influences specific molecular pathways and genes that Table 1 Effect of high glucose on cell permeability Ctrl +Glc p value Significance TEER (ohm/cm2) 934 ± 32.35 809.5 ± 39.29 0.0707 NS Normalized OCLN mRNA levels 1 ± 0.095 0.93 ± 0.10 0.6096 NS Normalized ZO1 mRNA levels 1 ± 0.064 1.05 ± 0.14 0.7336 NS Caco-2 cells were grown on collagen-coated inserts in 5.5 and 25 mM glucose containing DMEMs for 21 days. TEER value was measured before RNA isolation. Occludin (OCLN) and Zoolin1 (ZO1) mRNA levels were measured by RT-qPCR, and relative fold changes of gene expression levels were analyzed. Statistical significance was determined by Student’s t test. Values are expressed as the mean ± SD; n = 3 independent experiments with three technical replicates Caco-2 cells were grown on collagen-coated inserts in 5.5 and 25 mM glucose containing DMEMs for 21 days. TEER value was measured before RNA isolation. Occludin (OCLN) and Zoolin1 (ZO1) mRNA levels were measured by RT-qPCR, and relative fold changes of gene expression levels were analyzed. Statistical significance was determined by Student’s t test. Investigation of microarray data on glucose-regulated genes We also searched our gene expression array data to find relevant candidate gene(s) that may be important in en- terocyte glucose metabolism under high-glucose condi- tions. High glucose significantly induced levels of transcription of TXNIP (3.2-fold; p = 0.0001) and LCN15 Boztepe and Gulec Genes & Nutrition (2018) 13:11 Page 4 of 8 Page 4 of 8 Table 5 Confirmation of mRNA expression of genes from molecular pathways Genes Ctrl +Glc p value Significance Group1 SRP14 1.008 ± 0.046 1.419 ± 0.102 0.002 Yes SRP54 1.038 ± 0.101 1.804 ± 0.113 0.0003 Yes SRP72 1.095 ± 0.20 1.177 ± 0.101 0.7509 NS Group 2 UBE2L6 1.002 ± 0.151 2.034 ± 0,185 0.0007 Yes UBE2Q2 1.000 ± 0.136 1.578 ± 0.139 0.01 Yes SKP1 1.005 ± 0.167 1.516 ± 0.151 0.03 Yes Group 3 ALDOA 1.000 ± 0.087 0.6652 ± 0.042 0.006 Yes ALDH2 1.000 ± 0.138 0.3593 ± 0.146 0.01 Yes PFKP 1.000 ± 0.088 0.3360 ± 0.074 0.0002 Yes PGAM1 1.000 ± 0,108 0.5777 ± 0.138 0.03 Yes PGD 1.000 ± 0.101 0.4760 ± 0.066 0.002 Yes PFKFB3 1.000 ± 0.179 0.2334 ± 0,082 0.003 Yes The gene expression levels from molecular pathways were analyzed by RT- qPCR method. The relative changes of mRNA expression levels in the protein export (group1) and ubiquitin-mediated proteolysis (group2) pathways were indicated in the table. The mRNA expression of common genes from molecular pathways that were downregulated by high glucose treatment was shown as group 3 in the table. Statistical significance was determined by Student’s t test. Values are expressed as the mean ± SD; n = 3 independent experiments with three technical replicates barrier that was not disrupted by exposure to high glu- cose levels. Initial experiments to evaluate the effects of glucose on known glucose-regulated genes indicated that SGLT1, GLUT5, and GLUT2 were downregulated in response to high glucose exposure. The regulations of SGLT1 and GLUT5 genes in this paper are in contrast with Mahraoui et al. [17]. They reported that the glucose con- sumption of early (p29) or late (p198) passages of Caco-2 cells was different, and the mRNA regulations of SGLT1, GLUT5, and GLUT2 genes were affected by pas- sage number of the cells and basal glucose consumption rate. Investigation of microarray data on glucose-regulated genes Northern blot analysis from the same study indi- cated that 21-day high glucose (25 mM) treatment of Caco-2 cells can significantly induce SGLT1 and GLUT5 mRNA expression levels compared to low glucose- treated groups (1 mM). However, GLUT2 mRNA level was reduced by high glucose treatment, which we observed similar results for GLUT2 mRNA expression. There are different steps of experimental approaches between these two studies. We used polarized Caco-2 cells from 35 to 45 passages and maintained Caco-2 cells under high-glucose condition for longer time periods. Moreover, we compared mRNA expression results from 25 and 5.5 mM glucose treatment groups. It seems pos- sible that these factors might affect the regulation of SGLT1 and GLUT5 genes. It has been shown that SGLT1 and GLUT2 mRNA expression levels were induced when glucose level was increased into the lumen of the intes- tine in in vivo [18, 19]. It has been indicated that intra- cellular and extracellular factors are involved in the regulations of glucose transporters [20, 21]. The different mRNA regulations of SGLT1 and GLUT2 in in vitro and in in vivo might be due to those factors that are not predominantly regulated in in vitro models under high glucose treatment. Despite these differences, we may at least conclude that high glucose influences the genetic responses of Caco-2 cells (6-fold; p = 0.0016) genes whereas mRNA levels of ABCA1 (5.3-fold; p = 0.0004) and IRX3 (13-fold; p = 0.0001) were downregulated in the high glucose treatment group (Table 6). Conclusions An important limitation of this study was the use of the colon carcinoma cell line, Caco-2, as a model of the human intestine. Although Caco-2 is a cancer cell line, it is established in the literature as an in vitro model of the human intestine system, and there are no alternative non-cancerous human cell lines that function similarly. The scope of this study was limited to enterocyte glu- cose metabolism gene regulation during high glucose treatment; therefore, the study findings should be inter- preted with caution. However, the results provide valu- able information regarding the molecular and genetic responses of enterocytes to high glucose levels, which is the main nutritional contributor to the development of obesity and T2DM. Studies of the intestine and obesity have largely focused on intestinal glucose sensing and reduced intestinal glucose absorption through delaying the breakdown of carbohydrates in enterocytes, while the mechanisms by which enterocytes respond to high glucose levels have not been investigated previously. Thus, the identification of candidate genes (for example, TXNIP) involved in the control of intestinal glucose absorption during high glucose consumption is import- ant. Moreover, enterocytes exposed to high glucose may communicate, possibly via LCN15, with endocrine cells in the intestine or peripheral tissues. This screen of glucose-dependent genetic regulation in enterocytes has identified important target genes and will facilitate further investigation of the control of glucose metabol- ism in enterocytes during high glucose intake, which is a common cause of the progression of obesity and T2DM. Discussion However, high glucose reduces ABCA1 mRNA and protein levels in the macrophages, decreasing choles- terol flux from the macrophages to the blood; hence, glucose influences cholesterol metabolism via control of ABCA1 gene expression. Our results also demon- strate that high glucose can reduce ABCA1 mRNA expression levels in intestinal enterocytes, and levels of cholesterol efflux from the cells in our experimen- tal model should be measured to allow definitive con- clusions to be drawn from our results. Surprisingly, IRX mRNA expression was also regulated in Caco-2 cells under high glucose treatment, since IRX3 is highly expressed in the brain and involved in feeding behavior [24]. Single nucleotide polymorphisms in the IRX3 gene are reported as strongly associated with BMI in human subjects [24]. Moreover, IRX3 knock- out mice had body weights approximately 30% less than their wild-type littermates [24], suggesting that the IRX3 gene is closely related to obesity. Glucose- regulated IRX3 mRNA regulation was shown, for the first time in our study; however, the function of intes- tinal IRX3 is unknown, and regulation of IRX3 in the intestine under high glucose consumption requires further investigation. High glucose significantly in- duced mRNA levels of the TXNIP and LCN15 genes. TXNIP plays a role in glucose uptake and is involved in glucose production in the mouse liver [25, 26]. TXNIP-mutant mice become hypertriglyceridemic and hypoglycemic during fasting [27]. These observations suggest that TXNIP may be an important candidate intracellular regulator of enterocyte glucose metabol- ism. There is very limited information about LCN15 in the literature, and its physiological function is unknown; however, it is a member of the lipocalin gene family, of which LCN2 is a well-studied member involved in obesity and diabetes. LCN2, also known as an adipokine, is secreted from the adipose tissue, and its expression is elevated in obese humans and T2DM mouse models [28]. LCN2-null male mice are protected from high fat diet-induced obesity and insu- lin resistance [29]. It was concluded that LCN2 may be an adipose tissue regulator that modulates glucose metabolism in murine tissues and in the 3T3-L1 adi- pocyte cell culture model [30]. These studies clearly suggest that ABCA1, IRX3, TXNIP, and LCN15 may physiologically interact with high glucose in entero- cytes. An important finding of the current study is Discussion Our results also demon- strate that high glucose can reduce ABCA1 mRNA expression levels in intestinal enterocytes, and levels of cholesterol efflux from the cells in our experimen- tal model should be measured to allow definitive con- clusions to be drawn from our results. Surprisingly, IRX mRNA expression was also regulated in Caco-2 cells under high glucose treatment, since IRX3 is highly expressed in the brain and involved in feeding behavior [24]. Single nucleotide polymorphisms in the IRX3 gene are reported as strongly associated with BMI in human subjects [24]. Moreover, IRX3 knock- out mice had body weights approximately 30% less than their wild-type littermates [24], suggesting that the IRX3 gene is closely related to obesity. Glucose- regulated IRX3 mRNA regulation was shown, for the first time in our study; however, the function of intes- tinal IRX3 is unknown, and regulation of IRX3 in the intestine under high glucose consumption requires further investigation. High glucose significantly in- duced mRNA levels of the TXNIP and LCN15 genes. TXNIP plays a role in glucose uptake and is involved in glucose production in the mouse liver [25, 26]. TXNIP-mutant mice become hypertriglyceridemic and hypoglycemic during fasting [27]. These observations suggest that TXNIP may be an important candidate intracellular regulator of enterocyte glucose metabol- ism. There is very limited information about LCN15 in the literature, and its physiological function is unknown; however, it is a member of the lipocalin gene family, of which LCN2 is a well-studied member involved in obesity and diabetes. LCN2, also known as an adipokine, is secreted from the adipose tissue, and its expression is elevated in obese humans and T2DM mouse models [28]. LCN2-null male mice are protected from high fat diet-induced obesity and insu- lin resistance [29]. It was concluded that LCN2 may be an adipose tissue regulator that modulates glucose metabolism in murine tissues and in the 3T3-L1 adi- pocyte cell culture model [30]. These studies clearly suggest that ABCA1, IRX3, TXNIP, and LCN15 may that intestinal glucose absorption may be controlled via TXNIP and LCN15. provides cholesterol to high-density lipoproteins (HDL). The intestine expresses high levels of ABCA1, indicating that this tissue is a major cholesterol source for plasma HDL; indeed, intestinal ABCA1 contributes 30% of circulating HDL cholesterol [22]. High dietary cholesterol intake in the intestine increases basolateral ABCA1 mRNA and protein levels in enterocytes [23]. Discussion In this study, we used the established in vitro Caco-2 monolayer system to model the effects of high glucose exposure on intestinal enterocytes. The results of TEER measurement and evaluation of the expression of genes encoding the tight junction proteins, OCLN and ZO1, indicated that the cells successfully formed a monolayer Several molecular pathways, including intracellular protein export and ubiquitin-mediated proteolysis (upregulated), along with glycolysis/gluconeogenesis, pentose phosphate, and fructose-mannose metabolism (downregulated), were identified as responsive to high glucose in our in vitro enterocyte model. Changes in these molecular pathways under high-glucose conditions should also be evaluated at the functional level to deter- mine the physiological relevance of the findings of the current study to enterocyte glucose metabolism during high glucose treatment in vivo. Table 6 Glucose-regulated mRNA levels of individual genes Genes Ctrl +Glc p value Significance TXNIP 1.000 ± 0.140 3.170 ± 0.146 0.0001 Yes LCN15 1.000 ± 0.129 5.992 ± 1.270 0.0016 Yes ABCA1 1.000 ± 0.145 0.1939 ± 0.045 0.0004 Yes IRX3 1.000 ± 0.153 0.07723 ± 0.027 0.0001 Yes Microarray data indicated individual candidate genes that might be important for intestinal glucose metabolism during high glucose consumption. High glucose treatment significantly induced TXNIP and LCN15 mRNA expression levels relative to the control group while ABCA1 and IRX3 mRNA levels were downregulated in the high glucose-treated group. Statistical significance was determined by Student’s t test. Values are expressed as the mean ± SD; n = 3 independent experiments with three technical replicates A search for genes of particular interest among those identified as regulated by elevated glucose using genome-wide microarray analysis detected several strong candidates, including ABCA1, IRX3, TNXIP, and LCN15. ABCA1 is involved in cellular cholesterol efflux and Boztepe and Gulec Genes & Nutrition (2018) 13:11 Page 5 of 8 Page 5 of 8 provides cholesterol to high-density lipoproteins (HDL). The intestine expresses high levels of ABCA1, indicating that this tissue is a major cholesterol source for plasma HDL; indeed, intestinal ABCA1 contributes 30% of circulating HDL cholesterol [22]. High dietary cholesterol intake in the intestine increases basolateral ABCA1 mRNA and protein levels in enterocytes [23]. However, high glucose reduces ABCA1 mRNA and protein levels in the macrophages, decreasing choles- terol flux from the macrophages to the blood; hence, glucose influences cholesterol metabolism via control of ABCA1 gene expression. Culture of Caco-2 cells The human colorectal adenocarcinoma epithelial cell line, Caco-2, was purchased from the American Type Culture Collection (ATCC, HTB-37). Caco-2 cells were main- tained at 37 °C in a 5% CO2/95% O2 atmosphere. They were grown in DMEM supplemented with 15% (v:v) fetal bovine serum (Gibco, Cat. No.: 10500), 1% penicillin and streptomycin solution with antimycotic (Sigma, Cat. No.: A5955), and 1% (v:v) nonessential amino acids (Gibco, Cat. No.: 11140). Cells were separated into two groups: control (ctrl) and high glucose-treated (+Glc), which were grown in DMEM cell culture medium containing different glucose concentrations (selected based on a previous re- port [31]) as follows: ctrl group 5.5 mM glucose (Sigma, Cat. No.: D6046) and +Glc group 25 mM glucose (Sigma, Cat. No.: D6429). Cells were maintained in 5.5 and 25 mM DMEM cell culture media for three passages (28 days) to acclimate to the different glucose conditions. Page 6 of 8 Boztepe and Gulec Genes & Nutrition (2018) 13:11 The experiments were performed when Caco-2 cells were between passages 35 and 45. ABI StepOnePlus instrument (Lifetech, CA, USA) by SYBR-Green mix (Lifetech, Cat. No.: 4367659). The run- ning method of RT-qPCR is as follows: 95 °C/10 min, 40 cy- cles (95 °C/10 s, 60 °C/1 min), and melt curve stage (95 °C/ 15 s, 60 °C/1 min). Human cyclophilin A was used as a housekeeping gene for normalization of mRNA expression results. Primer sequences are listed in Additional file 1: Table S1. The 2−ΔΔCt analysis method was applied to calculate mean fold changes in mRNA levels [36]. Gene ontology and statistical analyses Significantly regulated mRNAs between the control and the high glucose-treated groups were conducted to DA- VID Functional Annotation Bioinformatics Microarray Analysis 6.7 (National Institute of Allergy and Infectious Diseases), to determine statistically overrepresented mo- lecular pathways. KEGG Pathways database was used for functional annotation enrichment analysis. Fisher exact test was used with selected value (p ≤0.05) in DAVID an- notation system. All the results were expressed as means ± SD. Student t test was performed for relative gene ex- pression to compare the two groups. GraphPad Prism (version 6.0 for Windows, GraphPad. CA, USA) was used to calculate significance between groups. We measured glucose efflux to the basolateral surface of the Caco-2 cell monolayer to test the function of the small intestine model. After ctrl and +Glc Caco-2 cells had been cultured for 21 days, the apical surfaces of the polarized cell monolayers were maintained in DMEM containing 5. 5 and 25 mM (modeling high dietary glucose consump- tion) glucose; the basolateral surfaces of both experimen- tal groups were incubated in DMEM containing 5.5 mM glucose. Samples were collected from the basolateral sur- faces of the two experimental groups at 0-, 30-, 60-, 120-, 180-, and 240-min time points. The glucose levels on the basolateral surfaces of the monolayers were measured using a Glucose Oxidase Enzymatic Assay kit, according to the manufacturer’s protocol (Sigma, Cat. No.: GAGO20). Data were normalized to total protein levels. Total proteins were extracted from cells using RIPA buffer [35] and their concentrations determined using a BCA protein assay kit (Pierce, Cat. No.: 23227). Genome-wide microarray analysis The integrity of RNA samples from each group was assessed using an Agilent 2100 Bioanalyzer and RNA 6000 Nano reagent (Agilent). RNA samples were converted to cRNA using an Ambion Illumina Total Prep RNA Ampli- fication Kit. cRNA samples were loaded onto a microarray (HumanHT-12 v4 Expression BeadChip) then left over- night for hybridization. Finally, the BeadChip was read and analyzed using a microarray laser reader. The signifi- cance of the mRNA expression between the control and the high glucose-treated groups was calculated Log2 fold change of ± 1.5 with p < 0.05. Modeling of the human intestinal system and glucose treatment When Caco-2 cells are grown in a bicameral cell cul- ture system, they can polarize to produce apical and basolateral surfaces [32], mimicking the human small intestine [33, 34]. Caco-2 cells (106/cm2) from the control and high glucose-treated groups were seeded on collagen-coated polytetrafluoroethylene membrane with 0.4-μm pore size and 1.12 cm2 diameter (Corning, Cat. No.: 3493). After 3 days of confluent culture, the cells were grown for an additional 21 days in the bicameral cell culture system in the indicated DMEM media. Media were changed every 2 days during the 21- day experimental period. Transepithelial electrical re- sistance (TEER) was measured using an Epithelial Volt/ Ohm Meter (EVOM; World Precision Instruments, USA) to confirm polarization of the monolayer and cell integrity. The TEER value of polarized Caco-2 cells should be at least 250 Ω/cm2 [34]. Additional file 1: Table S1. The Primer list for RT-qPCR. (PDF 856 kb) Additional file 2: Table S2. The list of the significantly downregulated genes under high glucose treatment condition (382 genes). (XLSX 89 kb) Additional file 3: Table S3. The list of the significantly upregulated genes under high glucose treatment condition (297 genes). (XLSX 85 kb) Additional file 4: Figure S1. Ubiquitin mediated proteolysis Pathway (Upregulated). (TIFF 497 kb) Additional file 5: Figure S2. Intracellular protein export pathway (Upregulated). (TIFF 872 kb) Additional file 6: Figure S3. Glycolysis/Gluconeogenesis Pathway (Downregulated). (TIFF 416 kb) Additional file 7: Figure S4. Pentose Phosphate Pathway (Downregulated). (TIFF 490 kb) Additional file 8: Figure S5. Fructose and Mannose Metabolism Pathway (Downregulated). (TIFF 857 kb) Competing interests h h d l h 21. Klip A, Tsakiridis T, Marette A, Ortiz PA. Regulation of expression of glucose transporters by glucose: a review of studies in vivo and in cell cultures. FASEB J. 1994;8(1):43–53. Both authors declare that they have no competing interests. Both authors declare that they have no competing interests. Funding This work was supported by a grant (TUBITAK, ID: 214Z217) from Scientific and Technological Research Council of Turkey. The funding bodies had no role in the design of the study, analysis, or interpretation of data. This work was supported by a grant (TUBITAK, ID: 214Z217) from Scientific and Technological Research Council of Turkey. The funding bodies had no role in the design of the study, analysis, or interpretation of data. 16. Morey JS, Ryan JC, Van Dolah FM. Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR. Biol Proced Online. 2006;8:175–93. Abbreviations 9. Dailey MJ. Nutrient-induced intestinal adaption and its effect in obesity. Physiol Behav. 2014;136:74–8. ABCA1: ATP-binding cassette, sub-family A member1; ALDH2: Aldehyde dehydrogenase 2; ALDOA: Aldolase A; Caco-2: Colorectal adenocarcinoma epithelial cell; GLUT2: Glucose transporter 2; GLUT5: Glucose transporter 5; IRX3: Iroquois homeobox 3; LCN15: Lipocalin 15; PFKFB3: 6-Phosphofructo-2- kinase/fructose-2,6-biphosphatase 3; PFKP: Phosphofructokinase platelet; PGAM1: Phosphoglycerate mutase 1; PGD: Phosphogluconate dehydrogenase; PTFE: Polytetrafluoroethylene; SGLT1: Sodium-dependent glucose transporter 1; SKP1: S-phase kinase-associated protein 1; SRP14/54/ 72: Signal recognition particle 14 kDa/54 kDa/72 kDa; T2DM: Type 2 diabetes mellitus; TEER: Transepithelial electrical resistance; TXNIP: Thioredoxin- interacting protein; UBE2L6: Ubiquitin-conjugating enzyme E2L 6; UBE2Q2: Ubiquitin-conjugating enzyme E2Q family member 2 10. Vereecke L, Beyaert R, van Loo G. Enterocyte death and intestinal barrier maintenance in homeostasis and disease. Trends Mol Med. 2011;17(10):584–93. 11. Roder PV, Geillinger KE, Zietek TS, Thorens B, Koepsell H, Daniel H. The role of SGLT1 and GLUT2 in intestinal glucose transport and sensing. PLoS One. 2014;9(2):e89977. 12. Dyer J, Vayro S, Shirazi-Beechey SP. Mechanism of glucose sensing in the small intestine. Biochem Soc Trans. 2003;31(Pt 6):1140–2. 13. Krotkiewski M, Bjorntorp P, Sjostrom L, Smith U. Impact of obesity on metabolism in men and women. Importance of regional adipose tissue distribution. J Clin Invest. 1983;72(3):1150–62. 14. Carrasco-Pozo C, Morales P, Gotteland M. Polyphenols protect the epithelial barrier function of Caco-2 cells exposed to indomethacin through the modulation of occludin and zonula occludens-1 expression. J Agric Food Chem. 2013;61(22):5291–7. Received: 10 November 2017 Accepted: 6 April 2018 24. Smemo S, Tena JJ, Kim KH, Gamazon ER, Sakabe NJ, Gomez-Marin C, Aneas I, Credidio FL, Sobreira DR, Wasserman NF, Lee JH, Puviindran V, Tam D, Shen M, Son JE, Vakili NA, Sung HK, Naranjo S, Acemel RD, Manzanares M, Nagy A, Cox NJ, Hui CC, Gomez-Skarmeta JL, Nobrega MA. Obesity- associated variants within FTO form long-range functional connections with IRX3. Nature. 2014;507(7492):371–5. Availability of data and materials 17. Mahraoui L, Rodolosse A, Barbat A, Dussaulx E, Zweibaum A, Rousset M, Brot- Laroche E. Presence and differential expression of SGLT1, GLUT1, GLUT2, GLUT3 and GLUT5 hexose-transporter mRNAs in Caco-2 cell clones in relation to cell growth and glucose consumption. Biochem J. 1994;298(Pt 3):629–33. The list of the high glucose-regulated genes can be found with GEO accession number: GSE97977. Accessed 7 December 2017. The list of the high glucose-regulated genes can be found with GEO accession number: GSE97977. Accessed 7 December 2017. RNA isolation and RT-qPCR Total RNA was isolated using RNAzol reagent (MRC, Cat. No.: RN190), according to the manufacturer’s protocol. Total RNA samples were dissolved in DEPC water, followed by measurement of their concentrations using a NanoDrop instrument (Thermofisher, MA, USA). Each RNA sample was separated into two aliquots for microarray analysis and RT-qPCR. One microgram of each RNA sam- ple was converted to cDNA using a cDNA synthesis kit (Lifetech, Cat. No.: 4368814). To determine the mRNA ex- pression levels of genes, RT-qPCR was performed on an Page 7 of 8 Page 7 of 8 Page 7 of 8 Boztepe and Gulec Genes & Nutrition (2018) 13:11 Acknowledgements h k h We thank the Izmir Institute of Technology, Biotechnology and Bioengineering Research and Application Center for providing their laboratory facilities. 15. Park HY, Kunitake Y, Hirasaki N, Tanaka M, Matsui T. Theaflavins enhance intestinal barrier of Caco-2 cell monolayers through the expression of AMP- activated protein kinase-mediated Occludin, Claudin-1, and ZO-1. Biosci Biotechnol Biochem. 2015;79(1):130–7. Authors’ contributions 18. Miyamoto K, Hase K, Takagi T, Fujii T, Taketani Y, Minami H, Oka T, Nakabou Y. Differential responses of intestinal glucose transporter mRNA transcripts to levels of dietary sugars. Biochem J. 1993;295(Pt 1):211–5. SG designed the investigation. TB and SG performed the experiments, interpreted the data, and wrote the manuscript. Both authors read and approved the final manuscript. 19. Ritze Y, Bardos G, D'Haese JG, Ernst B, Thurnheer M, Schultes B, Bischoff SC. Effect of high sugar intake on glucose transporter and weight regulating hormones in mice and humans. PLoS One. 2014;9(7):e101702. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. 20. Drozdowski LA, Thomson AB. Intestinal sugar transport. World J Gastroenterol. 2006;12(11):1657–70. Publisher’s Note 22. Brunham LR, Kruit JK, Iqbal J, Fievet C, Timmins JM, Pape TD, Coburn BA, Bissada N, Staels B, Groen AK, Hussain MM, Parks JS, Kuipers F, Hayden MR. Intestinal ABCA1 directly contributes to HDL biogenesis in vivo. J Clin Invest. 2006;116(4):1052–62. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 23. Field FJ, Watt K, Mathur SN. Origins of intestinal ABCA1-mediated HDL- cholesterol. J Lipid Res. 2008;49(12):2605–19. Received: 10 November 2017 Accepted: 6 April 2018 Received: 10 November 2017 Accepted: 6 April 2018 References 1. Raben A, Vasilaras TH, Moller AC, Astrup A. Sucrose compared with artificial sweeteners: different effects on ad libitum food intake and body weight after 10 wk of supplementation in overweight subjects. Am J Clin Nutr. 2002;76(4):721–9. 25. Yu FX, Goh SR, Dai RP, Luo Y. Adenosine-containing molecules amplify glucose signaling and enhance txnip expression. Mol Endocrinol. 2009; 23(6):932–42. 2. Rippe JM, Marcos A. Controversies about sugars consumption: state of the science. Eur J Nutr. 2016;55(Suppl 2):11–6. 3. W.H. Organization. Global report on diabetes: WHO Library Cataloguing-in- Publication Data. Switzerland; 2016. 26. Hui ST, Andres AM, Miller AK, Spann NJ, Potter DW, Post NM, Chen AZ, Sachithanantham S, Jung DY, Kim JK, Davis RA. Txnip balances metabolic and growth signaling via PTEN disulfide reduction. Proc Natl Acad Sci U S A. 2008;105(10):3921–6. 4. Raben A, Moller BK, Flint A, Vasilaris TH, Christina Moller A, Juul Holst J, Astrup A. Increased postprandial glycaemia, insulinemia, and lipidemia after 10 weeks’ sucrose-rich diet compared to an artificially sweetened diet: a randomised controlled trial. Food Nutr Res. 2011;55 27. Hui TY, Sheth SS, Diffley JM, Potter DW, Lusis AJ, Attie AD, Davis RA. Mice lacking thioredoxin-interacting protein provide evidence linking cellular redox state to appropriate response to nutritional signals. J Biol Chem. 2004; 279(23):24387–93. 5. Basu S, Yoffe P, Hills N, Lustig RH. The relationship of sugar to population- level diabetes prevalence: an econometric analysis of repeated cross- sectional data. PLoS One. 2013;8(2):e57873. 5. Basu S, Yoffe P, Hills N, Lustig RH. The relationship of sugar to population- level diabetes prevalence: an econometric analysis of repeated cross- sectional data. PLoS One. 2013;8(2):e57873. 28. Guo H, Jin D, Zhang Y, Wright W, Bazuine M, Brockman DA, Bernlohr DA, Chen X. Lipocalin-2 deficiency impairs thermogenesis and potentiates diet- induced insulin resistance in mice. Diabetes. 2010;59(6):1376–85. 6. Solini A, Rossi C, Mazzanti CM, Proietti A, Koepsell H, Ferrannini E. Sodium- glucose co-transporter (SGLT)2 and SGLT1 renal expression in patients with type 2 diabetes. Diabetes Obes Metab. 2017;19:1289-94 6. Solini A, Rossi C, Mazzanti CM, Proietti A, Koepsell H, Ferrannini E. Sodium- glucose co-transporter (SGLT)2 and SGLT1 renal expression in patients with type 2 diabetes. Diabetes Obes Metab. 2017;19:1289-94 29. Law IK, Xu A, Lam KS, Berger T, Mak TW, Vanhoutte PM, Liu JT, Sweeney G, Zhou M, Yang B, Wang Y. Lipocalin-2 deficiency attenuates insulin resistance associated with aging and obesity. Diabetes. 2010;59(4):872–82. 7. Boztepe and Gulec Genes & Nutrition (2018) 13:11 References Rines AK, Sharabi K, Tavares CD, Puigserver P. Targeting hepatic glucose metabolism in the treatment of type 2 diabetes. Nat Rev Drug Discov. 2016; 15(11):786–804. 7. Rines AK, Sharabi K, Tavares CD, Puigserver P. Targeting hepatic glucose metabolism in the treatment of type 2 diabetes. Nat Rev Drug Discov. 2016; 15(11):786–804. 30. Yan QW, Yang Q, Mody N, Graham TE, Hsu CH, Xu Z, Houstis NE, Kahn BB, Rosen ED. The adipokine lipocalin 2 is regulated by obesity and promotes insulin resistance. Diabetes. 2007;56(10):2533–40. 8. Rajas F, Bruni N, Montano S, Zitoun C, Mithieux G. The glucose-6 phosphatase gene is expressed in human and rat small intestine: regulation of expression in fasted and diabetic rats. Gastroenterology. 1999;117(1):132–9. Page 8 of 8 Page 8 of 8 Boztepe and Gulec Genes & Nutrition (2018) 13:11 Boztepe and Gulec Genes & Nutrition (2018) 13:11 31. D'Souza VM, Shertzer HG, Menon AG, Pauletti GM. High glucose concentration in isotonic media alters caco-2 cell permeability. AAPS PharmSci. 2003;5(3):E24. 31. D'Souza VM, Shertzer HG, Menon AG, Pauletti GM. High glucose 32. Hidalgo IJ, Raub TJ, Borchardt RT. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology. 1989;96(3):736–49. 33. Satake M, Enjoh M, Nakamura Y, Takano T, Kawamura Y, Arai S, Shimizu M. Transepithelial transport of the bioactive tripeptide, Val-Pro-Pro, in human intestinal Caco-2 cell monolayers. Biosci Biotechnol Biochem. 2002;66(2):378–84. 34. Sambuy Y, De Angelis I, Ranaldi G, Scarino ML, Stammati A, Zucco F. The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol. 2005;21(1):1–26. 35. Gulec S, Collins JF. Silencing of the Menkes copper-transporting ATPase (Atp7a) gene increases cyclin D1 protein expression and impairs proliferation of rat intestinal epithelial (IEC-6) cells. J Trace Elem Med Biol. 2014;28(4):459–64. 36. Hu Z, Gulec S, Collins JF. Cross-species comparison of genomewide gene expression profiles reveals induction of hypoxia-inducible factor-responsive genes in iron-deprived intestinal epithelial cells. Am J Physiol Cell Physiol. 2010;299(5):C930–8.
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Resistance of Xanthomonas campestris pv. vesicatoria isolates from Tanzania to copper and implications for bacterial spot management
African journal of microbiology research
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Received 18 May, 2013; Accepted 10 March, 2014 Received 18 May, 2013; Accepted 10 March, 2014 Thirty isolates of Xanthomonas campestris pv. vesicatoria (Xcv) from Tanzania were evaluated for sensitivity to copper sulphate (CuSO4:5H20) using dilution plate counts. Ninety-three percent (28/30) of the isolates produced countable colonies on nutrient glucose agar amended with 0.8 mM CuSO4. All the Xcv isolates (7/7) from Arusha (northern Tanzania) grew on the copper medium. Isolates of the pathogen from Iringa (southern Tanzania) produced variable results (15/21). However, neither of the two Xcv isolates from Morogoro (Eastern Tanzania) grew on the copper medium. These results indicate long-term exposure of Xcv isolates from Tanzania to selection pressure for copper tolerance. Key words: Xanthomonas campestris pv. vesicatoria, copper resistance, Tanzania. Kenneth C. Shenge1,5*, Robert B. Mabagala2, Carmen N. Mortensen3 and Kerstin Wydra4 2Department of Crop Science and Production, Sokoine University of Agriculture, P.O. Box 3005, Morogoro, Tanzania. 3Danish Seed Health Centre for Developing Countries, Department of Plant Biology, Plant Pathology Section, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, Frederiksberg C, DK-1871, Copenhagen, Denmark. 4Georg-August-University Göttingen, Centre for Tropical and Subtropical Agriculture and Forestry (CeTSAF), Buesgenweg 1 D 37077 Gottingen Germany g g g y n, Faculty of Agriculture/Institute of Agricultural Research, Ahmadu Bello University, Zaria, Nigeria. Full Length Research Paper Full Length Research Paper Resistance of Xanthomonas campestris pv. vesicatoria isolates from Tanzania to copper and implications for bacterial spot management African Journal of Microbiology Research African Journal of Microbiology Research INTRODUCTION Bacterial leaf spot (BLS) of tomato (Solanum lycopersicum L.), caused by Xanthomonas campestris pv. vesicatoria (Syn. Xanthomonas axonopodis pv. vesicatoria (Vauterin et al., 2000) or Xanthomonas euvesicatoria (Jones et al., 2004)) is one of the most destructive and widely distributed diseases of tomatoes (Hovarth et al., 2012; Yu et al., 1995). The disease affects every above-ground part of the tomato plant. Attack on leaves causes defoliation, resulting in exposure of fruits to sun scald (Dougherty, 1978; Pohronezny and Volin, 1983). However, the main economic effect of the disease is the reduction in fruit weight and quality. Bacterial spots on the fruits have been reported to account for up to 52% weight loss in infected fruits (Jones et al., 1986). Disease control is exceedingly difficult to achieve when environmental and weather conditions are conducive for pathogen proliferation (Jones et al., 1986). It is not clear when tomato bacterial spot was first Afr. J. Microbiol. Res. 2882 identified and recorded in Tanzania, but Black et al. (2001) reported that the disease was widespread in tomato and pepper fields in all the vegetable-growing regions in Northern and Southern mainland Tanzania. Other surveys by Kaaya et al. (2003) confirmed wide- spread occurrence of bacterial spot in the country. Although most tomato growers in Tanzania rely on fixed copper chemicals to manage tomato bacterial spot, the disease has continued to spread in spite of the chemical application (Shenge et al., 2007). In spite of these reports, the long-term impact of these chemicals on the disease, the environment and agricultural production in Tanzania has not been assessed. The present study, therefore, aimed at assessing isolates of Xcv from Tanzania for resistance to copper, and determining if copper-based chemicals were an effective way of managing the disease in Tanzania. CuSO4 is presented in Figure 2. Seventy-three percent of the isolates (22/30) produced countable colonies on the copper-amended medium, with all the isolates from Arusha Region (7/7) showing resistance to copper. Out of 21 isolates from Iringa Region, 15 grew on the copper- amended medium; neither of the isolates from Morogoro produced colonies on the copper medium. The findings of this study demonstrated that resistance to copper-based chemicals was widespread within Xcv populations from the northern and southern highlands of Tanzania. INTRODUCTION These regions are also the leading tomato production areas in Tanzania, indicating that long-term use of copper-based chemicals in tomato production inadvertently led to Xcv selection for resistance to copper in the regions. In addition to tomato production, Arusha Region also has a long history of coffee production, which also receives heavy applications of copper compounds for coffee disease management. p g Copper is required in trace quantities by many bacteria for their structural composition, as a co-factor in enzy- matic functions (Bai et al., 2007), and electron transport and redox reactions (Cervantes and Guitierrex-Corona, 1994). However, at high concentrations, copper mole- cules become toxic to bacteria, interfering with the energy transport system and disrupting enzyme active sites, as well as the integrity of cell membranes (Cervantes and Gutierrez-Corona, 1994; Garcia-Horsman et al., 1994). High cellular copper concentrations have also been shown to damage lipids, proteins and DNA (Bai et al., 2007). Owing to these toxic properties, copper formula- tions have been used against a wide range of bacterial and fungal plant pathogens for more than 100 years (Cooksey and Azad, 1992). However, copper ions are not degraded in soil and can accumulate to high levels at locations with a history of intensive copper application (Koller, 1998). Long-term microbial exposure to sub-lethal concentrations of the chemical leads to adaptation through the development of plasmid/chromo-somally- borne cellular copper sequestration (Cooksey, 1990) and detoxification systems that protect the bacteria from toxic concentration levels of copper, while at the same time ensuring that their nutritional copper require-ments are met (Voloudakis et al., 2005). MATERIALS AND METHODS Seven samples were collected from Arusha, 2 from Morogoro, and 21 from Iringa region (Figure 1). Presumptive Xanthomonas campestris pv. vesicatoria isolates from tomato fruit lesions, and one reference Xcv strain from The Göttingen Collection of Phytopathogenic Bacteria (GSPB), GSPB 2043 were tested for Xcv determinative characteristics, including Gram reaction, nitrate reduction, Kovac’s oxidase reaction, starch hydrolysis, pectin degradation, Biolog and hypersensitive reaction on tobacco leaves, following methods and classifications outlined in Schaad et al. (1988), Holt et al., (2000), O’Garro et al. (2003) and Woodland, (2004). Pathogenicity of the isolates was confirmed by misting an inoculum suspension of the bacteria adjusted to 108 cfu ml-1 onto 35-day-old tomato (cv. Tanya) plants and scoring disease symp- toms 14 days after inoculation. Suspensions of 48 h old bacterial isolates were prepared in phosphate buffered saline (PBS) and adjusted to an optical density (OD) of 0.06 at 620 nm, correspon- ding to ca 108 cfu ml-1. Twenty microliters of each suspension were spread evenly onto the 0.8 mM copper-containing medium (200 mg CuSO4.5H2O/1000 ml of NGA). NGA served as the untreated con- trol. Inoculated plates were incubated at 28°C and enumerated 36 to 48 h later. Counts on the copper-amended medium and NGA were compared statistically using t-tests at P≤0.05. RESULTS AND DISCUSSION Yellow-pigmented Xcv isolates from lesions on tomato fruit samples were rod-shaped, unipolar flagellated, Gram-negative, oxidase-negative and catalase-positive. All the isolates hydrolyzed starch and degraded pectin, but were unable to reduce nitrates. Tobacco leaves ino- culated with suspensions of the isolates showed a hyper- sensitive reaction within 24 h, and typical bacterial spot symptoms that were similar to natural symptoms deve- loped on inoculated tomato (cv. Tanya) plants following inoculation. The isolates differed widely in their carbon source utilization profiles in Biolog; the dendrogram showed three distinct clusters that confirmed a strong relationship with geographic origin (Shenge et al., 2007). In general, the ability of Xcv strains to tolerate copper in artificial growth media is used to measure copper resis- tance in the field. Several instances of this approach have been documented in literature (Cooksey et al., 1990; Gore and O’Garro, 1999; Tesoriero et al., 1997; Zevenhuizen et al., 1979; Martin and Hamilton, 2004) with similar levels of effectiveness. In studies by Zevenhuizen et al. (1979), strains of bacteria were con- sidered to be resistant to CuSO4.5H20 if they were able to survive in a medium containing 1.0 mM of the compound. In other studies, Gore and O’Garro (1999) reported that ability of Xcv strains to express confluent growth on NA amended with copper sulphate at a concentration of 200 µg ml-1 (0.80 mM) was an expression of resistance to the The response of Xcv isolates from Tanzania to 0.8 mM 2883 Shenge et al. Figure 1. Geopolitical map of Tanzania. Red stars indicate locations where Xanthomonas campestris pv. vesicatoria isolates were collected. The locations were selected based on a history of tomato production, and also to cover a wide range of ecological conditions. Figure 1. Geopolitical map of Tanzania. Red stars indicate locations where Xanthomonas campestris pv. vesicatoria isolates were collected. The locations were selected based on a history of tomato production, and also to cover a wide range of ecological conditions. environmental impact of long-term use of copper-based chemicals as a plant disease management option. With particular reference to Tanzania, these results de- monstrated that copper pesticides are no longer an effective means for tomato bacterial spot management. Identification of alternative natural and synthetic antimic- robial agents against Xcv is therefore, exigent. Recent reports by Mbega et al. (2012) indicated that some plant extracts were effective in reducing seed-borne xantho- monads associated with bacterial leaf spot. RESULTS AND DISCUSSION It seems that tomato bacterial spot management approaches that chemical. In the current study, 73% of Xcv isolates from Tanzania produced countable colonies in NGA amended with 0.8 mM CuSO4. Based on the conclusions of previous studies, the Xcv strains from Tanzania can, therefore, be classified as resistant to copper. The findings of this study are in agreement with other reports, which showed that increasing occurrence of cop- per resistance within populations of Xcv was becoming a serious problem in many tomato production areas (Gore and O’ Garro, 1999; Martin and Hamilton, 2004; Mirik et al., 2007). These findings highlight the negative 2884 Afr. J. Microbiol. Res. CuSo4 Figure 2. Resistance of Xanthomonas campestris pv. vesicatoria to CuSO4. Solid, black bars indicate the number of countable Xcv colony forming units (CFU) on nutrient glucose agar (NGA: Nutrient agar + 5% glucose) medium amended with 0.8 mM CuSO4:5H2O while unshaded bars indicate the number of Xcv CFU on NGA medium without CuSO4. Labels on the vertical axis represent isolates used in the study. Isolate names ending with A indicates that the isolates were collected from Arusha Region; Ir, IrA and IrB were collected from Iringa Region, while those ending with M were collected from Morogoro region. IrA and IrB indicate that the diseased tomato fruit samples from which the pathogens were isolated came from the same field. CuSo4 Figure 2. Resistance of Xanthomonas campestris pv. vesicatoria to CuSO4. Solid, black bars indicate the number of countable Xcv colony forming units (CFU) on nutrient glucose agar (NGA: Nutrient agar + 5% glucose) medium amended with 0.8 mM CuSO4:5H2O while unshaded bars indicate the number of Xcv CFU on NGA medium without CuSO4. Labels on the vertical axis represent isolates used in the study. Isolate names ending with A indicates that the isolates were collected from Arusha Region; Ir, IrA and IrB were collected from Iringa Region, while those ending with M were collected from Morogoro region. IrA and IrB indicate that the diseased tomato fruit samples from which the pathogens were isolated came from the same field. Shenge et al. 2885 integrate the use of such plant products, with synthetic chemicals, disease-free seeds, field sanitation and resistant tomato varieties would be an effective way of managing the disease in a sustainable manner. RESULTS AND DISCUSSION In gene- ral, successful management of bacterial leaf spot in Tanzania would require the design of spray programs that accommodate the pesticide sensitivity status of pathogen populations. One component of such a pro- gram should consist of regular field surveys to determine the likelihood that farmers will encounter resistant strains of the pathogens, and a system that combines one or two antimicrobial compounds to eliminate the likelihood of the pathogens developing resistance to any one of them. Jones JB, Pohnezny KL, Stall RE, Jones JP (1986). Xanthomonas campestris pv. vesicatoria on tomato crop residue, weeds, seeds and volunteer tomato plants. Phytopathol. 76:430- 34. Jones BJ, Lacy GH, Bouzar H, Stall RE, Schaad NW (2004). Reclassi- fication of the xanthomonads associated with bacterial spot disease of tomato and pepper. Syst. Appl. Microbiol. 27:755-762. Kaaya NKF, Mortensen CN, Mabagala RB, Massomo SMS (2003). A guide on seed borne bacterial diseases of tomato in Tanzania. Technical Bulletin, Danish Government Institute of Seed Pathology for Developing Countries (DGISP), Copenhagen, Denmark. 17p. Koller W (1998). Chemical approaches to managing plant pathogens. In: Handbook of Integrated pest Management, ed: J.R. Ruberson, Dekker, New York. 864 p. Martin HL, Hamilton VA (2004). Copper tolerance in Australian populations of Xanthomonas campestris pv. vesicatoria contributes to poor control of bacterial spot of pepper. Plant Dis. 88 (9):921-924. Mbega ER, Mortenson CN, Mabagala RB, Wulf EG (2012). The effect of plant extracts as seed treatments to control bacterial leaf spot of tomato in Tanzania. J. Gen. Plant Pathol. 78:277–286. Conflict of Interests Mirik M, Aysan Y, Cinar O (2007). Copper-resistant strains of Xanthomonas axonopodis pv. vesicatoria (Doidge) Dye in the eastern Mediterranean region of turkey. J. Plant Pathol. 89(1):153-154. The author(s) have not declared any conflict of interests. O’Garro LW, Gore JP, Ferguson E (1999). Races of Xanthomonas campestris pv. vesicatoria overcoming the gene Bs2 for bacterial spot resistance in pepper, prevalent on Capsicum chinense in Barbados and Grenada and weakly pathogenic on bell pepper and tomato in the field. Plant Pathol. 48:588–594 ACKNOWLEDGEMENTS Pohronezny K, Volin RB (1983). The effect of bacterial spot on yield and quality of fresh market tomatoes. HortScience 18:69-70. This study was funded by the German Academic Exchange Service (DAAD). The technical assistance of Mrs. Birgit Milde of the Phytobacteriology Laboratory, Institute of Plant Diseases and Plant Protection, University of Hannover is greatly appreciated. Voloudakis EA, Reignier TM, Cooksey DA (2005). Regulation of resistance to copper in Xanthomonas axonopodis pv. vesicatoria. App. Environ. Microbiol. 71:782–789. pp Schaad NW, Stall RE (1988). Xanthomonas. In: Schaad NW, ed., Laboratory Guide for Identification of Plant Pathogenic Bacteria. 2nd Schaad NW, Stall RE (1988). Xanthomonas. In: Schaad NW, ed., Laboratory Guide for Identification of Plant Pathogenic Bacteria. 2nd edn. St. Paul, MN: American Phytopathological Society, pp.81–94. y g edn. St. Paul, MN: American Phytopathological Society, pp.81–94. Shenge KC, Mabagala RB, Mortensen CN (2007). Identification and characterization of strains of Xanthomonas campestris pv. vesicatoria from Tanzania by Biolog system and sensitivity to antibiotics. Afr. J. Biotechnol. 6:15-22. REFERENCES Bai W, Zhao K, Asami K (2007). Effects of copper on dielectric properties of E. coli cells. Coll. Surf. B. Bioinfo. 58:105-115. Tesoriero L, Warren M, Minchin F (1997). Copper resistance in Australian isolates of Pseudomonas syringae pv. tomato. in: Proceeding of the Australian Plant Pathology Society, 44 th Biennial Conference, Perth Australia. p.311 Black R, Seal S, Abubakar Z, Nono-Womdim R, Swai I (2001). Black spot (Xanthomonas campestris pv. vesicatoria) of tomato and sweet pepper in Tanzania. Plant Pathol. 50:810. Venette JR, Lamey HA, Smith RC (1996). Bacterial spot and bacterial speck of Tomato. North Dakota State University Extension Services. 2p. p pp Cervantes C, Gutierrez-Corona F (1994). Copper resistance mechanisms in bacteria and fungi. FEMS Microbiol. Rev. 14:121-138. Cooksey DA (1990). Genetics of bactericide resistance in plant- pathogenic bacteria. Annual Rev. Phytopathol. 28:201-14. Woodland J (2004). Bacteriogy. In: Heil N (Ed) (2004). National Wild Fish Health Survey-Laboratory Procedures Manual 2.0. Edition. US Fish and Wildlife Service, Warm Springs, GA. 5:1-44. Cooksey DA, Azad HR (1992). Accumulation of copper and other metals of copper-resistant plant-pathogenic and saprophytic pseudomonads. App. Environ. Microbiol. 58:274-278. Yu ZH, Wang JF, Stall RE, Vallejos CC (1995). Genomic localization of tomato genes that control a hypersensitive reaction to Xanthomonas campestris pv. vesicatoria (Doidge) Dye. Genetics 141: 675-682. Dougherty DE (1978). Yield reduction in tomato caused by bacterial spot, and disease control with copper sprays. Proc. Florida State Hortic. Soc. 91:291-293. Zevenhuizen LPTM, Dolling J, Eshui EJ, Scholten-Koerselman IJ (1979). Inhibitory effects of copper on bacteria related to the free ion concentration. Microb. Ecol. 5: 139-146. Gore JP, O’Garro LW (1999). Xanthomonas campestris pv. vesicatoria from bell pepper and tomato in Barbados undergoes changes in race structure, virulence and sensitivity to chemical control agents. J. Phytopathol. 47:397-402. Garcia-Horsman JAB, Barquera R, Rumbley JM, Gennis RB (1994). The superfamily of heme-copper respiratory oxidases. J. Bacteriol. 176:5587–5600. Holt JG, Krieg NR, Sneath PH, Staley JT, Williams ST (2000). Bergey’s manual of determinative bacteriology, 9 th Edition. Lippincott Williams & Wilkins, Philadelphia. 787p. Hovath DM, Stall RE, Jones JB, Pauly, MH, Vallad, GE, Dahlbeck, D, Staskawicz BJ, Scott, JW (2012). Transgenic resistance confers effective field level control of bacterial spot disease in tomato. PLoS ONE 7(8): e42036. doi:10.1371/journal.pone.0042036
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Factors associated with the specialty choice of Korean medical students: a cross-sectional survey
Human resources for health
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RESEARCH Open Access Factors associated with the specialty choice of Korean medical students: a cross- sectional survey Yeon-Yong Kim1, Un-Na Kim1, Yon Su Kim2 and Jin-Seok Lee1,3* Yeon-Yong Kim1, Un-Na Kim1, Yon Su Kim2 and Jin-Seok Lee1,3* * Correspondence: phealth@snu.ac.kr 1Department of Health Policy and Management, Seoul National University College of Medicine, 110-799 103 Daehak-ro, Jongno-gu, Seoul, Republic of Korea 3Institute of Health Policy and Management, Medical Research Center, Seoul National University, Seoul, Republic of Korea Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: An imbalance of physician supply by medical specialty has been observed in most countries. In Korea, there is a greater tendency to avoid surgical specialties and specialty choices in nonclinical medicine, such as the basic science of medicine. In this study, we identified factors affecting the specialty choice of physicians in order to provide a basis for policies to address this problem. Methods: We used the results of a 2013 nationwide survey of 12 709 medical students (82.7 % responded) to analyze the data of 9499 students after excluding missing data. Descriptive analyses of all students’ specialty choice were performed. Logistic regression was performed by selecting gender, age, grade level, type of medical school, hometown, and the location of the medical school as the independent variables. Medical specialty was the dependent variable. The dependent variable, or specialty of medicine, was categorized into three groups: nonclinical/clinical medicine, surgical-medical specialty, and controllable lifestyle specialty. Results: The order of preferred medical specialties was internal medicine, psychiatry, and pediatrics; for surgical specialties, the order was orthopedic surgery, general surgery, and ophthalmology. Medical specialties were most favored by women and students in the third (men) and second (women) year of the medical program, whereas surgical specialties were most preferred by men and students in the first year of the program. Students in the third year mostly favored nonclinical medicine. Medical college students had a stronger preference for nonclinical medicine (odds ratio [OR] 1.625, 95 % confidence interval [CI] 1.139–2.318) than graduate medical school students. Surgical specialties were more favored by men (OR 2.537, 95 % CI 2.296–2.804) than by women. However, they were favored less by medical college students (OR 0.885, 95 % CI 0.790–0.991) than by graduate medical school students and by medical students in metropolitan areas (OR 0.892, 95 % CI 0.806–0.988) than by medical students in nonmetropolitan areas. A controllable lifestyle specialty was less favored by men (OR 0.802, 95 % CI 0.730–0.881) than by women. Conclusions: Based on these results, we can evaluate the effectiveness of the government’s educational policies for solving the imbalance of physician supply and provide empirical evidence to understand and solve this problem. Keywords: Health manpower, Medical education, Medical specialties, Specialty choice © 2016 The Author(s). Kim et al. Human Resources for Health (2016) 14:45 DOI 10.1186/s12960-016-0141-8 Kim et al. Human Resources for Health (2016) 14:45 DOI 10.1186/s12960-016-0141-8 * Correspondence: phealth@snu.ac.kr 1Department of Health Policy and Management, Seoul National University College of Medicine, 110-799 103 Daehak-ro, Jongno-gu, Seoul, Republic of Korea 3Institute of Health Policy and Management, Medical Research Center, Seoul National University, Seoul, Republic of Korea Full list of author information is available at the end of the article * Correspondence: phealth@snu.ac.kr 1Department of Health Policy and Management, Seoul National University College of Medicine, 110-799 103 Daehak-ro, Jongno-gu, Seoul, Republic of Korea 3Institute of Health Policy and Management, Medical Research Center, Seoul National University, Seoul, Republic of Korea Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background limited in Korea [9, 13, 14]. In addition, doubts have been raised about whether the policy for solving the imbalance has achieved its goal, and there have not been enough studies on this matter. There has not yet been a proper evaluation of the representative policy, introdu- cing a graduate medical school, which aimed to increase researchers in the basic science of medicine. In contrast, there is a substantial amount of relevant research, includ- ing surveys of the desired career paths of medical stu- dents, as well as matching programs in the United States and Japan [15–17]. limited in Korea [9, 13, 14]. In addition, doubts have been raised about whether the policy for solving the imbalance has achieved its goal, and there have not been enough studies on this matter. There has not yet been a proper evaluation of the representative policy, introdu- cing a graduate medical school, which aimed to increase researchers in the basic science of medicine. In contrast, there is a substantial amount of relevant research, includ- ing surveys of the desired career paths of medical stu- dents, as well as matching programs in the United States and Japan [15–17]. The imbalance of the supply of physicians has been reported among different specialties and regions and between the public and private sectors. The disparity has been an important issue in most countries [1, 2]. In Korea, the supply imbalance among different specialties is problematic since the percentage of specialists is high among physicians (approximately 73 %) [3] and there is a large income gap among the specialties [4]. As a result, some specialties consistently have a short supply of phy- sicians because of a lack of applicants relative to the number of openings, whereas for other specialties, it takes 2 or 3 years for some medical students to be admitted to the specialty due to the fierce competition. In this study, we aimed to analyze factors affecting the specialty choices of medical students, based on the results of a survey taken by all medical students in Korea in 2013. In particular, the analysis focused on the factors affecting the choice of nonclinical medicine, surgical specialties, and controllable lifestyle specialties. This classification has been adopted in previous studies [9, 13–17]. Abstract Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. Human Resources for Health (2016) 14:45 Kim et al. Human Resources for Health (2016) 14:45 Page 2 of 8 Page 2 of 8 Page 2 of 8 Methods Participants The total number of participants was 12 709; 10 514 persons participated in the survey, yielding a response rate of 82.7 %. Excluding missing data, analyses were per- formed on survey data from 9499 respondents. Specialty choice is also influenced by the curriculum. Courses on the basic science of medicine are primarily taught in the lower grade of medical education, while students in the upper grade mostly learn clinical medi- cine, which eventually leads to their decreased interest in the basic science of medicine [9]. Educational pro- grams for physicians also play an important role. Korea’s physician training system has been a 6-year undergradu- ate program in the medical college since 1946; however, a 4-year graduate program in the medical school, accept- ing graduates from different majors, was introduced in 2006 [10]. These two types of medical programs have similarities in their curricula but differences in the stu- dents’ experiences. The purpose of employing this sys- tem was based on the expectation that allowing the entrance of graduates who majored in diverse fields of study would enable diverse specialty choices other than clinical medicine [11, 12]. It was also expected that most of students who had majored in basic science before en- tering the graduate program of medical school would tend to choose specialties in nonclinical medicine in the hope of further developing their major studied at the undergraduate level [11]. Currently, both the 6-year medical college and 4-year graduate medical school pro- grams coexist in Korea. Design and survey This study used a cross-sectional design. A nationwide survey was distributed to medical college/graduate med- ical school students by the Ministry of Health and Welfare in 2013. The questionnaire included students’ school, grade, year of admission, gender, hometown, and desired medical specialty in the future. Background Based on this study, we aimed to identify factors affecting students’ specialty choices and provide evidence for related policies to solve the imbalance in the phys- ician supply. In Korea, nonclinical medicine, such as the basic sci- ence of medicine, and certain surgical specialties are traditionally avoided for economic reasons. As the ma- jority of healthcare institutions are private, economic factors have an important role in hospital management in Korea. Other than the economic aspects, the basic science of medicine is known to be avoided because of the instability of the position [5], and surgical specialties are avoided due to the high demand of physical intensity and stress [6]. Although it is impossible to measure the stability and stress of these positions, they are factors that significantly affect specialty choice [7] and down- grade lifetime utility [8]. Variables Gender, age, grade, type of medical school, hometown, and location of medical school were selected as independent variables. Type of medical school was divided into medical college and graduate medical school. Hometown was determined by the participant’s high school alma mater and classified as metropolitan (Seoul, Gyeonggi, and Incheon) or nonmetropolitan. The location of the med- ical school was also classified as metropolitan or nonmetro- politan. Grade was defined as the first, second, third, and fourth years of medical education. To test for the presence of multicollinearity, correlation analyses were performed with the independent variables, and it was confirmed that none of the variables had correlation coefficients above 0.8 for their relationships with any of the other variables (the Although the problem of an imbalance of specialty choices is serious, studies on this subject have been Kim et al. Human Resources for Health (2016) 14:45 Page 3 of 8 Page 3 of 8 correlation coefficient between type of medical school and age was the highest at 0.52). medicine, pediatrics, obstetrics-gynecology, orthopedic sur- gery, general surgery, and urology. For the latter definition, the specialties with more than 50 % of self-employed spe- cialists [18], such as dermatology, otolaryngology, plastic surgery, ophthalmology, urology, pediatrics, family medi- cine, obstetrics-gynecology, and tuberculosis, were included in the controllable lifestyle specialty category. g g The desired medical specialty for training was se- lected as the dependent variable. A total of 27 special- ties was divided based on three criteria. The first criterion was whether the specialty belonged to clinical medicine or nonclinical medicine. Nonclinical medicine included pathology, preventive medicine, and the basic science of medicine, whereas clinical medicine included the remaining subspecialties (internal medicine, neur- ology, psychiatry, pediatrics, dermatology, therapeutic radiology, rehabilitative medicine, family medicine, nu- clear medicine, emergency medicine, tuberculosis, occu- pational and environmental medicine, general surgery, orthopedic surgery, neurosurgery, plastic surgery, cardio- thoracic surgery, obstetrics-gynecology, ophthalmology, otolaryngology, urology, anesthesiology, diagnostic radi- ology, and clinical pathology). The second criterion distin- guished the medical and surgical specialties within clinical medicine. The surgical specialties included general sur- gery, orthopedic surgery, neurosurgery, plastic surgery, cardiothoracic surgery, obstetrics-gynecology, ophthal- mology, otolaryngology, and urology. The remaining areas of clinical medicine were categorized as a medical specialty. The third criterion was whether the specialty offered a controllable lifestyle. Data analysis A d i i A descriptive analysis of the clinical/nonclinical special- ties and an analysis of the controllable/noncontrollable lifestyle specialties in the self-employed specialties were performed on data from 9499 persons. An analysis of the surgical/nonsurgical specialties was performed on data from 9313 persons, excluding those who desired nonclinical medicine. An analysis of the controllable/ noncontrollable lifestyle specialties in a US-based study was conducted on the data from 8259 persons, excluding those who desired specialties that were not included in our categories. A descriptive analysis was performed on the specialty choices of all of the students, and logistic regression was conducted to find the factors influencing specialty choice. In the analyses of the nonclinical medicine and surgical specialties, a gender subgroup analysis was performed. SPSS 22.0 was used for the statistical analyses, and the significance level was set at P < 0.05. This study received an exemption from the Seoul National University Hospital Institutional Review Board (IRB No. 1506-132-683). Variables Controllable lifestyle special- ties were defined two ways and analyzed separately: special- ties previously identified as offering a controllable lifestyle in a US-based study [16] and specialties in which a majority of practitioners were self-employed in Korea. These classifi- cations are not well established in the literature; therefore, we did not attempt to investigate the data further with subgroup analyses. For the former definition, the specialties offering a controllable lifestyle included anesthesiology, dermatology, emergency medicine, neurology, ophthalmol- ogy, otolaryngology, pathology, psychiatry, and diagnostic radiology. Specialties with noncontrollable lifestyles in a US-based study included internal medicine, family General characteristics The general characteristics of the study participants are presented in Table 1. There were more men (60.9 %) than women (39.1 %), and the number of students from nonmetropolitan areas (57.6 %) was higher than that from metropolitan areas (42.4 %). There were more graduate medical school students (60.4 %) than medical Table 1 General characteristics (N = 9499) Table 1 General characteristics (N = 9499) Grade (year) 1st 2nd 3rd 4th Total P value % % % % % Gender Men 1 747 64.8 1 473 60.7 1 276 61.0 1 288 56.3 5 784 60.9 <0.001 Women 947 35.2 954 39.3 816 39.0 998 43.7 3 715 39.1 Hometown Metropolitan 1 160 43.1 1 028 42.4 860 41.1 981 42.9 4 029 42.4 0.541 Nonmetropolitan 1 534 56.9 1 399 57.6 1 232 58.9 1 305 57.1 5 470 57.6 Type of medical school Medical college 1 137 42.2 979 40.3 839 40.1 808 35.3 3 763 39.6 <0.001 Graduate medical school 1 557 57.8 1 448 59.7 1 253 59.9 1 478 64.7 5 736 60.4 Location of medical school Metropolitan 871 32.3 847 34.9 547 26.1 891 39.0 3 156 33.2 <0.001 Nonmetropolitan 1 823 67.7 1 580 65.1 1 545 73.9 1 395 61.0 6 343 66.8 Age (mean, SD) 23.82 (2.94) 25.00 (2.99) 26.34 (3.14) 27.37 (3.05) 25.53 (3.32) <0.001 Chi-square test was performed for gender, hometown, type of medical school, and location of medical school. Analysis of variance (ANOVA) was performed for age Kim et al. Human Resources for Health (2016) 14:45 Page 4 of 8 specialties was highest for internal medicine (29.8 %), followed by psychiatry (7.8 %), and pediatrics (6.8 %), whereas the preference among the surgical specialties was highest for orthopedic surgery (9.0 %), followed by general surgery (6.7 %), and ophthalmology (3.7 %) (Table 2). The percentage of students who preferred medical specialties was the highest (67.6 %), with the percentages of students choosing surgical specialties college students (39.6 %), and the number of students in nonmetropolitan medical schools (66.8 %) was higher than that in metropolitan medical schools (33.2 %). The average age was 25.5 years old. *P < 0.05, chi-square test was performed for specialty (grouped by grade and gender) Descriptive analysis Human Resources for Health (2016) 14:45 Page 5 of 8 Page 5 of 8 and nonclinical medicine being 30.4 % and 2.0 %, respectively. greater desire to specialize in nonclinical medicine than the graduate medical school students did, and the differ- ence was significant. Other variables were not statisti- cally significant. In the subgroup analysis by gender, men were more likely to favor nonclinical medicine when they were medical college students rather than graduate medical school students and if they were born in a metropolitan area than in a nonmetropolitan area. These differences were significant. In women, there was a sig- nificant difference by grade, with the first year students showing a higher preference for nonclinical medicine. The difference between medical college and graduate medical school was not significant. In all grades, medical specialties were preferred more by women than men, except rehabilitative medicine, emer- gency medicine, occupational and environmental medi- cine, nuclear medicine, and tuberculosis, which were favored more by men. The preference for the medical specialties by grade was highest in the third year in the men (62.0 %) and the second year in the women (80.9 %) and was lowest in the first year in both the men (59.5 %) and the women (76.3 %). However, psychiatry and re- habilitative medicine were most preferred by the first year students, whereas anesthesiology, family medicine, thera- peutic radiology, and occupational and environmental medicine were most preferred by the fourth year students in both men and women. The number of students desiring a surgical specialty was 2892 out of 9313, after excluding students who wished to specialize in nonclinical medicine. Among them, 2168 were men and 724 were women. The results of the logistic regression for factors affecting the choice of surgical specialties are presented in Table 4. A prefer- ence for surgical specialties was significantly higher in men than in women and in graduate medical school stu- dents than in medical college students. Other variables were not statistically significant. The subgroup analysis by gender revealed that, among the men, surgical spe- cialties were preferred by the younger students, by the graduate medical school students over medical college students, and by students at medical schools located in nonmetropolitan rather than metropolitan areas. For women, those in the first year preferred surgical special- ties. Other factors were not statistically significant. Descriptive analysis Surgical specialties were favored more by men than women in all grades, except obstetrics-gynecology, which was preferred by women. The highest preference for surgi- cal specialties was found in the first year in both men (38.7 %) and women (21.0%) and the lowest preference was found in the third year in men (36.0 %) and second year in women (17.6 %). However, general surgery was most preferred by men in the third year (8.5 %), and oto- laryngology and plastic surgery were most preferred by both the men and women in the fourth year. For nonclini- cal medicine, the highest preference was found in both men (2.0 %) and women (2.8 %) in the third year and the lowest preference was found in the first year men (1.8 %) and fourth year women (1.4 %). In the analysis of controllable lifestyle specialties, 2911 of the 8259 students (35.2 %) desired specialties with a controllable lifestyle, after excluding those who desired specialties not included in the existing controllable/non- controllable classification (1240 students). The results of the logistic regression for factors affecting specialty choice for controllable lifestyle specialties showed a Descriptive analysis In the descriptive analysis of the desired specialties of all the students, the preference among the medical Table 2 Specialty preferences of medical students (N = 9499) Grade/gender specialty 1st 2nd 3rd 4th Total Men Women Men Women Men Women Men Women Clinical Medical Internal medicine 478 (27.4)* 283 (29.9)* 461 (31.3)* 326 (34.2)* 368 (28.8)* 282 (34.6)* 350 (27.2)* 287 (28.8)* 2 835 (29.8) Psychiatry 144 (8.2)* 98 (10.3)* 94 (6.4) 84 (8.8) 82 (6.4) 68 (8.3) 83 (6.4) 91 (9.1) 744 (7.8) Pediatrics 51 (2.9)* 101 (10.7)* 45 (3.1)* 105 (11.0)* 58 (4.5)* 110 (13.5)* 50 (3.9)* 123 (12.3)* 643 (6.8) Dermatology 79 (4.5) 59 (6.2) 45 (3.1) 65 (6.8) 48 (3.8) 35 (4.3) 39 (3.0) 50 (5.0) 420 (4.4) Neurology 52 (3.0) 48 (5.1) 56 (3.8) 50 (5.2) 48 (3.8) 39 (4.8) 47 (3.6) 38 (3.8) 378 (4.0) Rehabilitative medicine 75 (4.3)* 36 (3.8)* 57 (3.9)* 24 (2.5)* 48 (3.8)* 14 (1.7)* 51 (4.0)* 32 (3.2)* 337 (3.5) Diagnostic radiology 57 (3.3)* 32 (3.4)* 37 (2.5) 43 (4.5) 35 (2.7) 26 (3.2) 33 (2.6) 46 (4.6) 309 (3.3) Anesthesiology 37 (2.1) 30 (3.2) 39 (2.6) 28 (2.9) 35 (2.7)* 20 (2.5)* 43 (3.3) 42 (4.2) 274 (2.9) Family medicine 22 (1.3) 20 (2.1) 23 (1.6) 33 (3.5) 23 (1.8) 23 (2.8) 24 (1.9) 37 (3.7) 205 (2.2) Emergency medicine 22 (1.3)* 8 (0.8)* 24 (1.6)* 8 (0.8)* 21 (1.6) 12 (1.5) 18 (1.4)* 8 (0.8)* 121 (1.3) Clinical pathology 6 (0.3) 7 (0.7) 3 (0.2) 4 (0.4) 6 (0.5) 5 (0.6) 2 (0.2)* 13 (1.3)* 46 (0.5) Therapeutic radiology 8 (0.5)* 1 (0.1)* 6 (0.4) 1 (0.1) 3 (0.2) 5 (0.6) 12 (0.9) 5 (0.5) 41 (0.4) Occupational and environmental medicine 5 (0.3) 0 (0.0) 4 (0.3) 0 (0.0) 8 (0.6) 3 (0.4) 12 (0.9) 5 (0.5) 37 (0.4) Nuclear medicine 3 (0.2) 0 (0.0) 3 (0.2) 0 (0.0) 7 (0.5) 0 (0.0) 3 (0.2) 0 (0.0) 16 (0.2) Tuberculosis 1 (0.1) 0 (0.0) 2 (0.1) 1 (0.1) 1 (0.1) 0 (0.0) 9 (0.7)* 1 (0.1)* 15 (0.2) Subtotal 1 040 (59.5)* 723 (76.3)* 899 (61.0)* 772 (80.9)* 791 (62.0)* 642 (78.7)* 776 (60.2)* 778 (78.0)* 6 421 (67.6) Surgical Orthopedic surgery 257 (14.7)* 14 (1.5)* 222 (15.1)* 4 (0.4)* 169 (13.2)* 7 (0.9)* 174 (13.5)* 6 (0.6)* 853 (9.0) General surgery 129 (7.4)* 67 (7.1)* 102 (6.9)* 46 (4.8)* 109 (8.5)* 50 (6.1)* 79 (6.1)* 51 (5.1)* 633 (6.7) Ophthalmology 83 (4.8)* 50 (5.3)* 57 (3.9)* 27 (2.8)* 21 (1.6) 20 (2.5) 52 (4.0) 41 (4.1) 351 (3.7) Neurosurgery 87 (5.0)* 20 (2.1)* 61 (4.1)* 21 (2.2)* 54 (4.2)* 5 (0.6)* 53 (4.1)* 7 (0.7)* 308 (3.2) Otolaryngology 50 (2.9)* 16 (1.7)* 37 (2.5)* 18 (1.9)* 35 (2.7)* 13 (1.6)* 41 (3.2) 28 (2.8) 238 (2.5) Plastic surgery 32 (1.8)* 2 (0.2)* 34 (2.3)* 14 (1.5)* 36 (2.8)* 12 (1.5)* 63 (4.9)* 16 (1.6)* 209 (2.2) Obstetrics-gynecology 8 (0.5) 15 (1.6) 3 (0.2)* 31 (3.2)* 9 (0.7)* 38 (4.7)* 8 (0.6)* 45 (4.5)* 157 (1.7) Cardiothoracic surgery 27 (1.5)* 14 (1.5)* 26 (1.8)* 6 (0.6)* 20 (1.6)* 6 (0.7)* 12 (0.9) 10 (1.0) 121 (1.3) Urology 3 (0.2) 1 (0.1) 4 (0.3) 1 (0.1) 6 (0.5) 0 (0.0) 5 (0.4) 2 (0.2) 22 (0.2) Subtotal 676 (38.7)* 199 (21.0)* 546 (37.1)* 168 (17.6)* 459 (36.0)* 151 (18.5)* 487 (37.8)* 206 (20.6)* 2 892 (30.4) Nonclinical Pathology 10 (0.6) 8 (0.8) 6 (0.4) 7 (0.7) 12 (0.9) 16 (2.0) 9 (0.7) 8 (0.8) 76 (0.8) Basic science of medicine 17 (1.0) 9 (1.0) 16 (1.1)* 3 (0.3)* 9 (0.7)* 2 (0.2)* 11 (0.9)* 3 (0.3)* 70 (0.7) Preventive medicine 4 (0.2) 8 (0.8) 6 (0.4) 4 (0.4) 5 (0.4) 5 (0.6) 5 (0.4) 3 (0.3) 40 (0.4) Subtotal 31 (1.8) 25 (2.6) 28 (1.9) 14 (1.5) 26 (2.0) 23 (2.8) 25 (1.9) 14 (1.4) 186 (2.0) *P hi f d f i l ( d b d d d ) Table 2 Specialty preferences of medical students (N = 9499) Table 2 Specialty preferences of medical students (N = 9499) Grade/gender specialty 1st M W Kim et al. Logistic regression results The number of students hoping to specialize in nonclini- cal medicine was 186 out of the 9499 (2.0 %); among these, 110 were men and 76 were women. The results of the logistic regression for nonclinical medicine are pre- sented in Table 3. The medical college students had a Table 3 Logistic regression for factors associated with nonclinical medicine specialty N = 9499) All Subgroup analysis aOR (95 % CI) Men Women aOR (95 % CI) aOR (95 % CI) Men (ref: women) 0.851 (0.628–1.152) Age 1.052 (0.998–1.110) 1.038 (0.971–1.110) 1.098 (0.999–1.207) Grade 1st Reference Reference Reference 2nd 0.780 (0.518–1.174) 1.040 (0.617–1.753) 0.488* (0.249–0.956) 3rd 1.011 (0.671–1.522) 1.047 (0.603–1.820) 0.857 (0.459–1.597) 4th 0.689 (0.438–1.084) 0.964 (0.541–1.717) 0.374* (0.176–0.793) Medical college (ref: graduate medical school) 1.625* (1.139–2.318) 1.768* (1.114–2.807) 1.484 (0.823–2.677) Metropolitan hometown (ref: nonmetropolitan) 1.161 (0.857–1.573) 1.513* (1.023–2.240) 0.783 (0.481–1.274) Metropolitan school (ref: nonmetropolitan) 1.183 (0.858–1.631) 1.198 (0.794–1.808) 1.189 (0.709–1.993) *P < 0.05 egression for factors associated with nonclinical medicine specialty N = 9499) Kim et al. Human Resources for Health (2016) 14:45 Page 6 of 8 Table 4 Logistic regression for factors associated with surgical specialty preference (N = 9313) All Subgroup analysis aOR (95 % CI) Men Women aOR (95 % CI) aOR (95 % CI) Men (ref: women) 2.537* (2.296–2.804) Age 0.985 (0.968–1.003) 0.974* (0.954–0.995) 1.024 (0.986–1.064) Grade 1st Reference Reference Reference 2nd 0.907 (0.802–1.027) 0.967 (0.836–1.119) 0.768* (0.608–0.970) 3rd 0.909 (0.795–1.040) 0.949 (0.809–1.113) 0.805 (0.625–1.038) 4th 1.022 (0.890–1.173) 1.065 (0.903–1.256) 0.876 (0.677–1.135) Medical college (ref: graduate medical school) 0.885* (0.790–0.991) 0.849* (0.743–0.970) 1.009 (0.810–1.256) Metropolitan hometown (ref: nonmetropolitan) 1.031 (0.938–1.134) 1.055 (0.941–1.182) 0.970 (0.815–1.154) Metropolitan school (ref: nonmetropolitan) 0.892* (0.806–0.988) 0.840* (0.743–0.949) 1.032 (0.858–1.241) *P < 0.05 Table 4 Logistic regression for factors associated with surgical specialty preference (N = 9313) logistic regression can be summarized as follows. Nonclin- ical medicine was more preferred by medical college stu- dents than by graduate medical school students, by students from metropolitan areas among the men, and by lower grade students among the women. Surgical special- ties were favored more by men than by women, by gradu- ate medical school students than by medical college students, and by medical students from medical schools in nonmetropolitan areas than in metropolitan areas. The women favored the controllable lifestyle specialties more than the men did. Logistic regression results significantly higher preference in women over men, first year over second and third years, and medical college students over graduate medical school students (Table 5). For further comparison, the analysis was performed after categorizing the specialties with more than 50 % of self- employed specialists as controllable lifestyle specialties (Table 5). The results showed that women, older students, students originally from a metropolitan area, students cur- rently attending a nonmetropolitan medical school, and fourth year students (reference first year) had a stronger preference for a controllable lifestyle specialty. According to the classification of specialties by Organ- isation for Economic Cooperation and Development (OECD) health data [3], the proportion of specialists in Korea in 2013 was found to be 72.7 %, including 34.7 % in a medical specialty, 32.9 % in a surgical specialty, and 5.1% in other specialties. Compared to the United States and United Kingdom, the percentage of medical special- ists in Korea appears to be similar, whereas the percent- age of surgical specialists is higher [3]. However, in a Discussion In this study, we examined the desired specialties of med- ical students and the factors affecting specialty choice. We found that medical specialties were most favored by women and middle grade students, whereas surgical spe- cialties were most preferred by men and lower grade stu- dents. Nonclinical medicine was most favored by lower grade students. The statistically significant results of the Table 5 Logistic regression for factors associated with controllable lifestyle specialty preference Controllable lifestyle Self-employed specialty [N = 8259] [N = 9499] aOR (95 % CI) aOR (95 % CI) Men (ref: women) 0.802* (0.730–0.881) 0.519* (0.470–0.573) Age 0.992 (0.974–1.011) 1.021* (1.002–1.041) Grade 1st Reference Reference 2nd 0.848* (0.749–0.961) 0.982 (0.858–1.124) 3rd 0.779* (0.679–0.894) 0.987 (0.853–1.142) 4th 0.965 (0.839–1.110) 1.229* (1.061–1.424) Medical college (ref: graduate medical school) 1.198* (1.067–1.344) 1.118 (0.990–1.263) Metropolitan hometown (ref: nonmetropolitan) 0.992 (0.901–1.092) 1.143* (1.033–1.264) Metropolitan school (ref: nonmetropolitan) 1.030 (0.930–1.140) 0.779* (0.698–0.870) *P < 0.05 Kim et al. Human Resources for Health (2016) 14:45 Page 7 of 8 Page 7 of 8 were preferred by men in both Korea and the United States. However, in the United States, some surgical sub- specialties have become more appealing to women in recent years [17]. Therefore, routine surveys investigat- ing trends in specialty preferences are needed. Yet, there was no difference in preferences between medical col- lege students and graduate medical students according to the popularity of the subspecialty. Therefore, the trend appears to be caused by the broad category of fea- tures of the surgical specialty, rather than by noncom- petitive subspecialties. However, in studies in the United States, MD-PhD students had a lower preference for sur- gical specialties, unlike those in Korea [25, 26]. The results are not easily compared with those of graduate medical schools in Korea, considering the environmental differences. It was found that nonmetropolitan medical students had a stronger preference for surgical special- ties than metropolitan medical students, and it can be assumed that there are relatively more job opportunities in nonmetropolitan hospitals than in metropolitan hos- pitals, where physician manpower is saturated [27, 28]. comparison with the United States [19] at the level of in- dividual specialties, Korea showed a relatively stronger preference for orthopedic surgery and neurosurgery ra- ther than general surgery, and this seems attributable to the fact that the income levels of orthopedic surgeons and neurosurgeons are much higher than that of others [20, 21]. Discussion The present application status for specialties in 2015, which consists of applications made by upper grade stu- dents who participated in the survey in 2013, revealed that the order of preferred specialties was internal medi- cine, orthopedic surgery, family medicine, pediatrics, anesthesiology, diagnostic radiology, and psychiatry [22]. Although it is impossible to make direct comparisons, similar tendencies were observed in the results of our survey. However, certain specialties, such as family medicine, were ranked higher among the actual applica- tions but lower in the survey, and other specialties showed the opposite pattern; therefore, further studies are needed to explain these differences. Nonclinical medicine was favored more by medical college students than by graduate medical school stu- dents, which is contrary to the government initiative of utilizing graduate medical schools. In the United States, medical scientist training programs have been used to nurture basic scientists with a medical background, and they have been effective to a certain extent [23]. A simi- lar program for nurturing basic scientists in medicine was employed in graduate medical school programs in Korea, but it was discontinued because of its lack of results [10]. According to this study, prioritizing policies for medical colleges rather than graduate medical schools, and to women rather than men, should be more effective. Grade-dependent patterns of preferences showed a clear difference among the women in this study; here, nonclinical medicine was preferred more by the students in the lower grade, which is when most of the courses in the basic science of medicine are taken. Therefore, the curriculum needs to be improved to enable students in the upper grade to experience the basic science of medi- cine as well. In particular, for the specialties requiring greater demands that are chosen least often by the students, the trends in specialty choice during the years as a medical student are more important [24]. Therefore, these implications should be addressed more seriously in the curriculum. Women were found to have a stronger preference for a controllable lifestyle specialty. The percentage of stu- dents (upper grade) selecting the specialties categorized as controllable lifestyle was higher in women (39.0 %) than in men (34.6 %), which is different from the results of a study by Lambert et al. [17], in which men had a stronger preference than women for a controllable life- style specialty (men 40.7 %, women 34.4 %). Conclusions In this study, a descriptive analysis was performed on the choice of specialty and we were able to identify factors affecting students’ choices of nonclinical medicine, surgical specialties, and controllable lifestyle specialties. Nonclinical medicine was more favored by medical college students, and surgical specialties were more favored by men, gradu- ate medical school students, and students from nonmetro- politan medical schools. Depending on the type of analysis (using the existing categorizations or the percentage of self-employed specialists), the factors influencing the choice of a controllable lifestyle specialty were different. However, in both approaches, women consistently showed a stronger preference for a controllable lifestyle specialty. Based on these results, we can evaluate the effectiveness of governmental policies to resolve the supply imbalance and provide theoretical evidence for a solution to it. 12. World Health Organization. World directory of medical schools. 6th ed. Geneva: World Health Organization; 1988. 13. Kim H, Park S, Kim J, Park E, Lee H. Factors influencing the specialty selection of medical students. Korean J Med Educ. 2003;15:151–61. 13. Kim H, Park S, Kim J, Park E, Lee H. Factors influencing the specialty selection of medical students. Korean J Med Educ. 2003;15:151–61. 14. Lee JH, Kim GI, Park KH, Yune S-J. Differences in factors affecting medical specialty choices between medical college students and graduate medical school students. Korean J Med Educ. 2009;21:393–402. 14. Lee JH, Kim GI, Park KH, Yune S-J. Differences in factors affecting medical specialty choices between medical college students and graduate medical school students. Korean J Med Educ. 2009;21:393–402. 15. Enari T, Hashimoto H. Does salary affect the choice of residency in non- university teaching hospitals? A panel analysis of Japan Residency Matching Programme data. Hum Resour Health. 2013;11:12. 16. Dorsey ER, Jarjoura D, Rutecki GW. Influence of controllable lifestyle on recent trends in specialty choice by US medical students. JAMA. 2003; 290:1173–8. 16. Dorsey ER, Jarjoura D, Rutecki GW. Influence of controllable lifestyle on recent trends in specialty choice by US medical students. JAMA. 2003; 290:1173–8. 17. Lambert EM, Holmboe ES. The relationship between specialty choice and gender of US medical students, 1990–2003. Acad Med. 2005;80:797–802. 18. Oh KK. Nationwide doctor status report 2013. Korean Medical Association Healthcare Policy Forum. 2014;12:78–88. 19. Jagsi R, Griffith KA, DeCastro RA, Ubel P. Sex, role models, and specialty choices among graduates of US medical schools in 2006–2008. J Am Coll Surgeons. 2014;218:345–52. 20. Author details 1D f Author details 1Department of Health Policy and Management, Seoul National University College of Medicine, 110-799 103 Daehak-ro, Jongno-gu, Seoul, Republic of Korea. 2Department of Internal Medicine, Seoul National University College of Medicine, 110-799 103 Daehak-ro, Jongno-gu, Seoul, Republic of Korea. 3Institute of Health Policy and Management, Medical Research Center, Seoul National University, Seoul, Republic of Korea. 27. Ahn D. The end of internship training in South Korea. J Korean Medical Association. 2013;56:352–4. 27. Ahn D. The end of internship training in South Korea. J Korean Medical Association. 2013;56:352–4. 28. Seo HJ, Ahn YJ. Changes in background and characteristics in freshmen in one graduate medical school from 2009 to 2011. Korean J Med Educ. 2012; 24:163–8. 28. Seo HJ, Ahn YJ. Changes in background and characteristics in freshmen in one graduate medical school from 2009 to 2011. Korean J Med Educ. 2012; 24:163–8. Received: 30 September 2015 Accepted: 13 July 2016 29. Hundley G. Why and when are the self‐employed more satisfied with their work? Ind Relat. 2001;40:293–316. 29. Hundley G. Why and when are the self‐employed more satisfied with their work? Ind Relat. 2001;40:293–316. Competing interests The authors declare that they have no competing interests. 25. Andriole DA, Whelan AJ, Jeffe DB. Characteristics and career intentions of the emerging MD/PhD workforce. JAMA. 2008;300:1165–73. 25. Andriole DA, Whelan AJ, Jeffe DB. Characteristics and career intentions of the emerging MD/PhD workforce. JAMA. 2008;300:1165–73. The authors declare that they have no competing interests. 26. Andriole DA, Klingensmith ME, Jeffe DB. Who are our future surgeons? Characteristics of medical school graduates planning surgical careers: analysis of the 1997 to 2004 Association of American Medical Colleges’ Graduation Questionnaire National Database. J Am Coll Surgeons. 2006; 203:177–85. Authors’ contributions YYK UNK d JSL d i Authors’ contributions YYK, UNK, and JSL designed the study and analyzed the data. YSK conducted the literature review and edited the manuscript. All authors reviewed and approved the final manuscript. 23. Zemlo TR, Garrison HH, Partridge NC, Ley TJ. The physician-scientist: career issues and challenges at the year 2000. FASEB J. 2000;14:221–30. 23. Zemlo TR, Garrison HH, Partridge NC, Ley TJ. The physician-scientist: career issues and challenges at the year 2000. FASEB J. 2000;14:221–30. 24. Compton MT, Frank E, Elon L, Carrera J. Changes in US medical students’ specialty interests over the course of medical school. J Gen Intern Med. 2008;23:1095–100. 24. Compton MT, Frank E, Elon L, Carrera J. Changes in US medical students’ specialty interests over the course of medical school. J Gen Intern Med. 2008;23:1095–100. Kim et al. Human Resources for Health (2016) 14:45 Kim et al. Human Resources for Health (2016) 14:45 Page 8 of 8 Page 8 of 8 able to identify factors affecting specialty choice and provide evidence for solutions to the imbalances in physician specialties. The study is limited by the failure to include a variety of variables relevant to specialty choice. Individual factors (e.g., marital status, number of dependent children, student academic performance, and financial status/amount of student debt) and specialty- related factors (e.g., labor demands, remuneration, spe- cialty prestige, and the length of training) might signifi- cantly influence specialty choice although they have not been fully examined in previous studies. However, the present study has significant implications as the sample included most of the medical students in Korea. Con- tinuous research on factors affecting specialty choice based on this study will be required in the future. 9. Kim KJ, Park JH, Lee YH, Choi K. What is different about medical students interested in non-clinical careers? BMC Med Educ. 2013;13:81. 10. Shin JS. A review on the courses of the introduction of post-baccalaureate basic medical education system in Korea. Korean J Med Educ. 2006;18:121–32. 11. Choi JS, Suh DJ, Chai JY, Ohrr H, Hwang IK, Kang DY. A study of medical education system in Korea. Korean J Med Educ. 1996;8:189–99. Conclusions Lim SM, Im GJ, Park KJ, Park YH. An analysis on patients trend and income of primary care clinic. Health Policy and Management. 2014;24:92–9. Acknowledgements We appreciate the kind support for the data collection and administrative assistance from the Ministry of Health and Welfare. Acknowledgements h k d 21. Park WS, Kim HJ, Sohn M, Park EC. Analysis of influencing factors on hospital-employed physician’s income. Health Policy and Management. 1999;9:1–20. 22. Korea Hospital Association. Residents match data 2015. [http://www. khanews.com/news/articleView.html?idxno=107211]. Accessed 20 Jul 2015 22. Korea Hospital Association. Residents match data 2015. [http://www. khanews.com/news/articleView.html?idxno=107211]. Accessed 20 Jul 2015 2. Diallo K, Zurn P, Gupta N, Dal Poz M. Monitoring and evaluation of human resources for health: an international perspective. Hum Resour Health. 2003;1:3. 3. Organisation for Economic Cooperation and Development. OECD health statistics 2015. [http://stats.oecd.org/index.aspx?DataSetCode=HEALTH_ STAT]. Accessed 19 Jul 2015. 4. Park WS, Kim HJ, Sohn MS, Park EC. Analysis of influencing factors on self- employed physician’s income. Korean J Prev Med. 1998;31:770–85. 5. Park JH, Kim KH, Jun HR, Lee JY. A national sample survey of medical students about their perception and evaluation on medical study, career plan, and medical care system: part 1. Survey methods and characteristics of sample. Korean J Med Educ. 1999;11:339–63. 6. Kang SH, Boo YJ, Han HJ, Jung CW, Kim CS. High occupational stress and low career satisfaction of Korean surgeons. J Korean Med Sci. 2015;30:133–9. 7. Gagné R, Léger PT. Determinants of physicians’ decisions to specialize. Health Econ. 2005;14:721–35. 8. Nicholson S, Souleles NS. Physician income expectations and specialty choice. National Bureau of Economic Research; 2001. 9. Kim KJ, Park JH, Lee YH, Choi K. What is different about medical students interested in non-clinical careers? BMC Med Educ. 2013;13:81. 10. Shin JS. A review on the courses of the introduction of post-baccalaureate basic medical education system in Korea. Korean J Med Educ. 2006;18:121–32. 11. Choi JS, Suh DJ, Chai JY, Ohrr H, Hwang IK, Kang DY. A study of medical education system in Korea. Korean J Med Educ. 1996;8:189–99. 12. World Health Organization. World directory of medical schools. 6th ed. Geneva: World Health Organization; 1988. 13. Kim H, Park S, Kim J, Park E, Lee H. Factors influencing the specialty selection of medical students. Korean J Med Educ. 2003;15:151–61. 14. Lee JH, Kim GI, Park KH, Yune S-J. Differences in factors affecting medical specialty choices between medical college students and graduate medical school students. Korean J Med Educ. 2009;21:393–402. 15. Enari T, Hashimoto H. Does salary affect the choice of residency in non- university teaching hospitals? A panel analysis of Japan Residency Matching Programme data. Hum Resour Health. 2013;11:12. 16. Dorsey ER, Jarjoura D, Rutecki GW. Influence of controllable lifestyle on recent trends in specialty choice by US medical students. JAMA. 2003; 290:1173–8. 17. Lambert EM, Holmboe ES. The relationship between specialty choice and gender of US medical students, 1990–2003. Acad Med. 2005;80:797–802. 18. Oh KK. Nationwide doctor status report 2013. Korean Medical Association . Zurn P, Dal Poz MR, Stilwell B, Adams O. Imbalance in the health workforce. Hum Resour Health. 2004;2:13. 5. Park JH, Kim KH, Jun HR, Lee JY. A national sample survey of medical students about their perception and evaluation on medical study, career plan, and medical care system: part 1. Survey methods and characteristics of sample. Korean J Med Educ. 1999;11:339–63. 8. Nicholson S, Souleles NS. Physician income expectations and specialty choice. National Bureau of Economic Research; 2001. 4. Park WS, Kim HJ, Sohn MS, Park EC. Analysis of influencing factors on self- employed physician’s income. Korean J Prev Med. 1998;31:770–85. 20. Lim SM, Im GJ, Park KJ, Park YH. An analysis on patients trend and income of primary care clinic. Health Policy and Management. 2014;24:92–9. Discussion It is impos- sible to explain fully the discrepancy in specialty preference according to gender, but it is possible that a characteristic of the Korean culture, in which women have more responsibility for the housekeeping burden, could have had an effect. Specialties for which more than 50 % of the specialists went into clinical practice were favored by students who were women, older, from a metropolitan area, currently attending a nonmetropoli- tan medical school, and in their fourth year of studies. Both analyses found that women had a stronger prefer- ence for a controllable lifestyle specialty. Controllable lifestyle specialties were defined as those with less stress, more free time, a lower amount of work-related activity, and fewer demands. Considering a report [29] showing that self-employed doctors are more satisfied because they have autonomy, flexibility, skill utilization, and higher job security, we can also regard this distinction as an applic- able standard for a controllable lifestyle specialty. Surgical specialties were more favored by men, which is consistent with a previous study [13, 17], which found that graduate medical school students had a stronger preference for surgical specialties than medical college students did. Surgical specialties are a mixture of highly competitive (plastic surgery and orthopedic surgery) and noncompetitive subspecialties (cardiothoracic surgery and general surgery). We found that surgical specialties In order to solve the imbalances of physician supply, it is essential to monitor major determining factors, in- cluding factors influencing specialty choice [30]. Spe- cialty choice varies according to the grade and the curriculum of the medical student. Therefore, the rou- tine monitoring of students’ preferences should help track preference changes. Through this study, we were 25. Andriole DA, Whelan AJ, Jeffe DB. Characteristics and career intentions of the emerging MD/PhD workforce. JAMA. 2008;300:1165–73. 27. Ahn D. The end of internship training in South Korea. J Korean Medical Association. 2013;56:352–4. 6. Kang SH, Boo YJ, Han HJ, Jung CW, Kim CS. High occupational stress and low career satisfaction of Korean surgeons. J Korean Med Sci. 2015;30:133–9. 7 G é R Lé PT D t i t f h i i ’ d i i t i li 9. Kim KJ, Park JH, Lee YH, Choi K. What is different about medical students interested in non-clinical careers? BMC Med Educ. 2013;13:81. 21. Park WS, Kim HJ, Sohn M, Park EC. Analysis of influencing factors on hospital-employed physician’s income. Health Policy and Management. 1999;9:1–20. References 30. Stordeur S, Léonard C. Challenges in physician supply planning: the case of Belgium. Hum Resour Health. 2010;8:28. 30. Stordeur S, Léonard C. Challenges in physician supply planning: the case of Belgium. Hum Resour Health. 2010;8:28.
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Density responses of lesser-studied carnivores to habitat and management strategies in southern Tanzania’s Ruaha-Rungwa landscape
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OPEN ACCESS Compared to emblematic large carnivores, most species of the order Carnivora receive little conservation attention despite increasing anthropogenic pressure and poor understanding of their status across much of their range. We employed systematic camera trapping and spatially explicit capture-recapture modelling to estimate variation in population density of serval, striped hyaena and aardwolf across the mixed-use Ruaha-Rungwa landscape in southern Tanzania. We selected three sites representative of different habitat types, man- agement strategies, and levels of anthropogenic pressure: Ruaha National Park’s core tour- ist area, dominated by Acacia-Commiphora bushlands and thickets; the Park’s miombo woodland; and the neighbouring community-run MBOMIPA Wildlife Management Area, also covered in Acacia-Commiphora. The Park’s miombo woodlands supported a higher serval density (5.56 [Standard Error = ±2.45] individuals per 100 km2) than either the core tourist area (3.45 [±1.04] individuals per 100 km2) or the Wildlife Management Area (2.08 [±0.74] individuals per 100 km2). Taken together, precipitation, the abundance of apex pred- ators, and the level of anthropogenic pressure likely drive such variation. Striped hyaena were detected only in the Wildlife Management Area and at low density (1.36 [±0.50] individ- uals per 100 km2), potentially due to the location of the surveyed sites at the edge of the spe- cies’ global range, high densities of sympatric competitors, and anthropogenic edge effects. Finally, aardwolf were captured in both the Park’s core tourist area and the Wildlife Manage- ment Area, with a higher density in the Wildlife Management Area (13.25 [±2.48] versus 9.19 [±1.66] individuals per 100 km2), possibly as a result of lower intraguild predation and late fire outbreaks in the area surveyed. By shedding light on three understudied African car- nivore species, this study highlights the importance of miombo woodland conservation and community-managed conservation, as well as the value of by-catch camera trap data to improve ecological knowledge of lesser-studied carnivores. Citation: Hardouin M, Searle CE, Strampelli P, Smit J, Dickman A, Lobora AL, et al. (2021) Density responses of lesser-studied carnivores to habitat and management strategies in southern Tanzania’s Ruaha-Rungwa landscape. PLoS ONE 16(3): e0242293. https://doi.org/10.1371/journal. PLOS ONE PLOS ONE RESEARCH ARTICLE Marie HardouinID1*, Charlotte E. Searle2, Paolo Strampelli2, Josephine Smit3,4, Amy Dickman2, Alex L. LoboraID5, J. Marcus Rowcliffe1,6 Marie HardouinID1*, Charlotte E. Searle2, Paolo Strampelli2, Josephine Smit3,4, Amy Dickman2, Alex L. LoboraID5, J. Marcus Rowcliffe1,6 1 Faculty of Natural Sciences, Imperial College London, Ascot, United Kingdom, 2 Wildlife Conservation Research Unit, Department of Zoology, The Recanati-Kaplan Centre, Tubney, United Kingdom, 3 Southern Tanzania Elephant Program, Iringa, Tanzania, 4 Department of Psychology, University of Stirling, Stirling, United Kingdom, 5 Tanzania Wildlife Research Institute, Arusha, Tanzania, 6 Institute of Zoology, Zoological Society of London, London, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * marie.acx.hardouin@gmail.com * marie.acx.hardouin@gmail.com Introduction Emblematic large carnivores often benefit from significant conservation investment and are prioritised in terms of resource allocation over more threatened species [1]. Such prioritisation stems in part from their essential role in shaping ecosystems [2], but also their charisma and cultural significance, which are used to highlight the problem of biodiversity loss to the public [3, 4]. In comparison, carnivores with smaller body size or less aesthetic appeal tend not to spark as much conservation interest and remain understudied globally [1, 5]. The African con- servation landscape conspicuously illustrates these disparities, with iconic apex predators such as lion (Panthera leo), leopard (Panthera pardus) and cheetah (Acinonyx jubatus) drawing sub- stantial research attention [6]. Overshadowed by these flagship species, many lesser-studied carnivores nonetheless face declining trends in number and range [7], with anthropogenic threats including habitat loss/fragmentation [8], and persecution [9] jeopardising their survival alongside a growing human population [10]. This lack of research hinders conservation status assessments for a number of African car- nivores, thus precluding effective conservation planning [11], notwithstanding the ecological importance and vulnerability to extinction of many of these species. For instance, striped hyaena (Hyaena hyaena) have received very little conservation attention across their African range, although they provide essential ecosystem services in consuming carcasses and dispers- ing their nutrients [12]. The species is thought to be declining globally, mainly caused by poi- soning and decreasing sources of carrion, leading to its classification as “Near Threatened” [13]. Moreover, little information exists on the population status of many mesocarnivores, despite their key contribution to regulating lower trophic levels and maintaining biodiversity [14]. Classified as “Least Concern”, serval (Leptailurus serval) and aardwolf (Proteles cristata) nevertheless face increasing pressure due to continued urbanisation and agricultural expan- sion [15, 16]. Serval are particularly vulnerable to the degradation of wetland and grassland habitat, as the species prefers well-watered environments which attract higher densities of small mammals [17]. The insectivorous diet of the aardwolf exposes the species to insecticide poisoning, an ever-increasing threat with the intensification of agriculture [15, 18]. Competing interests: The authors have declared that no competing interests exist. The IUCN recommends further research to bridge knowledge gaps for these lesser-studied carnivore species’ spatial ecology and population status [13, 15, 16]. OPEN ACCESS pone.0242293 Editor: Bi-Song Yue, Sichuan University, CHINA Received: October 28, 2020 Accepted: March 16, 2021 Published: March 30, 2021 Editor: Bi-Song Yue, Sichuan University, CHINA Received: October 28, 2020 Accepted: March 16, 2021 Published: March 30, 2021 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0242293 Copyright: © 2021 Hardouin 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: The data used in this study is freely available at https://doi.org/10.5281/ zenodo.4041430. 1 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 PLOS ONE Population densities of lesser-studied carnivores in southern Tanzania’s Ruaha-Rungwa landscape Funding: Scholarship funding for CS and PS is provided by the University of Oxford NERC Environmental Research DTP (https://www. environmental-research.ox.ac.uk). AD is funded by a Recanati-Kaplan Fellowship (https://www. wildcru.org). JMR is supported by Research England funding to the Institute of Zoology (https:// re.ukri.org/funding/). Additional funding was awarded to CS for this research from National Geographic Society Early Career Grants (https:// www.nationalgeographic.org/funding- opportunities/grants), Cleveland Metroparks Zoo Africa Seed Grants (https://www. clevelandmetroparks.com/zoo), Chicago Zoological Society Chicago Board of Trade (CBOT) Endangered Species Fund (https://www.czs.org/ Chicago[1]Zoological-Society/Conservation% 20Leadership/CBOT-Endangered-Species-Fund/), and Pittsburgh Zoo & PPG Aquarium Conservation & Sustainability Fund (https://www.pittsburghzoo. org/conservation/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 Introduction The distribution of serval, striped hyaena and aardwolf, as well as population abundances and densities across their range, need to be investigated and accurately estimated to inform effective management strate- gies and conservation planning [19]. The cryptic nature of these species often impedes direct observation, and camera trapping offers an efficient alternative for collecting data [20]. Over the past decade, spatial capture-recapture [21], a well-established method for estimating the density of species with individual markings such as patterned coats, has been successfully used for several populations of these three carnivore species. Most of the published estimates for striped hyaena, however, concern Asiatic populations [22–24], with a single study of an Afri- can population from Kenya [25]. Spatial capture-recapture studies of aardwolf density are sim- ilarly limited, with only two published estimates from Kenya and Botswana [25, 26]. A few more surveys have been carried out for serval in Southern Africa [26–29], and Western and Central Africa [30, 31], revealing large variations in density across the species’ range. The lack of estimates and the disparity in published results suggest the need to investigate additional populations of serval, striped hyaena and aardwolf in areas where robust estimates do not exist. Such information is essential to better understand how and why population status varies across the species’ geographic range [32, 33]. The second largest National Park in East Africa at over 20,000 km2, Tanzania’s Ruaha National Park (RNP) belongs to the greater Ruaha-Rungwa landscape, which harbours 2 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 PLOS ONE Population densities of lesser-studied carnivores in southern Tanzania’s Ruaha-Rungwa landscape important wildlife biodiversity, partly due to the convergence of Acacia-Commiphora and miombo woodland (Brachystegia-Jubelnardia) ecotypes [34]. The Ruaha-Rungwa landscape also encompasses a range of fully and partially protected areas, including Game Reserves, Game Controlled Areas, Open Areas, and community-managed Wildlife Management Areas (WMA). Established on communal lands, WMAs are one of the most novel features in Tanza- nia’s conservation landscape, which intend to promote a sustainable scheme involving local communities in wildlife protection and its economic benefits [35]. Various sources and levels of anthropogenic pressure add to the vulnerability of carnivores in the Ruaha-Rungwa land- scape, including: habitat conversion for agriculture, prey base depletion due to bushmeat poaching, and direct persecution by humans in retaliation to livestock predation [36, 37]. Ethics statement Data collection consisted of camera trapping, a non-invasive method which avoids contact with the study species and minimises interference with their natural behaviour. Fieldwork was carried out under research permits 2018-368-NA-2018-107, 2019-96-ER-97-20 and 2019- 424-NA-2018-184, granted by the Tanzania Commission for Science and Technology (COST- ECH; Dar es Salaam, Tanzania; rclearance@costech.or.tz) and Tanzania Wildlife Research Institute (TAWIRI; Arusha, Tanzania; researchclearance@tawiri.or.tz). Introduction RNP also lies at the southern range limit for both the global striped hyaena population and the East & Northeast African aardwolf population [13, 15]. This ecosystem is therefore of particular conservation interest for the species, since peripheral populations often differ in density response and appear more sensitive to habitat change than more centrally located populations [38]. In addition to providing baseline status estimates for three African carnivore species for which insufficient data currently exist, this study intended to investigate local variations in density across different habitats, management strategies, and levels of protection and anthro- pogenic pressure. To this end, we used camera trapping and spatially explicit capture-recap- ture (SECR) modelling [39] to estimate population densities of serval, striped hyaena and aardwolf at three sites in Ruaha-Rungwa, representative of different ecosystem types and levels of protection and anthropogenic pressure: the Acacia-Commiphora core tourist area of RNP; the miombo woodland of western RNP; and the community-run Acacia-Commiphora MBO- MIPA WMA, which adjoins Ruaha to the east and acts as a buffer between the Park and unprotected village lands. PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 Study area https://doi.org/10.1371/journal.pone.0242293.g001 https://doi.org/10.1371/journal.pone.0242293.g001 MBOMIPA WMA was established in 2007 based on the principles of community-based natu- ral resource management [35] and encounters greater levels of anthropogenic impacts than the core tourist area of RNP [46]. The landscape is unfenced, which facilitates free movement of wildlife. Study area The Ruaha-Rungwa ecosystem in southern Tanzania extends over 45,000 km2 across three ecoregions: Central Zambezian miombo woodlands, Eastern miombo woodlands, and Eastern African acacia savannas [40] (Fig 1A and 1B). RNP lies at the centre of this ecosystem and was the largest National Park in Tanzania until the formation of Nyerere National Park in 2019 [41]. With an altitude ranging from 716 m to 1,888 m [42], the terrain features low rolling hills overlooked by an escarpment to the north, and the Great Ruaha River runs along the RNP’s eastern border [43]. RNP experiences a semi-arid to arid climate and a unimodal rainfall regime, from December to April [44]. Acacia-Commiphora deciduous bushlands and thickets predominate in about two-thirds of the park, while miombo woodlands cover its western part [45] (Fig 1B). A number of Game Reserves, Game Controlled Areas, and Open Areas complete the Ruaha-Rungwa landscape to the north and the west of RNP, while the Matumizi Bora ya Malihai Idodi na Pawaga (MBOMIPA) and Waga WMAs stretch along its eastern border. 3 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 PLOS ONE Population densities of lesser-studied carnivores in southern Tanzania’s Ruaha-Rungwa landscape Fig 1. Ruaha-Rungwa landscape and spatial distribution of camera trap stations. (A) Location of the Ruaha-Rungwa landscape in Tanzania (made with Natural Earth). (B) Ruaha-Rungwa landscape’s ecotypes [40] and land uses. The map depicts, but does not explicitly name, boundaries of additional protected areas, and only shows villages and towns near protected areas. (C) Core RNP Acacia-Commiphora grid. (D) RNP miombo grid. (E) MBOMIPA WMA Acacia-Commiphora grid. Fig 1. Ruaha-Rungwa landscape and spatial distribution of camera trap stations. (A) Location of the Ruaha-Rungwa landscape in Tanzania (made with Natural Earth). (B) Ruaha-Rungwa landscape’s ecotypes [40] and land uses. The map depicts, but does not explicitly name, boundaries of additional protected areas, and only shows villages and towns near protected areas. (C) Core RNP Acacia-Commiphora grid. (D) RNP miombo grid. (E) MBOMIPA WMA Acacia-Commiphora grid. Fig 1. Ruaha-Rungwa landscape and spatial distribution of camera trap stations. (A) Location of the Ruaha-Rungwa landscape in Tanzania (made with Natural Earth). (B) Ruaha-Rungwa landscape’s ecotypes [40] and land uses. The map depicts, but does not explicitly name, boundaries of additional protected areas, and only shows villages and towns near protected areas. (C) Core RNP Acacia-Commiphora grid. (D) RNP miombo grid. (E) MBOMIPA WMA Acacia-Commiphora grid. Camera trap surveys The study used a photographic dataset collected between June and November 2018 in RNP and MBOMIPA WMA to assess the status of leopard [46]. We surveyed two sites within RNP, and one in MBOMIPA WMA (Fig 1C–1E). The first camera trap grid covered an area of 223 km2 in highly-productive riverine Acacia-Commiphora habitat [47], at the core of the park’s tourist area, with 45 camera stations set out for 83 days. The second grid, located in miombo woodland in the western part of RNP, consisted of 26 camera stations over an area of 152 km2 4 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 PLOS ONE Population densities of lesser-studied carnivores in southern Tanzania’s Ruaha-Rungwa landscape for 90 days. We deployed the third grid in highly-productive riverine Acacia-Commiphora habitat in MBOMIPA WMA, with 40 stations spanning a total area of 270 km2 for 69 days. The duration of each survey was kept below 90 days both to ensure sufficient captures/ recaptures and to approximate closed populations, as per previous studies [20, 21, 28, 48–52]. The design of the camera trap layout sought to optimise capture rates of large carnivores, facili- tate individual identification, and ensure no gaps in the trapping array. Camera placement prioritised roads and junctions, or game trails, as large carnivores preferentially travel along roads [53]. With a single road crossing the study area within the park’s miombo woodland, half of the cameras in the RNP miombo grid were set on game trails along semi-open mbugas (drainage lines). The mean distance between two cameras ranged from 1.88 km in RNP miombo to 1.96 km and 2.08 km in Core RNP Acacia-Commiphora and WMA Acacia-Com- miphora, respectively. Given the published home range estimates for the target species [54, 55], this spacing enabled to recapture individuals at multiple camera trap stations [21, 56], in compliance with SECR requirements for modelling space use. All but one of the stations consisted of paired cameras facing one another on either side of a road or trail, to increase the likelihood of capturing both flanks of passing animals [48]. Cam- eras were mounted on trees at approximately 40 cm height, with the surrounding vegetation regularly cleared to prevent any obstruction of the lens or sensor and reduce the risk of bush fire damaging the camera. Camera trap surveys The survey deployed several motion-activated camera models: Cud- deback Professional Color Model 1347 and Cuddeback X-Change Color Model 1279, Non Typical Inc., Wisconsin, USA; HC500 HyperFire, Reconyx, Wisconsin, USA. The majority of cameras featured xenon flashes, which produce higher definition images of animal markings than infrared LED flashes [56]. PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 Density estimation We estimated the population density (defined as the number of individuals per 100 km2) of serval, striped hyaena, and aardwolf at each survey site via Maximum Likelihood Estimation SECR analysis, with the package secr v3.2.0 [57] in R version 3.6.3 [58]. SECR combines two sub-models representing the spatial distribution of a population and the detection process respectively [59], and therefore does not require ad hoc estimation of the effective trapping area as per conventional capture-recapture methods [60]. Data inputs consisted of detection histories detailing sampling occasions and locations of captures for each individual throughout the survey, and a trap layout listing the coordinates of every camera trap station and their activity periods (S2 and S3 Appendices). We visually iden- tified individuals in camera trap images through their unique pelage markings [48], focusing on flanks for serval and fore-quarters and hind-quarters for striped hyaena and aardwolf, as detailed in S1 Appendix. We sexed individuals based on the unobstructed view of external gen- italia, late pregnancy signs such as weight gain and enlarged abdomen (see S1 Appendix), or the presence of cubs. Individuals whose sex could not be confidently distinguished were classi- fied as “unknown sex” (coded NA in the detection histories). We selected the flank with the greatest number of capture events for each species and grid to produce detection histories, using the following framework: (i) sampling occasions spanned 24-hours from midday to mid- day, to account for the nocturnal nature of the studied species; (ii) individuals could be recorded at different locations during a sampling occasion, but only once per given location (as per standard practice [61]). The SECR observation sub-model describes the individual detection probability as a monotonous function of the distance to home range centre, characterised by g0, the detection probability at the range centre, and σ, a spatial scale related to home range width [62]. We 5 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 PLOS ONE Population densities of lesser-studied carnivores in southern Tanzania’s Ruaha-Rungwa landscape tested three standard functions (half-normal, negative exponential and hazard rate) to model the decrease of the detection function with the distance to the home range centre. To minimise any bias in estimated densities, we selected a buffer width at which density estimates reached a plateau for each analysis. Results Survey effort. The sampling effort across the 111 stations deployed at the three study sites totalled 8,477 camera trap nights and yielded 226 images of serval, 125 images of striped hyaena and 1,119 images of aardwolf (see S4 Appendix). We could identify individuals in 86.3% of serval photographs, 72.8% of striped hyaena photographs, and 81.1% of aardwolf photographs. Detection histories for serval featured 38 independent capture events for 13 iden- tified individuals in Core RNP Acacia-Commiphora, 31 events for 10 identified individuals in WMA Acacia-Commiphora, and 23 events for 12 identified individuals in RNP miombo. Striped hyaena were captured in WMA Acacia-Commiphora only, with 42 independent cap- ture events and 12 identified individuals. Aardwolf were only captured at camera stations in Core RNP Acacia-Commiphora and WMA Acacia-Commiphora, with 36 and 37 individuals identified, accounting for a total of 240 and 185 independent capture events, respectively. Aardwolf displayed the highest recapture rate, i.e. the percentage of individuals captured more than once, across all grids (84.9%), followed by striped hyaena (58.3%) and serval (57.1%). All captured individuals were adults or subadults, with the exception of one female serval and one aardwolf accompanied by offspring. The target species showed predominantly nocturnal activity, with 86% of identified serval captures, 98% of striped hyaena captures, and 99.4% of aardwolf captures occurring between 7 pm and 6 am. The poor visibility of genitalia and lack of clear secondary sexual traits limited sex determination to 2.9% of the identified ser- vals and 16.7% of identified striped hyaenas, preventing the modelling of sex differences in the density estimation process. Aardwolf sexing proved more successful, with sex assigned to 35.6% of identified individuals, which allowed us to model sex differences in detection func- tion parameters. Survey effort. The sampling effort across the 111 stations deployed at the three study sites totalled 8,477 camera trap nights and yielded 226 images of serval, 125 images of striped hyaena and 1,119 images of aardwolf (see S4 Appendix). We could identify individuals in 86.3% of serval photographs, 72.8% of striped hyaena photographs, and 81.1% of aardwolf photographs. Detection histories for serval featured 38 independent capture events for 13 iden- tified individuals in Core RNP Acacia-Commiphora, 31 events for 10 identified individuals in WMA Acacia-Commiphora, and 23 events for 12 identified individuals in RNP miombo. Striped hyaena were captured in WMA Acacia-Commiphora only, with 42 independent cap- ture events and 12 identified individuals. Density estimation We tested for variation in the baseline encounter probability g0 through the use of embed- ded predictors and covariates hypothesised to influence detection probability [63]. Specifically, we investigated the behavioural response of individuals to capture events by fitting the global trap response model (b predictor) and the local trap response model (bk predictor), and assessed the influence of the type of flash at each station (xenon versus LED flash), and station location (on-road versus off-road) on detection. Finally, we examined the effect of sex on detection function parameters g0 and σ by fitting a hybrid mixture model, which accommo- dates individuals of unknown sex [64]. Model selection was carried out by ranking models based on their Akaike Information Criterion score, corrected for small sample size (AICc) [65]. When more than one candidate model had substantial empirical support (ΔAICc < 2) [65], a final density estimate was derived by applying model averaging based on AICc weights [21]. PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 Results Aardwolf were only captured at camera stations in Core RNP Acacia-Commiphora and WMA Acacia-Commiphora, with 36 and 37 individuals identified, accounting for a total of 240 and 185 independent capture events, respectively. Aardwolf displayed the highest recapture rate, i.e. the percentage of individuals captured more than once, across all grids (84.9%), followed by striped hyaena (58.3%) and serval (57.1%). All captured individuals were adults or subadults, with the exception of one female serval and one aardwolf accompanied by offspring. The target species showed predominantly nocturnal activity, with 86% of identified serval captures, 98% of striped hyaena captures, and 99.4% of aardwolf captures occurring between 7 pm and 6 am. The poor visibility of genitalia and lack of clear secondary sexual traits limited sex determination to 2.9% of the identified ser- vals and 16.7% of identified striped hyaenas, preventing the modelling of sex differences in the density estimation process. Aardwolf sexing proved more successful, with sex assigned to 35.6% of identified individuals, which allowed us to model sex differences in detection func- tion parameters. Population density. Of the three detection functions tested, the negative exponential function best fitted the datasets for all target species at the three sites. We found no evidence of learned behavioural response or influence of covariates on detection for serval in Core RNP Acacia-Commiphora and WMA Acacia-Commiphora. However, the model in which g0 varied with station location was supported in RNP miombo. S5 Appendix details the ranking results based on AICc score. RNP miombo yielded the highest serval density, with 5.56 [Standard Error = ±2.45] individuals per 100 km2, followed by 3.45 [±1.04] individuals per 100 km2 in PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 6 / 15 PLOS ONE NE Population densities of lesser-studied carnivores in southern Tanzania’s Ruaha-Rungwa landscape Population densities of lesser-studied carnivores in southern Tanzania’s Ruaha-Rungwa landscape Fig 2. Population density estimates for the target species at the three survey sites. Bars represent standard errors. https://doi org/10 1371/journal pone 0242293 g002 Fig 2. Population density estimates for the target species at the three survey sites. Bars represent standard errors. https://doi org/10 1371/journal pone 0242293 g002 Fig 2. Population density estimates for the target species at the three survey sites. Bars represent standard errors. https://doi.org/10.1371/journal.pone.0242293.g002 Core RNP Acacia-Commiphora, and 2.08 [±0.74] individuals per 100 km2 in WMA Acacia- Commiphora (Fig 2, Table 1). Discussion Density comparison. This study produced the first population densities estimated through spatial capture-recapture for three lesser-studied carnivores, serval, striped hyaena and aardwolf, in Tanzania. Using camera trap data from the Ruaha-Rungwa landscape, we shed light on the population density responses of these increasingly threatened species to habi- tat and land management strategies. Our results provide an important baseline and reference estimate that can be employed for future ecological monitoring of striped hyaena, aardwolf and serval. Serval densities were higher within the RNP grids than in WMA Acacia-Commiphora, and in particular within the miombo woodland. This density distribution follows the precipitation pattern over the Ruaha-Rungwa landscape, as the miombo woodlands in the south-western part of RNP receive the greatest amount of rainfall on average and WMA Acacia-Commiphora is the driest of the three surveyed areas [44]. The miombo woodlands feature open, grassy mbuga (“dambo”) drainage lines. Seasonal rains flood mbugas and water remains present year-round, usually in distinct springs or pools during the dry season. This result aligns with the preference of servals for well-watered environments such as wetlands and riparian habitat [17]. The difference between RNP miombo and Core RNP Acacia-Commiphora could also stem from the lower abundance of lion, leopard and spotted hyaena (Crocuta crocuta) in the miombo woodlands [46], as these apex predator species occasionally kill serval [66]. The lower serval population density in WMA Acacia-Commiphora likely results from the WMA’s prox- imity to unprotected land, inducing greater edge effects [67], and a more pronounced level of anthropogenic pressure. For instance, the camera stations in the WMA recorded nine illegal incursions, two of them with evidence of bushmeat hunting, as well as two spotted hyaenas and two striped hyaenas bearing marks of snare captures. The common use of dogs for bush- meat hunting may also negatively impact resident populations of serval, as occasional killings by domestic dogs have been observed elsewhere in Tanzania [68]. In contrast, the survey only recorded one illegal incursion in the RNP grids. Moreover, the lower abundance of small ungulates in the WMA driven by anthropogenic impacts and the lack of habitat immediately outside the WMA may intensify competition between serval and leopard [47]. In areas with fewer impala or other medium-sized antelopes, leopard may shift their diet towards smaller prey species, such as large rodents and birds, and thus increase their dietary overlap with serval [69]. Results For striped hyaena in WMA Acacia-Commiphora, two models received strong support (ΔAICc < 2, [65]); the model with constant g0 and sigma, and the local trap response mode Fig 2. Population density estimates for the target species at the three survey sites. Bars represent standard errors. https://doi.org/10.1371/journal.pone.0242293.g002 Core RNP Acacia-Commiphora, and 2.08 [±0.74] individuals per 100 km2 in WMA Acacia- Commiphora (Fig 2, Table 1). For striped hyaena in WMA Acacia-Commiphora, two models received strong support (ΔAICc < 2, [65]); the model with constant g0 and sigma, and the local trap response model Table 1. Population density and parameter estimates, with standard errors/95% confidence intervals for the target species at the three survey sites. Table 1. Population density and parameter estimates, with standard errors/95% confidence intervals for the target species at the three survey sites. Estimate ± SE Estimate ± SE 95% CI Species Site Predictor g0 σ(m) Density (ind/100 km2) Serval Core RNP Acacia-Commiphora 0.041 ± 0.014 1062 ± 171 3.45 ± 1.04 1.93–6.16 RNP miombo On road 0.033 ± 0.017 1249 ± 361 5.56 ± 2.45 Off road 0.006 ± 0.004 2.44–12.69 WMA Acacia-Commiphora 0.034 ± 0.013 1566 ± 299 2.08 ± 0.74 1.05–4.09 Striped hyaena WMA Acacia-Commiphora bk = 0 0.032 ± 0.016 2625 ± 521 1.36 ± 0.50 bk = 1 0.078 ± 0.029 0.68–2.72 Aardwolf Core RNP Acacia-Commiphora bk = 0 0.030 ± 0.009 1220 ± 131 9.19 ± 1.66 bk = 1 0.263 ± 0.038 6.46–13.06 WMA Acacia-Commiphora Female 0.135 ± 0.085 476 ± 111 13.25 ± 2.48 Male 0.323 ± 0.075 553 ± 42 9.22–19.05 Table 1. Population density and parameter estimates, with standard errors/95% confidence intervals for the target s eter estimates, with standard errors/95% confidence intervals for the target species at the three survey sites. PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 7 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 PLOS ONE Population densities of lesser-studied carnivores in southern Tanzania’s Ruaha-Rungwa landscape (bk). Model averaging returned an estimated density of 1.36 [±0.50] individuals per 100 km2. For aardwolf in Core RNP Acacia-Commiphora, learned behavioural response influenced capture probability, with the local trap response model (bk) ranking highest, while the models in which g0 or sigma varied with sex ranked highest in WMA Acacia-Commiphora. Aardwolf density was estimated at 9.19 [±1.66] individuals per 100 km2 in Core RNP Acacia-Commi- phora, and 13.25 [±2.48] individuals per 100 km2 in WMA Acacia-Commiphora. PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 Discussion Serval density estimates using spatial capture-recapture vary greatly across Africa, from 0.63 and 1.28 individuals per 100 km2 in northern Namibia [28] to 101.21 in Mpumalanga, South Africa [29]. The latter estimate comes from a heavily modified habitat where the absence of persecution and interspecific competition, combined with high rodent biomass, enables ser- val to attain such high numbers. In comparison, our results lie within the lower range of pub- lished densities and are close to estimates for the Niokolo Koba National Park, Senegal (3.49– 4.08 individuals per 100 km2) [31] and the Drakensberg Midlands, South Africa (6.0–8.3 indi- viduals per 100 km2) [27]. The only other density estimate available for the species in Tanzania 8 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 PLOS ONE Population densities of lesser-studied carnivores in southern Tanzania’s Ruaha-Rungwa landscape exceeds our results, with 10.9 [SE = 3.17] individuals per 100 km2 in Tarangire National Park [70]; however, the study employed conventional capture-recapture, which has been shown to overestimate density compared to spatial capture-recapture [71]. We could only estimate striped hyaena population density in WMA Acacia-Commiphora, as the camera trap survey did not detect any individuals in Core RNP Acacia-Commiphora or RNP miombo. The Ruaha-Rungwa landscape lies at the southern limit of the species’ global range [13]; deeper within the species’ range, in Laikipia County, Kenya, population density estimated through SECR was around six times higher than the density estimated in WMA Aca- cia-Commiphora [25]. Such a pattern is consistent with widespread biogeographic trends, typi- cally showing diminished abundance towards species range edges [72]. Furthermore, the species is closely associated with Acacia-Commiphora bushland within Africa [68], and the fact that our study landscape traverses a transition from this habitat to miombo woodland may point to a mechanism delimiting the range edge in this area. The low density observed in WMA Acacia-Commiphora may also ensue from high edge effects due to the area’s proximity to human-dominated areas [67], as highlighted by the record of two individuals showing evi- dence of wire snare capture during the study period. In contrast to serval, we estimated a higher aardwolf density in WMA Acacia-Commi- phora than in Core RNP Acacia-Commiphora. The difference probably results from milder intraguild predation in the WMA, which supports lower densities of apex predators than the core tourist area of RNP [46]. PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 Discussion Considering the logistical and budget PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 9 / 15 PLOS ONE Population densities of lesser-studied carnivores in southern Tanzania’s Ruaha-Rungwa landscape requirements of camera trapping, this study illustrates the value of by-catch data in terms of resource optimisation, provided that the survey design also complies with the ecology of the non-target species [75]. Our trap layout met spatial sampling recommendations for the species we considered: the array coverage areas were larger than single home range areas of all species [54, 55, 76, 77], and spacing was sufficient to yield recaptures of individuals across multiple sta- tions in all cases [21, 76, 78–80]. However, the trap layout configuration was tailored to maxi- mise leopard capture rate, with stations preferentially deployed on roads and junctions [53], which may not be optimum for serval, striped hyaena and aardwolf. For instance, striped hyaena have been recorded to move mostly cross-country rather than along roads in the Seren- geti [12]. Similarly, mesocarnivores such as serval and aardwolf may avoid areas commonly used by apex predators, which would lower their capture rate along roads [81]. Although the analysis did not find any evidence of road avoidance behaviour, we suggest that future work targeting these species might improve detectability by positioning camera stations around sightings of the animals or their signs. The higher population density estimated for serval in miombo woodland indicates that this is an important habitat for the species in the region. One of the most extensive ecosystems in Sub-Saharan Africa, miombo woodlands cover a substantial portion of Tanzania’s protected and unprotected land [82]. Miombo plays a key role in the livelihood of rural communities but faces unsustainable resource exploitation [83, 84]. As such, increased woodland degradation and deforestation would negatively impact serval as well as a range of other mammal species. As miombo woodlands have received little conservation attention compared to Acacia habitats in East Africa [82], we recommend additional survey work across their extent to improve the understanding of these systems and protect their unique biodiversity. Additionally, our results highlight the importance of the community-managed MBOMIPA WMA for the striped hyaena and aardwolf in the Ruaha-Rungwa landscape. Along with the adjacent RNP, MBOMIPA WMA provides the two hyaenids with suitable habitat at the edge of their global range. Discussion Moreover, as aardwolf primarily feed on harvester termites, depletion of ungulates would not trigger dietary overlap with larger carnivores [18]. In addi- tion, aardwolf may benefit from accidental fire outbreaks which often occur later in the dry season in the WMA; the reduced grass cover caused by this burning may force harvester ter- mites to range more widely in search of food, thus lengthening their exposure time to preda- tion on the ground [73, 74]. The reverse trend in aardwolf density compared to serval could indicate different sensitivities to anthropogenic pressures between the two species, although poaching activities in the area do not specifically target either species. Aardwolf may also be more prone to predation by apex predators, with greater stealth and agility giving serval an edge on the slower-moving aardwolf when threatened [17]. The absence of records of aardwolf in miombo woodland reflects the species’ known preference for Acacia–Commi- phora bushland [68]. Our results align with aardwolf population densities estimated through conventional capture-recapture and spatial capture-recapture in Tarangire National Park and Kenya’s Laikipia District, of 9.04 and 11.63 individuals per 100 km2, respectively [25, 70]. Camera trap location impacted detection probability for serval in RNP miombo. Rather than denoting serval’s preference for larger roads, the higher detection probability at stations located on roads probably results from the particular road network in this portion of RNP, consisting of a single road complemented by two infrequently-used vehicle tracks and numerous subsidiary game trails. Though we tried to select the most heavily used trails, their large number offers wildlife multiple equivalent options compared to the stations set on the most readily accessible road. For aardwolf in Core RNP Acacia-Commiphora and striped hyaena, the probability of detection at a camera trap station was higher for individuals that had already been captured at the same site. This suggests that individuals favour the core area of their home range more often than the periphery and are not deterred by camera trap flash- lights (xenon or LED flash). Sex appeared to have a mild impact on aardwolf capture proba- bility in WMA Acacia-Commiphora, with males having a higher probability of capture than females. Conservation implications and recommendations. This study made use of photographic by-catch data from a research project targeting leopard. PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 Author Contributions Conceptualization: Marie Hardouin, Charlotte E. Searle, Paolo Strampelli, J. Marcus Rowcliffe. Data curation: Charlotte E. Searle. Formal analysis: Marie Hardouin. Funding acquisition: Charlotte E. Searle, Paolo Strampelli, Josephine Smit, Amy Dickman. Investigation: Charlotte E. Searle, Paolo Strampelli, Josephine Smit. Methodology: Marie Hardouin. Supervision: Charlotte E. Searle, Paolo Strampelli, J. Marcus Rowcliffe. Writing – original draft: Marie Hardouin. Writing – review & editing: Marie Hardouin, Charlotte E. Searle, Paolo Strampelli, Josephin Smit, Amy Dickman, Alex L. Lobora, J. Marcus Rowcliffe. Data curation: Charlotte E. Searle. Data curation: Charlotte E. Searle. Formal analysis: Marie Hardouin. Formal analysis: Marie Hardouin. Funding acquisition: Charlotte E. Searle, Paolo Strampelli, Josephine Smit, Amy Dickman. Investigation: Charlotte E. Searle, Paolo Strampelli, Josephine Smit. Funding acquisition: Charlotte E. Searle, Paolo Strampelli, Josephine Smit, Amy Dickman. Investigation: Charlotte E. Searle, Paolo Strampelli, Josephine Smit. Funding acquisition: Charlotte E. Searle, Paolo Strampelli, Josephine Smit, Amy Dickman. Investigation: Charlotte E. Searle, Paolo Strampelli, Josephine Smit. Methodology: Marie Hardouin. Supervision: Charlotte E. Searle, Paolo Strampelli, J. Marcus Rowcliffe. Supervision: Charlotte E. Searle, Paolo Strampelli, J. Marcus Rowcliffe. Writing – original draft: Marie Hardouin. Writing – review & editing: Marie Hardouin, Charlotte E. Searle, Paolo Strampelli, Josephine Smit, Amy Dickman, Alex L. Lobora, J. Marcus Rowcliffe. S4 Appendix. Survey grid summary information. (PDF) S4 Appendix. Survey grid summary information. (PDF) S5 Appendix. SECR model ranking. (PDF) Acknowledgments We would like to thank the Government of Tanzania, Tanzania Wildlife Research Institute (TAWIRI), Tanzania Commission for Science and Technology (COSTECH), Tanzania National Parks Authority (TANAPA), Tanzania Wildlife Management Authority (TAWA), and Idodi-Pawaga MBOMIPA WMA. We also thank the field staff of the Southern Tanzania Elephant Program (STEP) and Ruaha Carnivore Project (RCP) for their assistance with data collection, all WMA Village Game Scouts, TANAPA Rangers, and TAWA Game Scouts who contributed to fieldwork. S1 Appendix. Individual identification and sexing. (PDF) S2 Appendix. Capture histories. (PDF) Discussion More generally, MBOMIPA WMA acts as a buffer zone between the highly protected RNP and the surrounding villages, and our results demonstrate the potential of community conservation in complementing more traditional conservation strategies, par- ticularly in areas with lower densities of vulnerable species such as striped hyaena. Neverthe- less, the camera trap survey found evidence of bushmeat hunting in the WMA; although pictures show that hyaenas can break free from snares and survive with amputated limbs, snar- ing and other non-selective poaching methods generally induce an increase in anthropogenic mortality of non-target species in savannah ecosystems [85, 86]. Considering the species’ low density in the Ruaha-Rungwa landscape, a conservation priority for striped hyaena should aim to reduce the impact of snaring on resident populations and assess the corresponding risk to their viability. In conclusion, our study illustrates how data from camera trap surveys targeting charis- matic large carnivores can shed light on populations of lesser-studied carnivores, broadening the scope of capture-recapture studies beyond focal species. While adjustments to sample design can help to extend the range of accessible species in surveys [26], in this case, we were able to generate useful data without such adjustments. Similar efforts, employing by-catch data, could be easily replicated elsewhere to provide information on the spatial and population ecology of a range of species. Finally, we recommend complementing the baseline estimates provided for serval, striped hyaena and aardwolf in the Ruaha-Rungwa landscape with investi- gations into spatial variation in site use across the landscape to determine the environmental and anthropogenic factors driving their habitat selection. 10 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0242293 March 30, 2021 PLOS ONE Population densities of lesser-studied carnivores in southern Tanzania’s Ruaha-Rungwa landscape Supporting information S1 Appendix. Individual identification and sexing. (PDF) S2 Appendix. Capture histories. (PDF) S3 Appendix. Trap layouts. (PDF) S4 Appendix. 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An Update of Transition Metal-Catalyzed Decarboxylative Transformations of Cyclic Carbonates and Carbamates
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Linhong Zuo, Teng Liu, Xiaowei Chang and Wusheng Guo * Frontier Institute of Science and Technology (FIST), Xi’an Jiaotong University, Xi’an 710045, China; zuolinhong890523@163.com (T.Z.); liuteng1992@stu.xjtu.edu.cn (T.L.); changxw16@163.com (X.C.) * Correspondence: wusheng.guo@mail.xjtu.edu.cn Frontier Institute of Science and Technology (FIST), Xi’an Jiaotong University, Xi’an 710045, China; zuolinhong890523@163.com (T.Z.); liuteng1992@stu.xjtu.edu.cn (T.L.); changxw16@163.com (X.C.) * Correspondence: wusheng.guo@mail.xjtu.edu.cn Academic Editor: Joaquín García Álvarez Received: 26 September 2019; Accepted: 25 October 2019; Published: 31 October 201 Academic Editor: Joaquín García Álvarez Received: 26 September 2019; Accepted: 25 October 2019; Published: 31 October 2019 Abstract: Functionalized cyclic organic carbonates and carbamates are frequently used in a number of transition metal-catalyzed decarboxylative reactions for the construction of interesting molecules. These decarboxylative transformations have attracted more and more research attention in recent years mainly due to their advantages of less waste generation and versatile reactivities. On the basis of previous reviews on this hot topic, the present review will focus on the development of transition metal-catalyzed decarboxylative transformations of functionalized cyclic carbonates and carbamates in the last two years. molecules molecules An Update of Transition Metal-Catalyzed Decarboxylative Transformations of Cyclic Carbonates and Carbamates Linhong Zuo, Teng Liu, Xiaowei Chang and Wusheng Guo * 2. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbamates 2 T i i M l C l d D b l i f C li C b Cyclic carbamates have been widely used in the synthesis of a range of N-heterocyclic compounds through a key aza-quinone methide (aza-QM) intermediate. In the presence of a suitable palladium catalyst, the decarboxylation of vinyl cyclic carbamate would generate a zwitterionic intermediate (Scheme 1a) [8]. In contrast, the decarboxylation of ethynyl cyclic carbamate toward the formation of a zwitterionic intermediate generally requires an appropriate copper catalyst (Scheme 1b) [9]. In the presence of appropriate acceptors, a cyclization process would occur affording various interesting and useful N-heterocycles. In this section, the examples that have been reviewed by Lu and co-workers will not be discussed [5]. 2. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbamates Cyclic carbamates have been widely used in the synthesis of a range of N-heterocyclic compounds through a key aza-quinone methide (aza-QM) intermediate. In the presence of a suitable palladium catalyst, the decarboxylation of vinyl cyclic carbamate would generate a zwitterionic intermediate (Scheme 1a) [8]. In contrast, the decarboxylation of ethynyl cyclic carbamate toward the formation of a zwitterionic intermediate generally requires an appropriate copper catalyst (Scheme 1b) [9]. In the presence of appropriate acceptors, a cyclization process would occur affording various interesting and useful N-heterocycles. In this section, the examples that have been reviewed by Lu d k ll b d d nd co workers will not be discussed [5]. Scheme 1. Palladium (a) or Copper (b) catalyzed decarboxylation of cyclic carbamates toward the formation of zwitterionic intermediates. Scheme 1. Palladium (a) or Copper (b) catalyzed decarboxylation of cyclic carbamates toward the formation of zwitterionic intermediates. Scheme 1. Palladium (a) or Copper (b) catalyzed decarboxylation of cyclic carbamates toward the formation of zwitterionic intermediates. Scheme 1. Palladium (a) or Copper (b) catalyzed decarboxylation of cyclic carbamates toward the formation of zwitterionic intermediates. Decarboxylative cyclization of vinyl cyclic carbamates and benzylidene malononitriles toward the formation of chiral tetrahydroquinolines was first reported by Tunge et al. in 2008 [8]. In the following decade, cycloaddition reactions of vinyl cyclic carbamates with a variety of different electrophiles and nucleophiles have been boosted. In addition to previous achievements [5], new opportunities with neoteric acceptors have been discovered (Scheme 2). For instance, the decarboxylative formation of dihydroquinazolinones through a Pd-catalyzed [4 + 2] cycloaddition using sulfonyl isocyanates as electrophiles was reported by Shi and co-workers [10]. 1. Introduction Decarboxylation is generally associated with steps of C–C bond cleavage and CO2 generation. As the sole byproduct of decarboxylative reactions, CO2 is non-flammable and non-toxic and can be easily removed from the reaction system; thus, decarboxylative chemistry has become a very important and attractive strategy in organic synthesis. Carboxylate groups have served as miscellaneous connection points in the construction of various organic compounds [1–6]. In contrast, cyclic organic carbonates and carbamates enabled new reactivities that go beyond classical carboxylic acid derivatives. In the last few years, the research on this topic has been flowered and remarkable progress has been made by different research groups. Specifically, vinyl and alkynyl substituted cyclic organic carbonates were used as allylic and propargyl surrogates upon decarboxylation toward the formation of various interesting O-heterocycles. In contrast, the corresponding vinyl and ethynyl cyclic organic carbamates also showed marvelous reactivities in the construction of a number of N-heterocycles. In early 2018, Lu [5], Guo [6] and co-workers conducted a detailed review in this area. In the presence of transition metal catalysts, a number of structurally diverse N-heterocycles were synthesized using vinyl or ethynyl cyclic carbamates as starting materials [5]. Decarboxylation of cyclic carbonates proved to be a powerful tool to achieve chemo-, regio-, stereo- and enantioselective synthesis of complex structures [6]. As a related research topic, the Harrity group recently released a review of Pd-catalyzed cyclization reactions via π-allyl-Pd zwitterionic intermediates [7]. In order to avoid unnecessary duplication, we herein would like to focus on reviewing the latest contributions in the last two years, especially in transition metal-catalyzed decarboxylative transformations of cyclic carbonates and carbamates. We will first discuss the decarboxylative chemistry with cyclic carbamates as starting materials. Molecules 2019, 24, 3930; doi:10.3390/molecules24213930 www.mdpi.com/journal/molecules www.mdpi.com/journal/molecules Molecules 2019, 24, 3930 2 of 16 2. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbamates 2 T i i M l C l d D b l i f C li C b With the introduction of cyclic imines or barbiturate-based olefin reactants, it is feasible to construct highly functionalized quinazolines through decarboxylative cycloadditions [11–13]. Most recently, the annulation of deconjugated butenolides or azlactones with vinyl carbamates towards highly functionalized chiral dihydroquinol-2-ones was released by the Xiao group [14]. It was found that the utilization of the newly exploited chiral P,S-ligand and hydrogen bonding is the key to control the regioselectivity for this reaction. Apart from palladium catalysis, iridium/Brønsted acid co- catalytic system was applied for the preparation of quinolinones through a formal [4 + 2] cycloaddition by the Shi group [15] Later the same group developed a palladium-catalyzed cascade Decarboxylative cyclization of vinyl cyclic carbamates and benzylidene malononitriles toward the formation of chiral tetrahydroquinolines was first reported by Tunge et al. in 2008 [8]. In the following decade, cycloaddition reactions of vinyl cyclic carbamates with a variety of different electrophiles and nucleophiles have been boosted. In addition to previous achievements [5], new opportunities with neoteric acceptors have been discovered (Scheme 2). For instance, the decarboxylative formation of dihydroquinazolinones through a Pd-catalyzed [4 + 2] cycloaddition using sulfonyl isocyanates as electrophiles was reported by Shi and co-workers [10]. With the introduction of cyclic imines or barbiturate-based olefin reactants, it is feasible to construct highly functionalized quinazolines through decarboxylative cycloadditions [11–13]. Most recently, the annulation of deconjugated butenolides or azlactones with vinyl carbamates towards highly functionalized chiral dihydroquinol-2-ones was released by the Xiao group [14]. It was found that the utilization of the newly exploited chiral P,S-ligand and hydrogen bonding is the key to control the regioselectivity for this reaction. Apart from palladium catalysis, iridium/Brønsted acid co-catalytic system was applied for the preparation of quinolinones through a formal [4 + 2] cycloaddition by the Shi group [15]. Later, the same group developed a palladium-catalyzed cascade cyclization reaction of para-quinone methides and cyclic carbamates [16]. 3 of 16 3 of 16 Molecules 2019, 24, 3930 Scheme 2. Pd-catalyzed decarboxylative cycloadditions of vinyl cyclic carbamates. Scheme 2. Pd-catalyzed decarboxylative cycloadditions of vinyl cyclic carbamates. Scheme 2. Pd-catalyzed decarboxylative cycloadditions of vinyl cyclic carbamates. Scheme 2. Pd-catalyzed decarboxylative cycloadditions of vinyl cyclic carbamates. Molecules 2019, 24, 3930 4 of 16 Interestingly, the Zhai group developed a palladium-catalyzed decarboxylative coupling of arynes and vinyl benzoxazinanones toward the formation of functionalized indoles (Scheme 3a). 2. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbamates 2 T i i M l C l d D b l i f C li C b In allylic chemistry, the nucleophilic attack of π-allyl palladium intermediate generally occurred at the terminal or internal carbon. However, this conversion features an intramolecular nucleophilic attack of the amide group at the central carbon of the π-allyl palladium intermediate, though a deeper understanding of the mechanism is still required [17]. The Shibata group reported an unprecedented intermolecular cyclization of cyclic carbamate and sulfur ylides toward the formation of 4-trifluoromethyl-dihyroquinolines (Scheme 3b, path a); without externally added acceptors, a cyclization process would also be possible through an intramolecular attack of the zwitterionic π-allyl intermediate (Scheme 3b, path b) [18]. In contrast, the same group also proved [4 + 1] diastereoselective intermolecular cyclization using sulfur ylides and non-vinyl-substituted benzoxazinanones, affording various trifluoromethyl-substituted indolines [19]. Moreover, a three-component domino reaction via decarboxylation, allylation, and N-H carbene insertion in the sequence was reported by Yang and co-workers toward the formation of various allylic sulfone-containing amino acid derivatives (Scheme 3c) [20]. Comparatively, the decarboxylative annulation of ethynyl cyclic carbamates has mostly been achieved through Cu-allenylidene intermediate. The Cu-allenylidene dipole species could be trapped by different nucleophiles toward the formation of various functionalized indole skeletons (Scheme 4). In the presence of phosphonate nucleophile, 2-phosphorylmethyl indoles could be produced with this strategy [21]. The use of an indole nucleophile allows the facile synthesis of a variety of 3,3’-biindoles [22]. In the presence of an appropriate copper catalyst and chiral urea-cinchona organocatalyst, enantioselective decarboxylative propargylation and hydroamination of ethynyl benzoxazinanones were established affording chiral 3-indolin malononitrile derivatives [23]. Notably, the Zhao group reported a metal-free decarboxylative protocol utilizing propargylic carbamates and aldehydes as substrates. In this methodology, a range of 4-alkynyl dihydroquinolinones and 2, 3-difunctionalized indoles could be readily synthesized under the catalysis of N-heterocyclic carbene [24]. Molecules 2019, 24, x FOR PEER REVIEW 4 of 16 benzoxazinanones, affording various trifluoromethyl-substituted indolines [19]. Moreover, a three- component domino reaction via decarboxylation, allylation, and N-H carbene insertion in the sequence was reported by Yang and co-workers toward the formation of various allylic sulfone- containing amino acid derivatives (Scheme 3c) [20]. Scheme 3. Palladium-catalyzed decarboxylative reaction of cyclic carbamates with different reaction partners: (a) with arynes; (b) with or without sulfur ylides and (c) three-component domino reactions. Scheme 3. Palladium-catalyzed decarboxylative reaction of cyclic carbamates with different reaction partners: (a) with arynes; (b) with or without sulfur ylides and (c) three-component domino reactions. Scheme 3. 2. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbamates 2 T i i M l C l d D b l i f C li C b Palladium-catalyzed decarboxylative reaction of cyclic carbamates with different reaction partners: (a) with arynes; (b) with or without sulfur ylides and (c) three-component domino reactions. Scheme 3. Palladium-catalyzed decarboxylative reaction of cyclic carbamates with different reaction partners: (a) with arynes; (b) with or without sulfur ylides and (c) three-component domino reactions. 5 of 16 Molecules 2019, 24, 3930 In the presence of suitable reagents, the decarboxylative annulation process using ethynyl cyclic carbamates proved to be a feasible method for the preparation of N-heterocycles. In this sense, different chemicals such as pyrazolones, hexahydro-1,3,5-triazines, azlactones, and C,N-cyclic azomethineimines were submitted for the reactions toward the formation of various N-heterocycles (Scheme 5) [25–28]. It is worth noting that the Wu group accomplished an enantioselective [4 + 2] cycloaddition reaction of ethynyl benzoxazinanones and silyloxyfurans toward the formation of tetrahydroquinolines featuring three stereo carbon centers [29]. Molecules 2019, 24, x FOR PEER REVIEW 5 of 16 Scheme 4. Decarboxylative conversions of ethynyl cyclic carbamates toward the formation of indole derivatives Scheme 4. Decarboxylative conversions of ethynyl cyclic carbamates toward the formation of i d l d i i Scheme 4. Decarboxylative conversions of ethynyl cyclic carbamates toward the formation of indole derivatives. Scheme 4. Decarboxylative conversions of ethynyl cyclic carbamates toward the formation of indole derivatives. Scheme 4. Decarboxylative conversions of ethynyl cyclic carbamates toward the formation of indole derivatives. Scheme 4. Decarboxylative conversions of ethynyl cyclic carbamates toward the formation of indole derivatives. 6 of 16 Molecules 2019, 24, 3930 Molecules 2019, 24, x FOR PEER REVIEW 6 o Scheme 5. Cu-catalyzed decarboxylative annulation of ethynyl carbamates with different reagents. 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates Scheme 5. Cu-catalyzed decarboxylative annulation of ethynyl carbamates with different reagents. 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates Scheme 5. Cu-catalyzed decarboxylative annulation of ethynyl carbamates with different reagents Scheme 5. Cu-catalyzed decarboxylative annulation of ethynyl carbamates with different reagents 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates As stable and readily accessible allylic and propargylic donors, cyclic carbonates have emerged as highly reactive substrates in various stereo- and enantioselective reactions. In the past ten years, the synthetic potential of these cyclic carbonates has been greatly demonstrated in a wide variety of decarboxylative processes. Decarboxylation of vinyl cyclic carbonate with a palladium catalyst would generate a zwitterionic intermediate featuring a nucleophilic alkoxide and an electrophilic π-allyl- palladium site. This reactive zwitterionic species can serve as 1,3- or 1,5-dipole. The reaction of this As stable and readily accessible allylic and propargylic donors, cyclic carbonates have emerged as highly reactive substrates in various stereo- and enantioselective reactions. In the past ten years, the synthetic potential of these cyclic carbonates has been greatly demonstrated in a wide variety of decarboxylative processes. Decarboxylation of vinyl cyclic carbonate with a palladium catalyst would generate a zwitterionic intermediate featuring a nucleophilic alkoxide and an electrophilic π-allyl-palladium site. This reactive zwitterionic species can serve as 1,3- or 1,5-dipole. The reaction of Molecules 2019, 24, 3930 7 of 16 this dipole species with different cyclization acceptors would lead to formal [3 + 2], [5 + 2], [5 + 3] or [5 + 4] annulation reactions. Molecules 2019, 24, x FOR PEER REVIEW 7 of 16 dipole species with different cyclization acceptors would lead to formal [3 + 2], [5 + 2], [5 + 3] or [5 annulation reactions. lecules 2019, 24, x FOR PEER REVIEW 7 of More recently, a palladium and squaramide co-catalyzed decarboxylative [3 + 2] cyclization of VCCs and β-nitroolefins was achieved by the Zhang group (Scheme 6a) [30]. The chiral squaramide proved to be vital for achieving high enantioselectivity. A similar strategy was utilized for the construction of furanbenzodihydropyran skeletons by the same group through a formal [3 + 2] cyclization with VCC as substrates [31]. With the introduction of coumalates as acceptors, the Guo group realized the chiral synthesis of nine-membered ethers with excellent enantioselectivities (mostly > 99% ee) through a tandem [3 + 2] cycloaddition followed by Cope rearrangement (Scheme 6b). The mechanism was further confirmed by DFT calculations [32]. More recently, a palladium and squaramide co-catalyzed decarboxylative [3 + 2] cyclization of VCCs and β-nitroolefins was achieved by the Zhang group (Scheme 6a) [30]. The chiral squaramide proved to be vital for achieving high enantioselectivity. 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates A similar strategy was utilized for the construction of furanbenzodihydropyran skeletons by the same group through a formal [3 + 2] cyclization with VCC as substrates [31]. With the introduction of coumalates as acceptors, the Guo group realized the chiral synthesis of nine-membered ethers with excellent enantioselectivities (mostly > 99% ee) through a tandem [3 + 2] cycloaddition followed by Cope rearrangement (Scheme 6b). The mechanism was further confirmed by DFT calculations [32]. Scheme 6. (a) Cooperative catalysis strategy for the cycloaddition of VECs with β-Nitroolefins and (b) [3 + 2] cycloaddition/Cope rearrangement reaction of VECs. Scheme 6. (a) Cooperative catalysis strategy for the cycloaddition of VECs with β-Nitroolefins and (b) [3 + 2] cycloaddition/Cope rearrangement reaction of VECs. Scheme 6. (a) Cooperative catalysis strategy for the cycloaddition of VECs with β-Nitroolefins and (b) [3 + 2] cycloaddition/Cope rearrangement reaction of VECs. Scheme 6. (a) Cooperative catalysis strategy for the cycloaddition of VECs with β-Nitroolefins and (b) [3 + 2] cycloaddition/Cope rearrangement reaction of VECs. In addition to these aforementioned [3 + 2] annulations, the decarboxylative formation o medium-membered heterocycles through a Pd-catalyzed [5 + n] annulation has been achieved by different research groups (Scheme 7) [33–38]. For example, the Xiao group realized an ti l ti [5 + 2] l dditi ti f VCC d di k t b i In addition to these aforementioned [3 + 2] annulations, the decarboxylative formation of medium-membered heterocycles through a Pd-catalyzed [5 + n] annulation has been achieved by different research groups (Scheme 7) [33–38]. For example, the Xiao group realized an enantioselective 8 of 16 Molecules 2019, 24, 3930 [5 + 2] cycloaddition reaction of VCCs and α-diazoketones by merging photoactivation and Pd catalysis and a variety of seven-membered lactones bearing chiral quaternary stereocenters with high enantioselectivity were delivered through this methodology [35]. Molecules 2019, 24, x FOR PEER REVIEW 8 of 16 [5 + 2] cycloaddition reaction of VCCs and α-diazoketones by merging photoactivation and Pd catalysis and a variety of seven-membered lactones bearing chiral quaternary stereocenters with high enantioselectivity were delivered through this methodology [35]. Molecules 2019, 24, x FOR PEER REVIEW 8 of 16 Scheme 7. Decarboxylative [5 + n] cycloadditions of VCCs with various electrophiles. Scheme 7. Decarboxylative [5 + n] cycloadditions of VCCs with various electrophiles. Scheme 7. Decarboxylative [5 + n] cycloadditions of VCCs with various electrophiles. Scheme 7. 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates Decarboxylative [5 + n] cycloadditions of VCCs with various electrophiles. Molecules 2019, 24, 3930 9 of 16 Umpolung reactivity of the π-allyl zwitterionic species was first noted by Guo and co-workers [39] and later, it was further explored by the Zhao group [40]. In the presence of a palladium and titanium catalyst, the umpolung annulation process occurred with the use of aurones and VCC as starting materials toward the formation of [6,5] and [5,5] spiro-heterocycles bearing three contiguous stereocenters (Scheme 8). Mechanistically, it was proposed that the Pd-π-allyl intermediate first reacted with Ti(OiPr)4, followed by ligand exchange between titanium and palladium resulting in a titanium-dienolate species through β elimination process. Molecules 2019, 24, x FOR PEER REVIEW 9 of 16 starting materials toward the formation of [6,5] and [5,5] spiro-heterocycles bearing three contiguous stereocenters (Scheme 8). Mechanistically, it was proposed that the Pd-π-allyl intermediate first reacted with Ti(OiPr)4, followed by ligand exchange between titanium and palladium resulting in a titanium-dienolate species through β elimination process. Molecules 2019, 24, x FOR PEER REVIEW 9 of 16 starting materials toward the formation of [6,5] and [5,5] spiro-heterocycles bearing three contiguous stereocenters (Scheme 8). Mechanistically, it was proposed that the Pd-π-allyl intermediate first reacted with Ti(OiPr)4, followed by ligand exchange between titanium and palladium resulting in a titanium-dienolate species through β elimination process. Scheme 8. Palladium-titanium relay catalysis enabled umpolung reactivity in the synthesis of spiro- heterocycles. Scheme 8. Palladium-titanium relay catalysis enabled umpolung reactivity in the synthesis of spiro-heterocycles. Scheme 8. Palladium-titanium relay catalysis enabled umpolung reactivity in the synthesis of spiro- heterocycles. Scheme 8. Palladium-titanium relay catalysis enabled umpolung reactivity in the synthesis of spiro- heterocycles. Scheme 8. Palladium-titanium relay catalysis enabled umpolung reactivity in the synthesis of spiro-heterocycles. Scheme 8. Palladium-titanium relay catalysis enabled umpolung reactivity in the synthesis of spiro- heterocycles. The Zhai group accomplished a Pd-catalyzed decarboxylative umpolung reaction of VCCs producing a variety of structurally diverse and synthetically useful all-carbon α-vinyl quaternary aldehydes (Scheme 9) [41]. The key step is that the π-allyl-Pd intermediate undergoes β-H elimination affording a nucleophilic dienolate. Most recently, a [3 + 3] cycloaddition of VCCs and triazinanes was achieved under the co-catalysis of palladium and Lewis acid towards the formation of polysubstituted tetrahydropyrimidines by the Yang group. 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates Interestingly in this case, the vinyl carbonates served as a 3-carbon synthon, which is unique in this kind of decarboxylative The Zhai group accomplished a Pd-catalyzed decarboxylative umpolung reaction of VCCs producing a variety of structurally diverse and synthetically useful all-carbon α-vinyl quaternary aldehydes (Scheme 9) [41]. The key step is that the π-allyl-Pd intermediate undergoes β-H elimination affording a nucleophilic dienolate. Most recently, a [3 + 3] cycloaddition of VCCs and triazinanes was achieved under the co-catalysis of palladium and Lewis acid towards the formation of polysubstituted tetrahydropyrimidines by the Yang group. Interestingly in this case, the vinyl carbonates served as a 3-carbon synthon, which is unique in this kind of decarboxylative cycloaddition process [42]. The Zhai group accomplished a Pd-catalyzed decarboxylative umpolung reaction of VCCs producing a variety of structurally diverse and synthetically useful all-carbon α-vinyl quaternary aldehydes (Scheme 9) [41]. The key step is that the π-allyl-Pd intermediate undergoes β-H elimination affording a nucleophilic dienolate. Most recently, a [3 + 3] cycloaddition of VCCs and triazinanes was achieved under the co-catalysis of palladium and Lewis acid towards the formation of polysubstituted tetrahydropyrimidines by the Yang group. Interestingly in this case, the vinyl carbonates served as a 3-carbon synthon, which is unique in this kind of decarboxylative cycloaddition process [42]. Scheme 9. Pd-catalyzed decarboxylative transformations of VCCs toward highly functionalized aldehydes bearing quaternary carbon centers. Scheme 9. Pd-catalyzed decarboxylative transformations of VCCs toward highly functionalized aldehydes bearing quaternary carbon centers. Scheme 9. Pd-catalyzed decarboxylative transformations of VCCs toward highly functionalized aldehydes bearing quaternary carbon centers. Scheme 9. Pd-catalyzed decarboxylative transformations of VCCs toward highly functionalized aldehydes bearing quaternary carbon centers. Scheme 9. Pd-catalyzed decarboxylative transformations of VCCs toward highly functionalized aldehydes bearing quaternary carbon centers. Scheme 9. Pd-catalyzed decarboxylative transformations of VCCs toward highly functionalized aldehydes bearing quaternary carbon centers. Molecules 2019, 24, 3930 10 of 16 Unlike the cyclization process mentioned above, in the presence of suitable palladium catalyst and ligand, the stereoselective synthesis of a variety of highly functionalized allylic alcohols can be realized. This concept was first proved by Guo and co-workers using aryl amine nucleophiles toward the formation of highly functionalized (Z)-configured allylic alcohols/amines; the DFT (Density Functional Theory) studies suggested that the formation of a six-membered palladacyclic intermediate is the key for excellent stereocontrol [43]. 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates Apart from amines, various other nucleophiles were demonstrated to be efficient for the syntheses of a huge number of allylic alcohols with excellent stereoselectivity (Scheme 10) [44–47]. By judicious choice of the palladium catalyst and ligand, the nucleophilic attack could be switched toward the sterically hindered carbon of the palladium allyl intermediate that derived from vinyl carbonate; this concept was first demonstrated by Guo and co-workers in the preparation of chiral α,α-disubstituted allylic aryl amines [48], and later it was further developed by the Khan group in the synthesis of chiral sulfones [49]. In the presence of naphthol nucleophile, the Liang group disclosed an unprecedented [3 + 2] or [3 + 3] cycloaddition reaction using VCC as substrates under palladium catalysis [50]. By simply switching the ligands, the cyclization mode can be readily controlled toward the formation of O-heterocycles. Molecules 2019, 24, x FOR PEER REVIEW 10 of 16 were demonstrated to be efficient for the syntheses of a huge number of allylic alcohols with excellent stereoselectivity (Scheme 10) [44–47]. By judicious choice of the palladium catalyst and ligand, the nucleophilic attack could be switched toward the sterically hindered carbon of the palladium allyl intermediate that derived from vinyl carbonate; this concept was first demonstrated by Guo and co- workers in the preparation of chiral α,α-disubstituted allylic aryl amines [48], and later it was further developed by the Khan group in the synthesis of chiral sulfones [49]. In the presence of naphthol nucleophile, the Liang group disclosed an unprecedented [3 + 2] or [3 + 3] cycloaddition reaction using VCC as substrates under palladium catalysis [50]. By simply switching the ligands, the cyclization mode can be readily controlled toward the formation of O-heterocycles. Scheme 10. Pd-catalyzed decarboxylative formation of highly substituted (Z)-configured allylic alcohols from VCCs. Scheme 10. Pd-catalyzed decarboxylative formation of highly substituted (Z)-configured allylic alcohols from VCCs. Scheme 10. Pd-catalyzed decarboxylative formation of highly substituted (Z)-configured allylic alcohols from VCCs. Scheme 10. Pd-catalyzed decarboxylative formation of highly substituted (Z)-configured allylic alcohols from VCCs. In contrast, the catalytic transformations of alkynyl-substituted cyclic carbonates were less investigated. The alkyne-functionalized carbonates were frequently used as important synthons in asymmetric propargylation reactions for the construction of quaternary stereocenters. In the presence of sodium sulfinate nucleophiles and copper catalyst, chiral propargylic sulfones can be synthesized In contrast, the catalytic transformations of alkynyl-substituted cyclic carbonates were less investigated. 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates 3. Transition Metal-Catalyzed Decarboxylation of Cyclic Carbonates The alkyne-functionalized carbonates were frequently used as important synthons in asymmetric propargylation reactions for the construction of quaternary stereocenters. In the presence of sodium sulfinate nucleophiles and copper catalyst, chiral propargylic sulfones can be synthesized 11 of 16 Molecules 2019, 24, 3930 with CO2 as the sole byproduct (Scheme 11a) [51]. Moreover, the coupling of malononitrile and alkynyl-carbonates proved to be feasible resulting in a series of chiral polysubstituted dihydrofurans with high enantioselectivities (up to 97% ee) (Scheme 11b) [52]. Most recently, the Gong group achieved the asymmetric synthesis of numerous spiro compounds with this strategy using both ethynylethylene carbonates and carbamates as starting materials through a NHC/copper cooperative catalytic system (Scheme 11c) [53]. Molecules 2019, 24, x FOR PEER REVIEW 11 of 16 Scheme 11. (a) Cu-catalyzed asymmetric propargylic sulfonylation; (b) asymmetric [3 + 2] reaction of carbamates with malononitrile and (c) [3 + 3] and [3 + 4] annulations of isatin-derived enals. Scheme 11. (a) Cu-catalyzed asymmetric propargylic sulfonylation; (b) asymmetric [3 + 2] reaction of carbamates with malononitrile and (c) [3 + 3] and [3 + 4] annulations of isatin-derived enals. Scheme 11. (a) Cu-catalyzed asymmetric propargylic sulfonylation; (b) asymmetric [3 + 2] reaction of carbamates with malononitrile and (c) [3 + 3] and [3 + 4] annulations of isatin-derived enals. Scheme 11. (a) Cu-catalyzed asymmetric propargylic sulfonylation; (b) asymmetric [3 + 2] reaction of carbamates with malononitrile and (c) [3 + 3] and [3 + 4] annulations of isatin-derived enals. 4. Miscellaneous Substrates. 4. Miscellaneous Substrates olecules 2019, 24, x FOR PEER REVIEW 12 Scheme 12. Selective formation of γ-lactams via C-H amidation. Scheme 12. Selective formation of γ-lactams via C-H amidation. Scheme 12. Selective formation of γ-lactams via C-H amidation. Scheme 12. Selective formation of γ-lactams via C-H amidation. A Rh-catalyzed three-component approach was reported for the synthesis of α-branched amines with terminal alkenes as substrates. The reactions could be carried out under mild conditions and tolerated well with different functionalities [59]. Regioselective amidation of allylic alkenes involving inert C-H activation with the use of 1,4,2-Dioxazol-5-ones as starting materials was also feasible utilizing Ir or Rh catalyst [60–62]. As analogs of cyclic carbamates, isatoic anhydrides have also emerged as powerful building blocks for the preparation of functionalized N-heterocycles. For example, Scheidt and co-workers disclosed an NHC-mediated [4 + 2] cycloaddition of isatoic anhydride and trifluoromethyl ketones. Various enantioenriched dihydrobenzoxazin-4-ones functionalized with a CF3 group can be produced with this protocol (Scheme 13a) [63]. In this transformation, the generation of an NHC-bonded intermediate is vital for the success of the reactions. Additionally, a Brønsted acid-catalyzed [4 + 3] cyclization of N,N’-cyclic azomethine imines with isatoic anhydrides was accomplished by the Shi group (Scheme 13b) [64]. A Rh-catalyzed three-component approach was reported for the synthesis of α-branched amines with terminal alkenes as substrates. The reactions could be carried out under mild conditions and tolerated well with different functionalities [59]. Regioselective amidation of allylic alkenes involving inert C-H activation with the use of 1,4,2-Dioxazol-5-ones as starting materials was also feasible utilizing Ir or Rh catalyst [60–62]. As analogs of cyclic carbamates, isatoic anhydrides have also emerged as powerful building blocks for the preparation of functionalized N-heterocycles. For example, Scheidt and co-workers disclosed an NHC-mediated [4 + 2] cycloaddition of isatoic anhydride and trifluoromethyl ketones. Various enantioenriched dihydrobenzoxazin-4-ones functionalized with a CF3 group can be produced with this protocol (Scheme 13a) [63]. In this transformation, the generation of an NHC-bonded intermediate is vital for the success of the reactions. Additionally, a Brønsted acid-catalyzed [4 + 3] cyclization of N,N’-cyclic azomethine imines with isatoic anhydrides was accomplished by the Shi group (Scheme 13b) [64]. A Rh-catalyzed three-component approach was reported for the synthesis of α-branched amines with terminal alkenes as substrates. The reactions could be carried out under mild conditions and tolerated well with different functionalities [59]. 4. Miscellaneous Substrates. 4. Miscellaneous Substrates 1,4,2-Dioxazol-5-ones, known as dioxazolones, can be easily prepared from the corresponding commercially available alkyl carboxylic acids. These substrates are relatively easy to be activated under mild reaction conditions due to the presence of a weak N-O bond in the heterocycle. In general, due to the inherent instability, carbonyl nitrenes are prone to undergo Curtius rearrangement affording isocyanates as the main products. Based on the computational calculations reported by the Chang group [54,55], it was believed that the Curtius rearrangement is more sensitive to the charge variations of the metal center than the C-H insertion, thus electron-donating ligands may increase the Curtius-rearrangement barrier to a larger extent than the C-H insertion barrier. In early 2018, Chang and co-workers developed a method for the regioselective formation of γ-lactams with iridium catalysis (Scheme 12). The reactions proceeded smoothly via sp3 and sp2 C-H amidation with exceptional selectivity. The application potential of the methodology was further demonstrated by the late-stage functionalization of different amino acid derivatives and other bioactive compounds [54]. Enantioselective nitrene insertion to C(sp3)-H bonds was also developed by different research 1,4,2-Dioxazol-5-ones, known as dioxazolones, can be easily prepared from the corresponding commercially available alkyl carboxylic acids. These substrates are relatively easy to be activated under mild reaction conditions due to the presence of a weak N-O bond in the heterocycle. In general, due to the inherent instability, carbonyl nitrenes are prone to undergo Curtius rearrangement affording isocyanates as the main products. Based on the computational calculations reported by the Chang group [54,55], it was believed that the Curtius rearrangement is more sensitive to the charge variations of the metal center than the C-H insertion, thus electron-donating ligands may increase the Curtius-rearrangement barrier to a larger extent than the C-H insertion barrier. In early 2018, Chang and co-workers developed a method for the regioselective formation of γ-lactams with iridium catalysis (Scheme 12). The reactions proceeded smoothly via sp3 and sp2 C-H amidation with exceptional selectivity. The application potential of the methodology was further demonstrated by the late-stage functionalization of different amino acid derivatives and other bioactive compounds [54]. Enantioselective nitrene insertion to C(sp3)-H bonds was also developed by different research groups to afford chiral γ-lactams [55–58]. 12 of 16 12 of 16 Molecules 2019, 24, 3930 Scheme 12. Selective formation of γ-lactams via C-H amidation. Scheme 12. Selective formation of γ-lactams via C-H amidation. 4. Miscellaneous Substrates. 4. Miscellaneous Substrates Regioselective amidation of allylic alkenes involving inert C-H activation with the use of 1,4,2-Dioxazol-5-ones as starting materials was also feasible utilizing Ir or Rh catalyst [60–62]. As analogs of cyclic carbamates, isatoic anhydrides have also emerged as powerful building blocks for the preparation of functionalized N-heterocycles. For example, Scheidt and co-workers disclosed an NHC-mediated [4 + 2] cycloaddition of isatoic anhydride and trifluoromethyl ketones. Various enantioenriched dihydrobenzoxazin-4-ones functionalized with a CF3 group can be produced with this protocol (Scheme 13a) [63]. In this transformation, the generation of an NHC-bonded intermediate is vital for the success of the reactions. Additionally, a Brønsted acid-catalyzed [4 + 3] cyclization of N,N’-cyclic azomethine imines with isatoic anhydrides was accomplished by the Shi group (Scheme 13b) [64]. Scheme 13. (a) NHC-catalyzed [4+2] cycloaddition of carbamates and CF3-containing partners and (b) brønsted acid-catalyzed [4 + 3] cyclization of carbamates with N,N’-cyclic azomethine imines. Conclusions and Outlook Scheme 13. (a) NHC-catalyzed [4+2] cycloaddition of carbamates and CF3-containing partners and (b) brønsted acid-catalyzed [4 + 3] cyclization of carbamates with N,N’-cyclic azomethine imines. Scheme 13. (a) NHC-catalyzed [4+2] cycloaddition of carbamates and CF3-containing partners and (b) brønsted acid-catalyzed [4 + 3] cyclization of carbamates with N,N’-cyclic azomethine imines. Scheme 13. (a) NHC catalyzed [4+2] cycloaddition of carbamates and CF3 containing partners and (b) brønsted acid-catalyzed [4 + 3] cyclization of carbamates with N,N’-cyclic azomethine imines. Scheme 13. (a) NHC-catalyzed [4+2] cycloaddition of carbamates and CF3-containing partners and (b) brønsted acid-catalyzed [4 + 3] cyclization of carbamates with N,N’-cyclic azomethine imines. Scheme 13. (a) NHC-catalyzed [4+2] cycloaddition of carbamates and CF3-containing partners and (b) brønsted acid-catalyzed [4 + 3] cyclization of carbamates with N,N’-cyclic azomethine imines. References 1. Weaver, J.D.; Recio III, A.; Grenning, A.J.; Tunge, J.A. Transition Metal-Catalyzed Decarboxylative Allylation and Benzylation Reactions. Chem. Rev. 2011, 111, 1846–1913. [CrossRef] [PubMed] 1. Weaver, J.D.; Recio III, A.; Grenning, A.J.; Tunge, J.A. Transition Metal-Catalyzed Decarboxylative Allylation and Benzylation Reactions. Chem. Rev. 2011, 111, 1846–1913. [CrossRef] [PubMed] 2. Rodríguez, N.; Goossen, L.J. Decarboxylative Coupling Reactions: A Modern Strategy for C–C-Bond Formation. Chem. Soc. Rev. 2011, 40, 5030–5048. [CrossRef] [PubMed] 3. Xuan, J.; Zhang, Z.-G.; Xiao, W.-J. Visible-Light-Induced Decarboxylative Functionalization of Carboxylic Acids and Their Derivatives. Angew. Chem. Int. Ed. 2015, 54, 15632–15641. [CrossRef] [PubMed] , J ; g, ; , J g y y Acids and Their Derivatives. Angew. Chem. Int. Ed. 2015, 54, 15632–15641. [CrossRef] [PubMed] 4. Wei, Y.; Hu, P.; Zhang, M.; Su, W. Metal-Catalyzed Decarboxylative C–H Functionalization. Chem. Rev. 2017, 117, 8864–8907. [CrossRef] 4. Wei, Y.; Hu, P.; Zhang, M.; Su, W. Metal-Catalyzed Decarboxylative C–H Functionalization. Chem. Rev. 2017, 117, 8864–8907. [CrossRef] 5. Li, T.-R.; Wang, Y.-N.; Xiao, W.-J.; Lu, L.-Q. Transition-Metal-Catalyzed Cyclization Reactions Using Vinyl and Ethynyl Benzoxazinones as Dipole Precursors. Tetrahedron Lett. 2018, 59, 1521–1530. [CrossRef] 5. Li, T.-R.; Wang, Y.-N.; Xiao, W.-J.; Lu, L.-Q. Transition-Metal-Catalyzed Cyclization Reactions Using Vinyl and Ethynyl Benzoxazinones as Dipole Precursors. Tetrahedron Lett. 2018, 59, 1521–1530. [CrossRef] 6. Guo, W.; Gómez, J.E.; Cristòfol, À.; Xie, J.; Kleij, A.W. Catalytic Transformations of Functionalized Cyclic Organic Carbonates. Angew. Chem. Int. Ed. 2018, 57, 13735–13747. [CrossRef] 6. Guo, W.; Gómez, J.E.; Cristòfol, À.; Xie, J.; Kleij, A.W. Catalytic Transformations of Functionalized Cyclic Organic Carbonates. Angew. Chem. Int. Ed. 2018, 57, 13735–13747. [CrossRef] 7. Allen, B.D.W.; Lakeland, C.P.; Harrity, J.P.A. 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Conclusions and Outlook Funding: This work is supported by the starting funding scheme of Xi’an Jiaotong University Conflicts of Interest: The authors declare that there are no conflicts of interest. 5. Conclusions and Outlook 5. Conclusions and Outlook 5. Conclusions and Outlook This review briefly summarizes the synthetic application of cyclic organic carbonates and carbamates with transition metal catalysis in the last two years. More and more novel catalytic transformations have been realized with efficient and vibrant reactivity and selectivity. The combination of transition metal catalysis with the use of cyclic carbamates as starting materials provides new strategies for the preparation of structurally diverse N-heterocycles. Cyclic carbonates played an important role in the synthesis of O-heterocycles. The merging of dioxazolones and transition metal catalysts proved to be a powerful tool to achieve challenging C–H bond This review briefly summarizes the synthetic application of cyclic organic carbonates and carbamates with transition metal catalysis in the last two years. More and more novel catalytic transformations have been realized with efficient and vibrant reactivity and selectivity. The combination of transition metal catalysis with the use of cyclic carbamates as starting materials provides new strategies for the preparation of structurally diverse N-heterocycles. Cyclic carbonates played an important role in the synthesis of O-heterocycles. The merging of dioxazolones and This review briefly summarizes the synthetic application of cyclic organic carbonates and carbamates with transition metal catalysis in the last two years. More and more novel catalytic transformations have been realized with efficient and vibrant reactivity and selectivity. The combination of transition metal catalysis with the use of cyclic carbamates as starting materials provides new strategies for the preparation of structurally diverse N-heterocycles. Cyclic carbonates played an important role in the synthesis of O-heterocycles. The merging of dioxazolones and transition metal 13 of 16 Molecules 2019, 24, 3930 catalysts proved to be a powerful tool to achieve challenging C–H bond functionalization toward the formation of interesting compounds. However, most of the decarboxylative transformations with cyclic carbamate and/or carbonate substrates require expensive transition metal catalysts. The development of interesting decarboxylative reactions with these cyclic structures as starting materials with earth-abundant metal alternatives as catalysts would be highly desired and of significance. Alternatively, the exploration of a metal-free strategy would be highly attractive for pharmaceutical purposes. Furthermore, the combination of transition metal catalysts with photochemistry, radical chemistry or organocatalyst may bring new opportunities during the exploration of novel and interesting chemistry. Author Contributions: All the authors participated in drafting the manuscript. Funding: This work is supported by the starting funding scheme of Xi’an Jiaotong University. References Enantioselective Construction of Tetrahydroquinazoline Motifs via Palladium-Catalyzed [4 + 2] Cycloaddition of Vinyl Benzoxazinones with Sulfamate-Derived Cyclic Imines. Org. Lett. 2018, 20, 2880–2883. [CrossRef] [PubMed] 12. Mun, D.; Kim, E.; Kim, S.-G. Palladium-Catalyzed Decarboxylative [4 + 2] Cycloaddition of Vinyl Benzoxazinanones with Cyclic N-Sulfimines: Stereoselective Synthesis of Benzosulfamidate-Fused Tetrahydroquinazolines. Synthesis 2019, 51, 2359–2370. [CrossRef] 13. Zhao, H.-W.; Feng, N.-N.; Guo, J.-M.; Du, J.; Ding, W.-Q.; Wang, L.-R.; Song, X.-Q. 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Org. Lett. 2019, 21, 598–602. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Community gardening and wellbeing: The understandings of organisers and their implications for gardening for health
Health and place/Health & place (Online)
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1. Introduction There is a long history of utilising greenspace to meet public health objectives (Carpenter 2013) and public health has played a role in supporting both the expansion of community gardens, and the ways in which they are conceived and evaluated. A growing body of evidence attests to the potential for community gardening to improve health. Community gardening has been described as having biopsychosocial benefits (George 2013), and as “a unique activity for individuals to enhance physical activity levels and improve their diet” (Heise et al., 2017: 1). Greenspace access is positively associated with healthy life expectancy (Jonker et al., 2014). Public funds, via local authorities and health trusts, make up a portion of community gardening funding (Sempik and Aldridge 2006; Milbourne 2012), and public bodies may initiate or contribute to existing community gardening projects. Com­ munity gardening has been incorporated into healthcare strategies, both to improve health outcomes and combat health inequalities (Witheridge and Morris 2016). i Community gardening involves the communal cultivation of plants, varying in form according to local contexts and the needs and desires of gardening spaces and local residents. It includes collective gardening undertaken for community development, food production, health pro­ motion, horticultural therapy, collective action, and environmental and permaculture education (Nettle 2014). An archetypal British community garden might involve a small-to-medium sized plot of land, containing raised beds, crops of potatoes, peas, and tomatoes, and benches, tended to by residents, volunteers, and (or) those working in the third sector. Community gardening activities occur in a wider range of spaces, from streetside planters to sections of public parks, and may focus more on, for example, ornamentation or biodiversity. In recent years, community gardening has increasingly attracted the attention of those in public health and adjacent fields, where it has been viewed primarily as a health behaviour; a means of fostering particular health outcomes. It has otherwise been framed as activism driven by political goals (Follman and Viehoff 2015), as a therapeutic landscape (Sanchez and Liamput­ tong 2017), and as a means of mitigating experience of environmental or socioeconomic injustices (Earle 2011). While the stated aims may be diverse, evaluation is often specifically framed in terms of obesity prevention via diet and exercise, reflecting much of the biomedically-oriented literature. There is a paradigmatic tendency for public health to frame health promotion, and community gardening, in terms of behaviour or lifestyle change (Skrabanek 1994). Community gardening and wellbeing: The understandings of organisers and their implications for gardening for health Laura McGuire *, Stephanine L. Morris, Tessa M. Pollard Department of Anthropology, Durham University, Dawson Building, South Road, Durham, DH1 3LE, UK Laura McGuire *, Stephanine L. Morris, Tessa M. Pollard Laura McGuire *, Stephanine L. Morris, Tessa M. Pollard Laura McGuire , Stephanine L. Morris, Tessa M. Pollard Department of Anthropology, Durham University, Dawson Building, South Road, Durham, DH1 3LE, UK Department of Anthropology, Durham University, Dawson Building, South Road, Durham, DH1 3LE, UK A R T I C L E I N F O Keywords: Community gardening Therapeutic landscapes Nature-based social prescribing Medicalisation Public health Community gardening is increasingly framed and promoted as a way to foster healthful behaviours, as a well­ being practice, and as a public health tool. This paper draws on semi-structured interviews with community gardening organisers (n = 9) in the North East of England, who were engaged in translating and transforming discourses and ideas about community gardening into places and practices that people can draw benefit from. Here, community gardening can be understood as a bricolage of ideas, resources, and skills at the nexus of several influences and movements, assembled to produce a localised, everyday sort of social change. We conclude that framing community gardening as a simple solution to be harnessed in the promotion of health and wellbeing undermines the richness that sustains it and may lead to disenchantment within health services and community gardening organisations that could threaten the future of ‘green social prescribing’. * Corresponding author. E-mail address: laura.e.mcguire@durham.ac.uk (L. McGuire). Contents lists available at ScienceDirect Contents lists available at ScienceDirect * Corresponding author. E-mail address: laura.e.m Available online 11 March 2022 1353-8292/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.healthplace.2022.102773 Received 28 July 2021; Received in revised form 1 November 2021; Accepted 18 February 2022 Health & Place 75 (2022) 102773 Health & Place 75 (2022) 102773 Health & Place 75 (2022) 102773 Contents lists available at ScienceDirect Health and Place journal homepage: www.elsevier.com/locate/healthplace Available online 11 March 2022 1353-8292/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author. E-mail address: laura.e.mcguire@durham.ac.uk (L. McGuire). https://doi.org/10.1016/j.healthplace.2022.102773 Received 28 July 2021; Received in revised form 1 November 2021; Accepted 18 February 2022 1. Introduction From this relational perspective, a person’s experience of gardening is shaped partly by past nature-based experiences alongside others, the ‘green’ cultural narratives one adopts through the life course (Bell et al., 2014), and the social and material affordances of greenspaces. And gardening is of course, about more than being in a garden; it is a meaning-laden (Ong et al., 2019), dynamic practice that can play a di­ versity of roles in one’s life. ‘Gardener’ is a lifestyle and identity; such lifestyle projects be a source of pleasure amid busy and changing lives (Chaney, 2002; Taylor 2002). Material semioticians and multispecies ethnographers have captured how garden-dwelling beings ‘become-­ with’ (Haraway 2016) one another through generative affective en­ counters. Both gardeners and plants are shaped through their interactions, in “ontological relations in their own rights” (Archambault 2016: 265–266) sometimes as friends (Degnen 2009), and analytically, as actants or agents. There is a sense that community gardens ‘have it all’; their ‘holistic’ orientation is one of their appeals. In these gardens, people may gain skills (Crossan et al., 2016), develop attachment to place and commu­ nity, and flourish (Whatley et al., 2015). However, their therapeutic potential may not be available to all, depending partly on differing biographical experiences of nature and gardening (Bell et al., 2018), and not all of the discourses that circulate within community gardens are philanthropic or benign. Gardens and greenspaces may be spoken about in ways that can perpetuate harmful discourses, often related to xeno­ phobia or exclusionary perceptions about heritage (Degnen 2009; Cloke, 2013). Both negative and positive aspects of the socio-ecological (Tid­ ball and Krasny 2007) space of the garden affect participation in gardening practices; there is ongoing debate around the enclosure and exclusivity of community gardens, and the equitable spread of the social capital they may foster (Glover 2004; Traill, 2021). The promotion of gardening for health overlaps with trends in per­ sonalised and person-centred healthcare involving adapting healthcare offerings and advice to the desires and needs of individuals. These converging movements aspire to a locally-tailored healthcare, that considers patient preferences, characteristics, and illness experiences. Their establishment is linked to efforts to improve healthcare efficiency, patient and nurse advocacy (Ghebrehiwet 2011), and neoliberal con­ ceptions of the responsibilised patient-as-consumer (Day et al., 2017; Van den Akker, 2019; Savard 2013). Social prescribing shares values with this movement (Polley et al., 2017). 1. Introduction Community gardening is framed as a relatively cheap (Lovell et al., Health and Place 75 (2022) 102773 L. McGuire et al. everyday spaces with health-promoting qualities (Bell et al., 2018). Gardeners have frequently described gardens as ‘therapeutic’, ‘rewarding’ (Sonti and Svendsen 2018) oases (McCabe 2014; Slabinski 2012), connoting a space of calmness amidst chaos. In contrast to rushed temporality of the workplace or home, gardens can offer a slower, restorative time that promotes restful psychosocial states (Bell et al., 2017; de Oliveira et al., 2013). Later writing in the therapeutic land­ scapes literature has questioned the inherent healthfulness of particular landscapes, instead emphasising that therapeutic landscape experiences (Williams 1998; Conradson 2005) are a contingent outcome of situated person-place interactions (Bell et al., 2019). 2014) prophylactic, that can increase fruit and vegetable intake (Algert et al., 2016; Hale et al., 2011) while providing an opportunity for moderate intensity physical activity (Park et al., 2014; Hawkins et al., 2015). Health professionals are enjoined to encourage widespread development of gardens as a means for fostering healthful behaviours (Thompson 2018), while developments in social prescribing lay a framework for expanding gardening for health. p g g g Increasingly, mental health is conceived of in similar terms: as sus­ tained by healthful behaviours and conscious caretaking. This iterates wider efforts to be more attentive to mental wellbeing, to encourage ‘connecting to others’ and ‘taking notice’ of one’s surroundings (Aked et al., 2008). The broader psychosocial benefits of community gardening are becoming more widely promoted, harnessed, and researched (Phil­ lips et al., 2015). A meta-analysis (Soga et al., 2017) of quantitative studies found that gardening could reduce symptoms of depression, anxiety, stress, and mood disturbance, and, qualitatively, community gardeners report improved feelings of mental wellness (Stuart 2005). Mental health-oriented organisations have promoted nature contact (MHF, 2021), while gardening-based organisations articulate the bene­ fits of horticulture through the language of mental wellness. Rather than merely hosting physical activity and encouraging vegetable consump­ tion, community gardens have been promoted as multifunctional spaces, carrying the positive valence of ‘greenness’. Healthcare practitioners have advocated for a ‘dose’ of gardening to improve mental health (Thompson 2018), while horticulture as both a therapy and a broader therapeutic activity (Sempik and Alridge 2006) grows. 1.2. Relational approaches to therapeutic experiences in greenspace Gardening, then, has been understood as a desirable, motivating, and sustainable activity for the horticulturally inclined to meet health goals. This framing both dovetails and diverges with other more relational understandings of links between community gardening and wellbeing. Most proximate are understandings of community gardening as a ther­ apeutic practice that also enhances mental wellbeing. More sociopolit­ ical and relational understandings of community gardening are mobilised in considering how these gardens can cohere communities, and enhance social, cultural, and economic capital. Entailed within this are questions of greenspace accessibility, and nuanced conceptions of therapeutic place. The therapeutic landscapes (Gesler 1996) literature increasingly addresses the complex, contingent, and therapeutic nature of place. Bell et al. (2017) define therapeutic landscapes as those that provide op­ portunities for meaningful activity, embodied restoration, sociality, and safety. The concept of therapeutic landscapes initially described extraordinary spaces of healing, but has more recently been applied to 1.3. Aims In the context of varied and sometimes contested understandings of the relationships between community gardening and health and well­ being, this study explores the beliefs, values, and motivations of com­ munity gardening organisers, in relation to their biographical, historical, and geographical contexts and wider discourses. Community gardens are evidently not only material, but social environments; by focusing upon community gardening organisers, we build upon research (Dinnie et al., 2013) that considers how people’s greenspace experi­ ences, intentionally and otherwise, shape and mediate those of others. As both facilitators and possible opinion leaders (Katz and Lazarsfeld, 1955), organisers have an important role in shaping activities and attendee bases, attracting resources, and gatekeeping access. We discuss the rich emerging picture of the experiences and understandings of community gardening organisers in relation to the growing con­ ceptualisation of, and institutional support for, gardening for health and wellbeing. 1. Introduction ‘Social prescribing’ describes the referral of people, by health professionals, to ‘non-medical’ activ­ ities. This broad practice occurs in different forms, both within and outside of the UK. However, a particular type of social prescribing is increasingly recognised and integrated within the UK healthcare system, one in which referrals are mediated via a ‘link worker’ who supports people in identifying their needs and benefiting from their prescription. Nature-based or ‘green’ social prescribing is a rapidly growing practice, and has been framed in terms of improving health, tackling health in­ equalities, and aiding those “hardest hit by coronavirus” (DEFRA et al., 2020). 1.3. Aims 2.3. Sample In May and June of 2020, LM conducted nine interviews with nine community gardening organisers, from seven organisations that facili­ tated community gardening (See Table 1 for contexual and demographic information). Interviews lasted between 57 and 93 min, with an average time of 74 min. Participants are referred to as organisers, while those who come to the community gardening sessions are called attendees. 2.4. Data analysis The recorded interviews were manually transcribed by LM, to enhance familiarity with the data (Reissman 1993). Data were analysed using thematic analysis, “a method for identifying, analysing, and reporting patterns (themes) in the data” (Braun and Clarke 2006: 79). This method accommodates constructionist epistemology, as themes are induced and interpreted from manifest and latent aspects of the inter­ view. Using the software NVivo 12, the transcriptions were inductively coded by LM, with accompanying memos. The stages of familiarisation, code generation, and finding and defining themes (ibid.) were followed recursively. This involved the grouping of the organisers’ actions and aims under more encompassing, conceptual codes, which were dis­ cussed by all authors. 2.5. Ethics This study received ethical approval from the [Anonymised] Uni­ versity Anthropology Department Ethics Committee. Participants received their information sheet, privacy notice, and consent form at least one week prior to their interview, allowing time for them to read the information and ask questions. In the information sheet, and at the beginning of interviews, participants were reminded that their consent to participate was revocable. All transcripts were anonymised, and participants were given pseudonyms. 2. Methods This study sought to explore the understandings of organisers, and the experience of community gardening, in the North East of England, through semi-structured interviews (n = 9). 2 L. McGuire et al. L. McGuire et al. Health and Place 75 (2022) 102773 3. Results In contexts where interview-like scenarios are a familiar part of life, the research interview can constitute a potentially “experience near” (Hockey 2002: 218) method. The experiences of organisers with funding-based evaluations, intermittent encounters with researchers, and a coronavirus-necessitated familiarity with time-bounded distance interactions, suggest this method is practical and elucidative. We We first describe the gardens and backgrounds of organisers before moving on to explore how organisers understood community gardening itself, considering what they were trying to do or foster through their work. 2.2. Data collection In-depth, semi-structured interviews with organisers from organisa­ tions working within North East England were conducted and audio- recorded. In the context of social distancing in response to the COVID pandemic, most of the interviews were conducted via online video- calling service. Video-calling allowed for non-verbal communication, and aided in the development of rapport and intimacy.. Reciprocity and flexibility can increase validity (Oakley 1981; Hyman et al., 1954), and make possible the probing for more in-depth information around bi­ ography, values, and wider cultural influences. An interview schedule was loosely followed, in a conversationally flexible (Currivan 2008) manner. Questions were directed towards eliciting the promises and challenges of community gardening, the practical matters of sustaining these practices, and organisers’ beliefs and values. For the three par­ ticipants who declined to use video-calling software (citing unfamiliar­ ity with or aversion to the software), telephone interviews were conducted. To provide a greater sense of immersion and context, social media pages (for gardens using them) were regularly checked weeks prior to interviewing, and throughout data analysis. Accompanying documents were read, including newsletters and organisational files. These documents gave a sense of the logistics and nature of the groups, while being interpretable artefacts elucidative of the enduring meanings circulating within the groups. b Index of multiple deprivation (IMD) decile of Lower Layer Super Output Area (as of 2019) of gardening location. IMD is a measure of relative deprivation of small areas within the UK. a Community Interest Company. b 2.1. Recruitment acknowledge interviews as an interactive site of meaning construction, and data were analysed with reflexivity surrounding how the situation and researcher subjectivity shape data production. Community gardens, and their contact email addresses, were located using the websites seedsofeden.org, farmgarden.org.uk, rhs.org.uk, and google.com, and facebook.com. Most participants were recruited via emails sent to these addresses, some of whom forwarded this informa­ tion on to other organisers. We employed purposive sampling, selecting participants practicing in socioeconomically diverse areas, and urban and rural areas. This sampling method was used in combination with snowball sampling due to challenges of paid staff being furloughed, which also provided information about the connectedness and social capital of participants. The unequal burden of coronavirus may bias the sample against groups who are disproportionately impacted, such as those of an ethnic minority (Liverpool 2020) or of ‘low’ socioeconomic status (Blundell et al., 2020). 2.3. Sample Community Interest Company. b Index of multiple deprivation (IMD) decile of Lower Layer Super Output Area (as of 2019) of gardening location. IMD is a measure of relative deprivation of small areas within the UK. c Community gardening initiative spans multiple locations, value given for one site. “I want to enable people to discover nature, to love it and understand it.. once you see something and you love it, you take care of it” (Caitlyn) “This is normal to us, we know it makes us feel great and we love it, because we have positive association with it.” ‘Nature’ did not refer only to flora or fauna, but also to temporal, processual, and almost cognitive elements too. Participants described nature as “cleverer than you think it is” (Jacob), having “her own seasons” (Oliver). They portrayed nature as having patterns and systems that should be followed: “there’s a value of being, of running alongside nature” (Oliver). This pace was something to synchronise with, to find peace within, and give people “something to hold on to” (Jessica). Activities at the gardens would be planned around seasonality; summer gatherings were both weather-afforded opportunity, and a conscious effort to make attendees aware of the flow of nature, and thus be ‘in touch’ with one’s surroundings in a way that was considered beneficial. For some, this respect and reverie for ‘nature’ (occasionally used interchangeably with ‘creation’) was described as a form of spirituality. For Oliver and Cait­ lyn, community gardening was an enactment of an environmentalist religiosity, rooted in the growing wider critiques of anthropocentrism and modernity surrounding their religions (see White 1967). Other formative experiences included volunteering and activism. Civic engagement was an important value for some of the participants. Activist and environmentalist identities were described as emerging through participation in urban agriculture (for some, across continents), experiences with permaculture and climate action groups, formal and informal study, and interactions with environmentalist coreligionists. The participants recounted the founding stories of their gardens, involving friends coming together in response to concerns about com­ munity sufficiency, environmental sustainability, austerity, or wishes to promote calm or contemplation. A desire to rehabilitate local green­ spaces and facilities was common. Martin, Andrew and Bradley had been spurred into action following public meetings to discuss this perceived neglect. The participants’ current social positions and connections proved important for their organising roles. These social connections allowed participants to mobilise resources that supported their community gardening projects. Social cause groups could be vital sources of infor­ mation and ideas, as well as opening channels for recruitment of at­ tendees, and even providing the land upon which the gardens were created. Caitlyn told of “[pinching] some policies and procedures from a local charity”, and Sarah described how she modelled the “systems” she created in the garden upon those taught in permaculture courses. “I want to enable people to discover nature, to love it and understand it.. once you see something and you love it, you take care of it” (Caitlyn) Ma­ terial and socioeconomic factors were important in determining the connections one could make. One of the gardens was in an affluent area, and Martin commented that: “in some ways, the Christian religion has been responsible for the..pillage of the environment..And we’ve kind of, humanity has trashed the earth … humans are part of the whole creation, in that we have a responsibility to look after the rest of creation” (Caitlyn) Table 1 participant information. Participant Age Gender Type of organisation(s) Highest level of formal education Type of organiser role Index of multiple deprivation decile of garden location (1 is most deprived)b Caitlyn 59 Female Church-affiliated Postgraduate degree Volunteer 5 Martin 75 Male Charity Postgraduate degree Volunteer 10 Andrew 79 Male Charity Postgraduate degree Volunteer 10 Oliver 47 Male Church-affiliated Undergraduate degree Volunteer, trustee 2 Sarah 37 Female CICa Undergraduate degree Employee 3 Jessica 31 Female CIC; charity Undergraduate degree Self-employed 4c Jacob 53 Male Charity Post-secondary vocational qualification Employee 5c Emily Data missing Female Charity Post-secondary vocational qualification Employee 5c Bradley 72 Male CIC Postgraduate degree Volunteer, director 5c a Community Interest Company. b Index of multiple deprivation (IMD) decile of Lower Layer Super Output Area (as of 2019) of gardening location. IMD is a measure of relative deprivation of small areas within the UK. c Community gardening initiative spans multiple locations, value given for one site. y p y b Index of multiple deprivation (IMD) decile of Lower Layer Super Output Area (as of 2019) of gardening location. IMD is a measure of relative deprivation of small areas within the UK. c Community gardening initiative spans multiple locations, value given for one site. 3 L. McGuire et al. Health and Place 75 (2022) 102773 organiser biographies and the social and environmental surrounds of the gardens. organiser biographies and the social and environmental surrounds of the gardens. 3.1. The gardens and the people All of the participants held organisational roles in organisations that facilitated community gardening. Three participants (Caitlyn, Oliver, and Sarah) worked on single-site community gardens (two of which occupied church-owned and adjacent land), whereas others worked across several sites. Participants dedicated their efforts to facilitating food growing, teaching, beekeeping, litter-picking, exploring, social­ ising, and eating, across gardens, parks, orchards, and woodlands. Some of the participants also worked to encourage gardening in homes, schools, and alleyways. For some of the organisations, community gardening was one of several projects that the organisation fostered. The sometimes-dispersed nature of activities, and sometimes-peripatetic working of organisers, is a little captured element within the existing literature. 3.2. Enabling people to “discover nature” 3.2. Enabling people to “discover nature” A dominant theme running through every interview to differing ex­ tents was that of ‘nature’, variously conceived. Nature was construed as a wise, coherent, and even spiritual or magical force. Participants described plants in animistic and relational ways. Plants would receive “almost parental” (Oliver) care. Instead of positioning humans and ‘na­ ture’ in antagonistic relation, participants stressed their essential inter­ connection. Litter-picking and plant cultivation became intentional pedagogies of interconnection, as participants tried to help attendees “understand … the very delicate relationship everything has with each other” (Emily) through these activities. As in Nettle’s (2010) research, the or­ ganisers’ working alongside nature was undergirded by ethics of com­ munity and cultivation, more than a romantic and protectionist view of nature. For Sarah, learning to value and caretake plants and animals was part of a process of learning to better treat and value herself, according with an understanding of the self as a component in a shared and interconnected ecosystem. Environmental caretaking was, for some, a matter of mobilising emotion - care, empathy, and wonder: Common to most organisers was a biography rich with green space activity; they had “always spent a lot of time in nature” (Sarah). They recounted memories of accompanying grandparents to allotments, and growing plants in parents’ gardens, revealing their encultured and socialised affinities for nature. Gardening was a familiar part of the rhythms of their lives; a routine and reliable source of calm and comfort. Speaking of her, and her colleagues’, familiarity with being in green spaces, Jessica said: “I want to enable people to discover nature, to love it and understand it.. once you see something and you love it, you take care of it” (Caitlyn) 3.3. Fostering wellbeing Most of the participants understood community gardening as something which enhanced their wellbeing. They recounted how it had helped them through difficult life events and mental unwellness, and provided a sense of purpose, enjoyment, and calm. They stressed the importance of the social, psychological, eudaimonic aspects of well­ being, and their passion for improving the confidence and skillsets of attendees - particularly those in more socioeconomically deprived areas. “there’s a lot of professionals, and over the years down the park we found some people with great expertise”. Their tools were serviced by engineer attendees, freely. This group had one of the largest attendee bases, consisting of many retirees. Oliver worked in a professional role, which was useful for both managing group finances and for utilising his social network for “funding and going to other professionals”. In contrast, Sarah, organising in a relatively deprived area, described how fellow organisers and volunteers recur­ rently fell away due to time and resource constraints, which limited the ability of such (former) attendees to contribute to, or benefit from, these gardening social networks. l Holistic, multifaceted conceptions of wellbeing mirrored ideas about community gardens, as: “[working] at so many different levels … the physical activity, which is good, the being outside, getting some vitamin D and some sunshine. It’s the social, and the spiritual bits … it ticks lots of boxes.” (Caitlyn) Fostering a sense of “collective … ownership” (Oliver), or having a stake in a shared local environment, was understood by many as vital for “unlocking” the benefits of community gardening. Feelings of ‘being part’ Community gardening activities were influenced by the interests, skills, knowledge, and connections of organisers, themselves shaped by 4 L. McGuire et al. Health and Place 75 (2022) 102773 traditional conceptions of health oriented around eating healthily, ex­ ercise, and medication. The participants’ experiences accord well with the concept of communal therapeutic mobility (Pollard et al., 2020), in which people involved in communal activity experience emergent therapeutic benefit. This is best captured by Caitlyn’s belief that the “magic” of the garden was a combination of the environment and the sociality it afforded: “it isn’t any one person that makes that happen”. and belonging, on abstract and physical levels, can lay the foundations for stronger feelings of connecting with other people (Priest, 2007). Relatedly, many participants made efforts to create an atmosphere of non-hierarchy and equal stake-holding. 3.4. Modelling and manifesting change Rather than being primarily about plant cultivation, several partic­ ipants saw community gardening as an enactment of social change. For some, this change was a local effort to cohere the local community, improve the life chances of the disadvantaged, or to beautify local greenspace for the benefits of residents nearby. Emily’s wish, in a context of high socioeconomic deprivation, was to help young people develop skills, confidence, and to “provide some greenery and some salad to go with a meal”. Others had more outwardly political and ‘subversive’ ideas and motivations, using community gardens as spaces to model and manifest practices and ideas they believed society should adopt more widely. In this way, a community garden might be viewed as a means for creating change, rather than an end in itself. Sarah and Oliver voiced critiques of capitalism and patriarchy, and concerns for social justice influenced the practices of the gardens, which were viewed as a: It is not only the properties of the environment that were perceived to be salutogenic, but the activities that it afforded. Gardening involves processes and movements, of tidying, watering, sowing, and monitoring, while transforming seeds into plants and punnets. These processes involve “quite a bit of buddying up” (Caitlyn); they provided opportu­ nities to be more or less socially involved while remaining physically involved, which can relax pressure around socialising (Doughty 2013). For Oliver, the yields of the garden were not just: “forum for having [political] discussions. The kind of space where con­ versations can be open and candid, and based around … love and kind­ ness” (Oliver). “the flowers, the produce … but the human interaction and the social inclusion and the social development is another yield you can get from the garden”. His perspective resonates with theorisations of community gardens as potential sites of ‘conscientisation’ to social inequities (Barron 2017; Freire 1972; Staeheli et al., 2002). While less inclined to describe themselves as activists, other participants voiced a sense that society was not as green, nor healthful, as it should be: Participants expressed the healthfulness of gardening using vary­ ingly weighted mixtures of ‘lifestyleist’ (Powell et al., 2017) ideas and language typical of biomedical understandings, and those rooted in a more “holistic” (Sarah) understanding. 3.3. Fostering wellbeing y q g Many participants gave a sense that some of the salutogenic prop­ erties of community gardening were rooted in both its materiality and meaning, according with Bhatti et al.‘s (2009: 68–69) ideas about the power of “literal and metaphorical ‘mixing with the earth’“. The phys­ icality of gardening was perceived as “[helping] to ground somebody in a difficult situation” (Sarah), and “having my hands in the soil, quietly talking to others, and seeing things” (Oliver) was felt to be a vital part of recovery from mental unwellness. Participants relayed the pride and satisfaction they felt they saw in attendees, as they harvested the “fruits of the la­ bour” (Emily). In this way, the production of plants and the garden seemed to materialise and evidence the qualities of the attendees, in ways that participants believed to improve self-worth. While the phys­ icality of gardening could be problematic – the wet could discourage, and the cold could be challenging for attendees struggling with fatigue – organisers would try to mitigate these issues, and saw this as building attendees’ resilience. 3.4. Modelling and manifesting change Tackling obesity was a target in many of his charity’s (often externally-funded) projects, yet Jacob said they struggled to engage “the obese and the really unfit”, who he perceived to be “for want of a better word, terminally lazy” and apathetic. These ideas were mixed with a more empathic understanding, which his colleague, Emily, tended towards. Jacob described how attendees referred from a mental health charity enjoyed socialising through the project, but did not want to do the “work”. Of them, he said that gardening was: “[gardening] should be part of everything, it should be part of hospital treatments, it should be a part of schools … Because, it’s not human to be cooped up, staring at a screen” (Jessica) These desires to manifest social change seemed to be rooted, for some, in disillusionment with politics, modern living, and other forms of activism. They described community gardening as a way to manifest their beliefs, that was realistic and tangible. Community gardens appeared as an avenue where participants could enact agency and ‘get things done’. This contrasted with their experiences in other domains, including employment and politics, in which they felt their agency was constrained. Their community gardening was both continuous and discontinuous with their prior experiences with international move­ ments (such as Extinction Rebellion, Transition Towns, and permacul­ ture) and local groups. While often still informed by the ideals of such groups, many had become disillusioned with trying to create large-scale change, and saw community gardening as a way to create local, palpable social change. For some, this meant a shift away from radical environ­ mentalisms, towards ordinary, everyday environmentalisms and activ­ isms (Milbourne 2012; Nettle 2010). Our interviews evidence resistance to a neoliberal ‘system’, alongside a somewhat reluctant redirection of activistic efforts towards the local. “good for them in that it got them out the house, and got socialised and got chatting and met new people, so it was a success in that respect.” This suggests that a mixture of views could be found at the level of organisers, and within community gardening organisations (see Powell et al., 2017). Several participants voiced critiques of mainstream or biomedical approaches to health, derived from personal experience, and, for Cait­ lyn, her pre-retirement experience as a healthcare practitioner: “all I had to offer was referral or erm, tablets, or listening. 3.4. Modelling and manifesting change Most participants tried to ensure that activities were free, and tried to match activities to different ability levels. This might mean giving more strenuous tasks to the more physically able, or breaking activities into “small-bite tasks” (Emily) for attendees who might struggle with con­ centration. They identified factors that discouraged people from participating in community gardening: teenagers were viewed as often seeing the activities as “uncool” (Emily; see the ‘teenage dip’, Richardson et al., 2019; MEF, 2021), while parents sometimes saw the activities as infantile – an unfortunate corollary of encouraging gardening among children. Exciting, higher-risk activities were mixed in to encourage participation, and Emily described how the success of a group activity could depend upon whether she could get the “leader” to decide it was worthwhile. This attests to the importance of organisers. Experiencing the tangible, material benefit of eating and sharing food encouraged further participation. Yet, despite their efforts, there were people who the organisers could not engage. It was accepted that not everybody would be interested. Some residents actively hindered the organisers’ work, through recur­ ring vandalism. This was more commonly mentioned by those working in more socioeconomically deprived areas, and it garnered empathy, frustration, and anger to varying degrees. Oliver described this as an expression of “urban pressure”, a term circulating in his activist circles, while Bradley discussed it alongside the demise of services for youth. There was thus a sense amongst several organisers that the success of community gardening was interlinked with the wider context. If community gardening is ‘to work’ for public health, a process of bidirectional influence and mutual accommodation might contribute to more successful outcomes. Organisers might appropriately and benefi­ cially be recognised as skilled and influential practitioners, with a closeness to those in the communities in which they work, and with which healthcare professionals work.. Marsh (2020) believes that community gardening can be a form of public health intervention, by virtue of its socio-ecological aims, and concern with the multiple, widespread and interacting factors that impact the wellbeing of com­ munities. This may apply to some strands of public health – and some community gardens - more than others. As the organisers’ un­ derstandings demonstrate, the aims (and capacities) of community gardening projects can be diverse. 3.4. Modelling and manifesting change What they really needed was social contact and friendship and, activity … I wanted to … practice, but in a more holistic way.” As discussed, the organisers were passionate about the therapeutic and eudaimonic potential of community gardens, and encouraged the participation of a diversity of people. Thus, it was important that the gardens were welcoming, inclusive, and non-judgemental social spaces. Community gardens may allow for the building of community cohesion across boundaries (Hite et al., 2017). Social justice was a key concern and value for many, and they saw their community gardening as a way to address social exclusions – be they to do with ability, access to greenspace, wellbeing, or education. Oliver saw this as integral to what the garden, and community gardening, was: Oliver espoused a non-individualised, multi-level conception of wellbeing, and viewed system change as part of the “long-term cure” for mental unwellness: “which is, you know, which is looking after the system, trying to fix the system, and fixing that person around, and looking after that person’s, you know, whole wellbeing”. Community gardening was understood as extending beyond 5 5 Health and Place 75 (2022) 102773 L. McGuire et al. “we’re an inclusive space, we’re against all forms of discrimination … By nature.” themselves. This is relevant to the upscaling of green social prescribing within the UK national health system (DEFRA et al. 2020; NHS 2019), and the broader promotion of gardening as a health practice or behav­ iour. Social Farms and Gardens (2019) expresses worry that, depending upon how social prescribing is enacted, community gardens could risk “losing their very essence”; the richness of this essence(s) is evident in the organisers’ understandings and experiences. As public health im­ peratives increasingly shape community gardening through funding, referrals, and traffic of ideas, it must be considered how this may represent a medicalising encroachment upon these practices. Provider organisations may ‘drift’ from their prior objectives and concerns (Bennett and Savani 2011), and towards lifestyleist conceptions of health and wellbeing (Williams and Fullagar 2019). Incentives to target particular ‘risk categories’ – as suggested in Jacob’s comments - may engender stigmatising attitudes that discourage participation, and reduce its health-giving potential (Meadows and Bombak 2019; Thomas 2015; Drury and Louis 2002). A mismatch in the expectations and pri­ orities of gardeners or referrers and community gardening organisers may disorient or alienate those referred. 4. Conclusions: community gardening for health - promise and caution Echoed in the accounts of the organisers are some of the themes and ideas from public health discourses. For the participants, community gardening was indeed a practice that formed part of their routines of wellness, socialising, leisure, and community building, and they were enthusiastic in their commitments to foster these experiences for those with whom they worked and volunteered. The influence of public health discourses was most evident in language relating to diet and exercise. Yet, both those who did and did not espouse such views saw the well­ being benefits of community gardening as exceeding these parameters. For some, their views were rooted in beliefs expressed about the de­ ficiencies and reductionism of mainstream approaches to health. For most, the benefits and roles of community gardening in these North East England sites were myriad and complex. Organisers’ community gardening was, variously, an enactment of civic duty, a holistic and therapeutic practice, a radical social action, and a way to cohere and improve the life chances of residents. These meanings and imperatives seem to both motivate and constitute some of the biopsychosocial benefits of community gardening for participants. Relatedly, creating gardening spaces and facilitating these practices was a sedulous process, requiring knowledge, time, and care. The success and sustainability of gardening sites depended upon the skills, social capital, resources and experiences of organisers and local residents. Organising community gardening built upon biographies that encultured affinities for nature, and taught organisers how to nourish green spaces and engage residents. To varying degrees, the discourses and beliefs that inspired these ac­ tivities shaped and circulated through the spaces in which organisers worked and volunteered. Community gardens can be understood as a bricolage of ideas, resources and skills at the nexus of several influences and movements, assembled to produce a localised, everyday sort of change. p q p y The organisers’ understandings of the benefits and roles of commu­ nity gardening, and of the factors that aid and hinder its success and sustainability, have implications for its utilisation in improving health and wellbeing outcomes. Organisers play a significant role in adapting the socio-ecological environment of the garden to the interests, experi­ ences, and skills (Howarth et al., 2021) of attendees, in the interest of making these therapeutic experiences available to others. 3.4. Modelling and manifesting change Australian-based research suggests that community gardening may be most successful in contributing to­ wards the aims of public health when gardens and their organisers are well connected, well resourced, and when supported by other organi­ sations and services (Marsh et al., 2018). Yet, community gardening cannot act upon all that determines wellbeing, nor always in a wide­ spread, equitable, and upstream way. i References Heise, T.L., Romppel, M., Molnar, S., Buchberger, B., van den Berg, A., Gartlehner, G., Lhachimi, S.K., 2017. Community gardening, community farming and other local community-based gardening interventions to prevent overweight and obesity in high-income and middle-income countries: protocol for a systematic review. BMJ Open 7, e016237. Aked, J., Marks, N., Cordon, C., 2008. Five Ways to Wellbeing. New Economics Foundation, London. Algert, S., Diekmann, L., Renvall, M., Gray, L., 2016. Community and home gardens increase vegetable intake and food security of residents in San Jose, California. Calif. Agr. 70 (2), 77–82. p Hite, E.B., Perez, D., D’Ingeo, D., Boston, Q., Mitchell, M., 2017. Intersecting race, space, and place through community gardens. Ann. Anthrop. Pract. 41 (2), 55–66. Archambault, J.S., 2016. Taking love seriously in human-plant relations in Mozambique: toward an Anthropology of affective encounters. Cult. Anthropol. 31 (2), 244–271. Hockey, J., 2002. Interviews as ethnography? Disembodied social interaction in britain. In: Rapport, N. (Ed.), British Subjects. Berghahn Books, Oxford, pp. 209–222. H H H C bb W F ld J J Cl d W H S b C H 1954 I i i i Hockey, J., 2002. Interviews as ethnography? Disembodied social interaction in britain. In: Rapport N (Ed ) British Subjects Berghahn Books Oxford pp 209 222 Barron, J., 2017. Community gardening: cultivating subjectivities, space, and justice. Local Environ. 22 (9), 1142–1158. In: Rapport, N. (Ed.), British Subjects. Berghahn Books, Oxford, pp. 209–222. Hyman, H.H., Cobb, W., Feldman, J.J., Clyde, W.H., Stember, C.H., 1954. Interviewing in Social Research. Chicago University Press, Chicago. Bell, S.L., Phoenix, C., Lovell, R., Wheeler, B.W., 2014. Green space, health and wellbeing: making space for individual agency. Health Place 30, 287–292. B ll S L Wh l B W Ph i C 2017 U i i l h di g y g Jonker, M.F., Lenthe, F. J. van, Donkers, B., Mackenbach, J.P., Burdorf, A., 2014. The effect of urban green on small-area (healthy) life expectancy. J. Epidemiol. Commun. H. 68 (10), 999–1002. l Bell, S.L., Wheeler, B.W., Phoenix, C., 2017. Using geonarratives to explore the diverse temporalities of therapeutic landscapes: perspectives from “green” and “blue” settings. Ann. Am. Assoc. Geogr. 107 (1), 93–108. Katz, L., Lazarsfeld, P., 1955. Personal Influence: the Part Played by People in the Flow of Mass Communications. Free Press, New York. Bell, S.L., Foley, R., Houghton, F., Maddrell, A., Williams, A.M., 2018. 4. Conclusions: community gardening for health - promise and caution They under­ stood nature as a wise and wondrous force, that they worked to encourage encounters with – sometimes, against the competing mean­ ings it held for those who came upon the garden. Wellbeing is “ines­ capably mediated by social relations (and associated networks, meanings, and practices)” (Dinnie et al., 2013: 2). The joy, company, calm, purpose, and movement that community gardening engendered for organisers, and that they hoped to facilitate for gardening attendees, was rooted in both the sociomaterial characteristics of practice and place, and their own attuning life experiences. Organisers’ roles in sustaining these spaces and engaging the disinclined might also usefully be considered in research on the use of gardens in relation to health. Their accounts suggest that perceptions of gardens as unattractive, scary or exclusive (Kessel et al., 2009; Milligan and Bingley, 2007) are not necessarily insurmountable, and highlights the important work of experienced and empathetic organisers in making these spaces appealing, and adapting practices to differing needs. Those who wish to implement or support gardening-based health initiatives must consider how this work will be sustainably funded, the need for which is likely to grow alongside referral numbers and diversity of referee needs. This understanding of the perspectives of organisers raises wider questions around community gardening’s compatibility with sometimes-disenchanting and reductionist biomedical orientations to health and wellbeing and the consequences of a mismatch for gardens While community gardening can be a salutogenic practice, our study suggests that it cannot necessarily or straightforwardly be co-opted to 6 L. McGuire et al. Health and Place 75 (2022) 102773 meet public health imperatives. Improving access to community gardens may allow an increasing number of individuals to incorporate these potentially healthful, sociable, and meaningful activities within their routines. Yet, sustained and beneficial participation with such activities is contingent. Our findings support a complex definition of ‘accessi­ bility’, which includes accommodative gardening spaces and supportive social and economic structures both in and outside of the garden (of which organisers and organisations are a part). Thus, while community gardening can be a valuable tool towards many ends, its framing as a simple solution to complex problems undermines the richness that un­ dergirds its sustained practice. Currivan, D., 2008. Conversational interviewing. In: Lavrakas, P.J. (Ed.), Encyclopedia of Survey Research Methods, vol. 1. SAGE Publications, London, 152–152. i Day, S., Coombes, R.C., McGrath-Lone, L., Schoenborn, C., Ward, H., 2017. Stratified, precision or personalised medicine? The authors have no conflicts of interest to declare. Gesler, W., 1996. Lourdes: healing in a place of pilgrimage. Health Place 2 (2), 95–105. Ghebrehiwet, T., 2011. Nurses and person-centred care. Int. J. Pers. Cent. Med. 1 (1), References From therapeutic landscapes to healthy spaces, places and practices: a scoping review. Soc. Sci. Med. 196, 123–130. Kessel, A., Green, J., Wilkinson, P., Grundy, C., Lachowycz, K., 2009. Multidisciplinary research in public health: a case study on research on access to green space. Public Health 123 (1), 32–38. Bell, S.L., Leyshon, C., Foley, R., Kearns, R.A., 2019. The “healthy dose” of nature: a cautionary tale. Geogr. Compass 13 (e12415.). Liverpool, L., 2020. Why are ethnic minorities worse affected? New Sci. 246 (3279), 11. ll k h l d h h h l h d ll b Bennett, R., Savani, S., 2011. Surviving mission drift: how charities can turn dependence on government contract funding to their own advantage. Nonprofit Manag. Lead. 22 (2), 217–231. Lovell, R., Husk, K., Bethel, A., Garside, R., 2014. What are the health and well-being impacts of community gardening for adults and children: a mixed method systematic review protocol. Environ. Evid. 3 (1), 20. Bhatti, M., Church, A., Claremont, A., Stenner, P., 2009. ‘I love being in the garden’: enchanting encounters in everyday life. Soc. Cult. Geogr. 10 (1), 61–76. Marsh, P., 2020. Agroecology as public health: the island example of Tasmania. In: Egerer, M., Hamutahl, C. (Eds.), Urban Agroecology: Interdisciplinary Research and Future Directions. CRC Press, Boca Raton, FL, pp. 143–153. Blundell, R., Costa Dias, M., Joyce, R., Xu, X., 2020. COVID-19 and inequalities. Fisc. Stud. 41 (2), 291–319. Marsh, P., Brennan, S., Vandenberg, M., 2018. ‘It’s not therapy, it’s gardening’: community gardens as sites of comprehensive primary healthcare. Aust. J. Prim. Health 24, 337–342. i Braun, V., Clarke, V., 2006. Using thematic analysis in psychology. Qual. Res. Psychol. 3 (2), 77–101. Carpenter, M., 2013. From ‘healthful exercise’ to ‘nature on prescription’: the politics of urban green spaces and walking for health. Landscape Urban Plan. 118, 120–127. Ch D C 2002 F f lif lif l hi ki l id l d Carpenter, M., 2013. From ‘healthful exercise’ to ‘nature on prescription’: the politics of urban green spaces and walking for health. Landscape Urban Plan. 118, 120–127. Chaney, D.C., 2002. From ways of life to lifestyle: rethinking culture as ideology and sensibility. In: Lull, J. (Ed.), Culture in the Communication Age. Routledge, London, pp. 75–88. l k l d l d h f l h d McCabe, A., 2014. Community gardens to fight urban youth crime and stabilize neighborhoods. Int. J. Child Health Hum. Dev. 4. Conclusions: community gardening for health - promise and caution Cancer services in the ‘real world’ of a London hospital. Sociol. Health Ill. 39 (1), 143–158. de Oliveira, E.S., Aspinall, P., Briggs, A., Cummins, S., Leyland, A.H., Mitchell, R., Roe, J., Thompson, C.W., 2013. How effective is the Forestry Commission Scotland’s woodland improvement programme—‘Woods In and Around Towns’ (WIAT)—at improving psychological well-being in deprived urban communities? A quasi- experimental study. BMJ Open 3, e003648. DEFRA (Department for Environment, Food and Rural Affairs)., DHSC (Department for Health and Social Care)., Pow, R., 2020. New sites to test how connecting people with nature can improve mental health [press release]. 19 December. Available online at: https://www.gov.uk/government/news/new-sites-to-test-how-connect ing-people-with-nature-can-improve-mental-health. (Accessed 16 May 2021). ing-people-with-nature-can-improve-mental-health. (Accessed 16 May 2021). D C 2009 O t bl l d i l t d l i th th f Degnen, C., 2009. On vegetable love: gardening, plants, and people in the north of England. J. Roy. Anthropol. Inst. 15 (1), 151–167. l Funding or sources of support Dinnie, E., Brown, K.M., Morris, S., 2013. Community, cooperation and conflict: negotiating the social well-being benefits of urban greenspace experiences. Landscape Urban Plan. 112, 1–9. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Doughty, K., 2013. Walking together: the embodied and mobile production of a therapeutic landscape. Health Place 24, 140–146. i Ethical approval was gained from the Durham University Ethics Committee and all participant quotes are anonymised in this paper using participant codes and pseudonyms. Drury, C.A.A., Louis, M., 2002. Exploring the association between body weight, stigma of obesity, and health care avoidance. J. Am. Acad. Nurse Prac. 14 (12), 554–561. Earle, M.D., 2011. Cultivating Health: Community Gardening as a Public Health Intervention [MPH thesis]. University of Otago. Follman, A., Viehoff, V., 2015. A green garden on red clay: creating a new urban common as a form of political gardening in Cologne, Germany. Local Environ. 20 (10), 1148–1174. Acknowledgements Gesler, W., 1996. Lourdes: healing in a place of pilgrimage. Health Place 2 (2), 95–105. Ghebrehiwet, T., 2011. Nurses and person-centred care. Int. J. Pers. Cent. Med. 1 (1), 20–22. Ghebrehiwet, T., 2011. Nurses and person-centred care. Int. J. Pers. Cent. Med. 1 (1), 20–22. 20–22. The authors would like to thank those who agreed to participate in this project for their time and generosity, for which we are grateful. We thank Dr Roslyn Malcolm for her contributions and comments, given at multiple points in this project. LM is supported by an ESRC doctoral studentship through the NINE-Doctoral Training Programme (ES/ P000762/1). Glover, T.D., 2004. Social capital in the lived experiences of community gardeners. Leisure Sci. 26 (2), 143–162. Hale, J., Knapp, C., Bardwell, L., Buchenau, M., Marshall, J., Sancar, F., Litt, J.S., 2011. Connecting food environments and health through the relational nature of aesthetics: gaining insight through the community gardening experience. Soc. Sci. Med. 72 (11), 1853–1863. Haraway, D.J., 2016. Staying with the Trouble: Making Kin in the Chthulucene. Duke University Press, London. Hawkins, J.L., Smith, A., Backx, K., Clayton, D.A., 2015. Exercise intensities of gardening tasks within older adult allotment gardeners in wales. J. Aging Phys. Activ. 23 (2), 161–168. Declaration of competing interest Freire, P., 1972. Pedagogy of the Oppressed. Penguin, London. George, D.R., 2013. Harvesting the biopsychosocial benefits of community gardens. Am. J. Public Health 103 (8). The authors have no conflicts of interest to declare. References 7 (3), 1–14. Chaney, D.C., 2002. From ways of life to lifestyle: rethinking culture as ideology and sensibility. In: Lull, J. (Ed.), Culture in the Communication Age. Routledge, London, pp. 75–88. Meadows, A., Bombak, A.E., 2019. Yes, we can (No, you can’t): weight stigma, exercise self-efficacy, and active fat identity development. Fat Stud. 8 (2), 135–153. i i MHF, 2021. Nature: How Connecting with Nature Benefits Our Mental Health. London. Mental Health Foundation. Available online at: https://www.mentalhealth.org. k/ i / t / t h (A d 16 M 2021) Cloke, P., 2013. Rurality and racialised others: out of place in the countryside? In: Chakraborti, N., Garland, J. (Eds.), Rural Racism. Willan, Cullompton, pp. 17–35. Conradson, D., 2005. Landscape, care and the relational self: therapeutic encounters in l l d l h l 11 (4) 33 348 Cloke, P., 2013. Rurality and racialised others: out of place in the countryside? In: Chakraborti, N., Garland, J. (Eds.), Rural Racism. Willan, Cullompton, pp. 17–35. Chakraborti, N., Garland, J. (Eds.), Rural Racism. Willan, Cullompton, pp. 17–35. Conradson, D., 2005. Landscape, care and the relational self: therapeutic encounters in rural England. Health Place 11 (4), 337–348. p uk/campaigns/nature/nature-research. (Accessed 16 May 2021). p g ( y ) Milbourne, P., 2012. Everyday (in)justices and ordinary environmentalisms: community gardening in disadvantaged urban neighbourhoods. Local Environ. 17 (9), 943–957. Milligan, C., Bingley, A., 2007. Restorative places or scary spaces? The impact of woodland on the mental well-being of young adults. Health Place 13 (4), 799–811. Milbourne, P., 2012. Everyday (in)justices and ordinary environmentalisms: community gardening in disadvantaged urban neighbourhoods. Local Environ. 17 (9), 943–957. Milligan C Bingley A 2007 Restorative places or scary spaces? The impact of Milbourne, P., 2012. Everyday (in)justices and ordinary environmentalisms: community gardening in disadvantaged urban neighbourhoods. Local Environ. 17 (9), 943–957. Milligan, C., Bingley, A., 2007. Restorative places or scary spaces? The impact of woodland on the mental well-being of young adults. Health Place 13 (4), 799–811. Crossan, J., Cumbers, A., McMaster, R., Shaw, D., 2016. Contesting neoliberal urbanism in glasgow’s community gardens: the practice of DIY citizenship. Antipode 48 (4), 937–955. Milligan, C., Bingley, A., 2007. Restorative places or scary spaces? The impact of woodland on the mental well-being of young adults. Health Place 13 (4), 799–811. 7 L. McGuire et al. Health and Place 75 (2022) 102773 Nettle, C., 2010. 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https://openalex.org/W4210292232
http://ejurnal.undana.ac.id/index.php/CMJ/article/download/5985/3317
English
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Awareness And Knowledge Of Glaucoma Among Workers In A Primary Public Health Centre In Rote Ndao
Cendana Medical Journal
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cc-by
2,548
Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Awareness And Knowledge AWARENESS AND KNOWLEDGE OF GLAUCOMA AMONG WORKERS IN A PRIMARY PUBLIC HEALTH CENTRE IN ROTE NDAO Setya Ningrum Tefbana, Joy Aprianis Haning ABSTRACT Glaucoma was one of the biggest causes of blindness in the world. Early detection of glaucoma is important to reduce the morbidity of visual impairments and blindness. Primary Public Health Centre is expected to be able to educate people about visual impairment and blindness due to glaucoma. This study aims to evaluate the level of awareness and knowledge about glaucoma in the staff of UPTD Puskesmas Delha as a way to increase the public awareness campaigns of glaucoma. Methods An observational study took place in the primary public health centre, UPTD Puskesmas Delha in West Rote District, Rote Ndao Regency, East Nusa Tenggara Province. The population was all staff, divided into two groups that were health workers and non-health workers. The subjects filled a questionnaire about the awareness and knowledge of glaucoma that was consisting of 12 questions. Data collected were analyzed by SPSS software version 21 using the Independent T-Test method with a significance level of 5%. Result A total of 51 staff participated in this study consisting of 7 males (13.7%) and 44 females (86.3%), age distribution between 22-54 years old. A total of 44 health workers (86.3%) and 7 non-health workers (13.7%), 84.3% aware of glaucoma, but only 62.7% has good knowledge. The result of Independent-T test P-value=0.084 (p >0.05). Conclusion there is no significant difference in awareness and knowledge of glaucoma between health workers and non-health workers. Continuing education is needed to improve the awareness and knowledge of glaucoma. Keywords: Awareness, Knowledge, Glaucoma, Primary Public Health Centre. eywords: Awareness, Knowledge, Glaucoma, Primary Public Health Centre. In 2015, there was estimated 253 million people (3.38%) suffer from visual impairments from 7.33 trillion people in the world, 36 million people are blind and 217 million have moderate to severe visual impairments.1 A total of 2.78% of visual impairments in the world were caused by glaucoma. In the case of blindness, glaucoma was one of the biggest causes (8.30%), after cataracts (34.47%) and refraction disorders (20.26%) in the world.1,2 Basic Health Research 2007 showed glaucoma prevalence in Indonesia was 0.45% that means 4 to 5 people from 1000 Indonesians suffer from glaucoma.2 not realized by the patients or likely to other disease symptoms. Most of the people with glaucoma are diagnosed after being in the last stadium or totally blind.2 The main mechanism of vision loss in glaucoma is the apoptosis of retina ganglion cells, which cause the thinning of retinal nerve fiber of the nuclear layer and axonal loss on nerve fiber.4 The damage is irreversible or can not recover. Therefore, glaucoma is one of the priority diseases in the management of visual impairment in Indonesia today. To reduce the morbidity of visual impairments and blindness due to glaucoma, it is very important to detect glaucoma as soon as possible.5 The early detection can be done every 2 to 4 years on the age group of under 40 years old, every 2 years on the age over 40 years old, and every year on the group with a family history of glaucoma.2 Glaucoma is the damage of the optic nerve that is characterized by the increase of eye pressure, optic nerve papillary atrophy, the narrowing of the visual field, which even leads to loss of visual function.2,3,4 Glaucoma symptoms are often 315 Universitas Nusa Cendana Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Awareness And Knowledge However, to increase the awareness to do the early detection, one needs proper knowledge about glaucoma. Several studies showed low awareness and knowledge of glaucoma. While a study in Nigerian tertiary health care showed an insignificant result of awereness in medical and nonmedical workers.6 diagnosed or treated with glaucoma were excluded from this research. All staff was divided into two groups that were health workers and non-health workers. The subjects of this study fill the questionnaire about the subject’s identity and the awareness and knowledge of glaucoma. RESULT A total of 56 staff of UPTD Puskesmas Delha were invited for this survey, 51 participants were appropriate with the inclusion and exclusion criteria, 3 staff have not worked more than 6 months, 2 staff were changed workplace, while no one staff has been diagnosed or treated of glaucoma. eywords: Awareness, Knowledge, Glaucoma, Primary Public Health Centre. The awareness is known by a question of “Have you ever heard about glaucoma?” The knowledge of glaucoma was known by the score of questionnaire answers. The subjects firstly filled the informed consent form and were willing to be the subjects. The questionnaire was consisting of 12 questions that were adapted from several previous studies and have been validated. Every right answer gets 1 points and the false answer gets 0 points. Primary Public Health Centre is one of the health centre facilities that organize environment health and individual health, emphasis on promotive and preventive strategies. This is expected to be able to become a place and or a facility to educate people about visual impairment and blindness due to glaucoma.5 A good education will be delivered if the health workers have good awareness and knowledge about glaucoma. With the background of health education, every worker is expected to have good awareness and knowledge. The collected data were analyzed by SPSS software version 21 using the Independent T-Test method with a significance level of 5%. The analysis was aimed to find out the difference of awareness and knowledge about glaucoma between health workers and non-health workers in UPTD Puskesmas Delha. This study aims to evaluate the level of awareness and knowledge about glaucoma in the staff of UPTD Puskesmas Delha. It is assumed that the staff with medical background has a higher awareness and knowledge of glaucoma than other non-medical workers. This is expected as one of the ways to increase the public awareness campaigns of glaucoma. METHODS This observational study took place in a Primary Public Health Centre, UPTD Puskesmas Delha in West Rote District, Rote Ndao Regency, East Nusa Tenggara Province. The population in this study were all 56 staff of UPTD Puskesmas Delha consisting of medical, paramedical, and administrative staff, and other supporting staff. The sampling method was a total sample. Inclusion criteria were the staff who has work more than 6 months, the staff were not on a long vacation or were changed workplace when the questionnaire collected, and willing to be subject of study. While the staff who has been Universitas Nusa Cendana 316 Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Awareness And Knowledge Table 1. Frequency Distribution based on Gender, Age, Profession, and Education Level of Respondents. Total (n=53) Percentage 100% Gender Female Male 44 7 86.3 13.7 Age 20-29 years old 30-39 years old 40-49 years old 50-59 years old 22 20 7 2 43.1 39.2 13.7 3.9 Profession Doctor Dentist Nurse Midwives Laboratory Staff Nutritionist Pharmacy Sanitarian 2 1 13 19 1 4 1 3 4 2 25 37 2 2 2 6 Administrative Staff Other Staff 3 4 6 2 Education Level High School Diploma 3 Diploma 4 Bachelor Profession 4 35 1 8 3 7.8 68.6 2.0 15.7 5.9 There were 7 males (13.7%) and 44 females (86.3%). The questionnaire was responded by 2 doctors, 1 dentist, 13 nurses, 19 midwives, 1 laboratory staff, 4 nutritionists, 1 pharmacy, 3 sanitarians, 3 administrative staff, and 4 other staff. The mean age of the subjects was 32.88 years with an age distribution between 22-54 years old. Table 1 The subjects were divided into two groups of 44 health workers and 7 non- health workers. Total of 84.3% ever heard about glaucoma but only 62.7% has good knowledge of glaucoma or can answer more than 60% of question. Table 2 Table 2. The awareness of glaucoma among workers Have the Respondent ever heard about glaucoma? Ever Never Total N % N % Health workers 39 88.6 5 11.4 44 Non-health workers 4 57.1 3 42.9 7 Total 43 84.3 8 15.7 51 Table 2. METHODS The awareness of glaucoma among workers 317 Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Awareness And Knowledge The most unknown question from the 12 questions of the questionnaire was “glaucoma can runs in families.” 35 respondents answer false (68.6%), “glaucoma is irreversible or can not be treated. The treatment just avoids worseness.” 28 respondents answer false (54.9%), and “steroid cause glaucoma” 27 respondents answer false (52.9%). Table 3 Response of staff to questions on Knowledge and Awareness of glaucoma Table 3. The Response of staff to questions on Knowledge and Awareness of glaucoma Question True False N % n % Glaucoma increase eye pressure, affects vision, clog eye fluid 44 86.3 7 13.7 Did you know that glaucoma can run in families 16 31.4 35 68.6 Does glaucoma only occur in elderly? 38 74.5 13 25.5 Can glaucoma affect babies? 26 51.0 25 49.0 Does glaucoma cause blindness 44 86.3 7 13.7 Did you know that diabetic patients are more prone to glaucoma? 32 62.7 19 37.3 Glaucoma is irreversible (can not be recovered). Treatment just prevents from getting worse 23 45.1 28 54.9 Did you know that glaucoma can be operated on? 31 60.8 20 39.2 Did you know that laser treatment can be used to treat glaucoma? 33 64.7 1 35.3 Is it necessary to do regular follow-up in treating glaucoma? 45 88.2 6 11.8 Did you know that the steroid use can cause glaucoma? 24 47.1 27 52.9 Universitas Nusa Cendana 318 Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Awareness And Knowledge Table 4. The difference of Awareness and Knowledge about Glaucoma between Health Workers and Non-Health Workers Table 4. The difference of Awareness and Knowledge about Glaucoma between Health Workers and Non-Health Workers ≥ 60% < 60% Total P-Value on T-Test N % N % Health workers 29 65.9 15 34.1 44 0.084 Non-health workers 3 42.9 4 57.1 7 Total 32 62.7 19 37.3 51 Table 4. The difference of Awareness and Knowledge about Glaucoma between Health Workers and Non-Health Workers detection to prevent the blindness. While education will increase the awareness itsself.7 There is no significant difference between health workers and non-health workers about the knowledge of glaucoma P-value= 0.084 (p>0.05). METHODS Table 4 There were 68.6% respondent did not know that glaucoma can run in families, 54.9% did not know that glaucoma is irreversible or can not be treated, and 52.9% did not know that steroid cause glaucoma. This despite to the study in tertiary centre in Karnataka, the knowledge of diabetes and family history of glaucoma as risk factors for glaucoma was high (84.4%). There were 60.60% know that glaucoma is an irreparable damage of vision. But the knowledge of corticosteroid as a risk factor was the same low (28.65%).7 Universitas Nusa Cendana DISCUSSION This study was held to assess the awareness and knowledge about glaucoma among the workers in a Primary Health Centre. The focus of this study was to evaluate the understanding about the glaucoma and the importance of screening as a way to prevent an irreversible loss of vision. The level of awareness of glaucoma among the respondents was 84.3%, both health workers (88.6%) and non-health workers (57.1%) had ever heard about glaucoma. This was different with the study of Comolafe O.O et al, Only 29.2% participants from the administrative division had heard of glaucoma. The low awareness can occur of poor access to information and lack of education media.6 The result of T-test showed p- value 0.084 > 0.05. That means that there was no significant difference of awareness and knowledge of glaucoma between health workers and non-health workers in UPTD Puskesmas Delha. The insignificance level in this study can occur because of the number of non-health workers were significant less than the health workers. In other hand, the minimal education of this population was high school. People with at least primary education found better aware of glaucoma, where better education positive related to awareness of glaucoma.7 A study of Alemu D.S et al in population on Northwest Ethiopia, there was 48.5% respondents had heard of glaucoma. It was 246 respondents were aware of glaucoma, 122 (49.6%) respondents had good knowledge and 124 (50.4%) respondents had poor knowledge.8 The result was despite with this study were 62.7% had knowledge more than 60% and 37.3% had knowledge less than 60%. Awareness of glaucoma can lead to early Being a health care, this study firstly showed the picture of health care workers awareness and knowledge of glaucoma in a primary public health centre in Rote Ndao. Universitas Nusa Cendana 319 Awareness And Knowledge Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Indonesia. Jakarta: Kementerian Kesehatan RI; 2019. Additionally, this study used a total sample to ensure the real picture of the population. While the study had limitation, even though the questionnaire has been adapted and validated, it just considering a close answers which is possible to misunderstanding of the respondents. 3. Ilyas S. Ilmu Penyakit Mata. Edisi Ke-3. Jakarta: Badan Penerbit FKUI; 2008.p.167-172,212-216. 4. Riordan-Eva P, Whitcher JP. Vaughan & Asbury’s General Ophthalmology. 17th ed. New York: The Mc Graw Hill; 2007;11. DISCUSSION As the public health centre, it is important to aware of the conditions which cause irreversible damage. Therefore, regular programs could be conducted for workers to increase the knowledge of glaucoma and the early detection. As the first point of contact for the patient, health education is a key in reducing morbidity to chronic diseases mainly the asymptomatic at an early stage. Hence, the health centre personnel should effectively educate patients regarding glaucoma requires a good understanding of the disease process.6 5. Kementerian Kesehatan RI. Direktorat Jenderal Pencegahan dan Pengendalian Penyakit Kementerian Kesehatan RI: Peta Jalan Penanggulangan Gangguan Penglihatan di Indonesia Tahun 2017-2030. Jakarta: Kementerian Kesehatan RI; 2018. CONCLUSION 6. Komolafe OO, Omolase CO, Bekibele CO, et al. Awareness and Knowledge of Glaucoma among Workers in a Nigerian Tertiary Health Care Institution. Middle East African Journal of Ophthalmologi 2013;20:167-163. 6. Based on this study, there is no significant difference between health workers and non-health workers about the awareness and knowledge of glaucoma in UPTD Puskesmas Delha. Even though the awareness about glaucoma is good but the knowledge of glaucoma is still limited. Continuing education is needed to improve the knowledge of all the staff especially health workers in the way to educate the public or patients in campaigns of glaucoma. 7. Padmajothi M.S, Chaitra M.C, Anneshi R.C. Awareness and Knowledge of Glaucoma Among Hospital Personnel in A Tertiary Care Center in Rural Karnataka. Tropical Journal of Ophthalmology and Otolaryngology 2019;4:130-126. Universitas Nusa Cendana REFERENCES 1. Kementerian Kesehatan RI. Pusat Data dan Informasi Kementerian Kesehatan RI: Situasi Gangguan Penglihatan. Jakarta: Kementerian Kesehatan RI; 2018. 1. Kementerian Kesehatan RI. Pusat Data dan Informasi Kementerian Kesehatan RI: Situasi Gangguan Penglihatan. Jakarta: Kementerian Kesehatan RI; 2018. 8. Alemu D.S, Gudeta A.D, Gebreselassie K.L. Awareness and Knowledge of Glaucoma and Associated Factors Among Adults: A Cross Sectional Study in Gondar Town, Northwest Ethiopia 2017;17:12-1. 8. 2. Kementerian Kesehatan RI. Pusat Data dan Informasi Kementerian Kesehatan RI: Situasi Glaukoma di Universitas Nusa Cendana 320
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Prediction Scores for Acute Kidney Injury following Adult Cardiac Surgery
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Prediction Scores for Acute Kidney Injury follow Adult Cardiac Surgery Yu Tian  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Wei Zhao  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Yuefu Wang  (  wangyuefu@hotmail.com ) Chinese Academy of Medical Sciences and Peking Union Medical College Fuwai Hospital https://orcid.org/0000-0003-4776-9266 Chunrong Wang  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Xiaolin Diao  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Weiwei Wang  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Yuchen Gao  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Sudena Wang  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Research Keywords: adult cardiac surgery, acute kidney injury, risk prediction score systems Posted Date: October 9th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-88293/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International Li Read Full License Prediction Scores for Acute Kidney Injury follow Adult Cardiac Surgery Yu Tian  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Wei Zhao  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Yuefu Wang  (  wangyuefu@hotmail.com ) Chinese Academy of Medical Sciences and Peking Union Medical College Fuwai Hospital https://orcid.org/0000-0003-4776-9266 Chunrong Wang  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Xiaolin Diao  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Weiwei Wang  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Yuchen Gao  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Sudena Wang  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Research Keywords: adult cardiac surgery, acute kidney injury, risk prediction score systems Posted Date: October 9th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-88293/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International Li Read Full License Prediction Scores for Acute Kidney Injury follo Adult Cardiac Surgery Yu Tian  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Wei Zhao  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Yuefu Wang  (  wangyuefu@hotmail.com ) Chinese Academy of Medical Sciences and Peking Union Medical College Fuwai Hospital https://orcid.org/0000-0003-4776-9266 Chunrong Wang  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Xiaolin Diao  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Weiwei Wang  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Yuchen Gao  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Sudena Wang  Chinese Academy of Medical Sciences & Peking Union Medical College Fuwai Hospital Research License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Page 1/23 Abstract Background In the development of scoring systems for acute kidney injury (AKI) following cardiac surgery, previous investigations have primarily and solely attached importance to preoperative associated risk factors without any consideration for surgery-derived physiopathology. We sought to internally derive and then validate risk score systems using pre- and intraoperative variables to predict the occurrence of any- stage (stage 1-3) and stage-3 AKI within 7 days. Methods Patients undergoing cardiac surgery from Jan 1, 2012, to Jan 1, 2019, were enrolled in our retrospective study. The clinical data were divided into a derivation cohort (n= 43799) and a validation cohort (n= 14600). Multivariable logistic regression analysis was used to develop the prediction models. Results The overall prevalence of any-stage and stage-3 AKI after cardiac surgery was 34.3% and 1.7%, respectively. Any-stage AKI prediction-model discrimination measured by the area under the curve (AUC) was acceptable (AUC = 0.69, 95% CI: 0.68, 0.69), and the prediction model calibration measured by the Hosmer-Lemshow test was good (P = 0.95). The stage-3 AKI prediction model had an AUC of 0.84 (95% CI 0.83, 0.85) and good calibration according to the Hosmer-Lemshow test (P = 0.73). Conclusions Using pre- and intraoperative data, we developed two scoring systems for any-stage AKI and stage-3 AKI in a cardiac surgery population. These scoring systems can potentially be adopted clinically in the field of AKI recognition and therapeutic intervention. Background Acute kidney injury (AKI) following cardiac surgery is a common and serious complication[1, 2]. It can evolve into a variety of chronic kidney diseases, prolong the length of intensive care unit (ICU) and hospital stay, and carry a high risk of increased medical expenditure and even mortality[3]. The incidence of postoperative any-stage AKI (stage 1, 2, 3) is 25% -35%[4, 5]. Renal replacement therapies (RRTs) following cardiac surgery secondary to AKI are required for approximately 1% of adult patients but are associated with in-hospital mortality rates of 50–80%[6, 7]. It has been postulated that any-stage AKI, even mild, reversible AKI, has important clinical indications, including increased short- and long-term poor outcomes after cardiac surgery[8]. Currently, efforts to alleviate AKI and its associated poor outcomes are primarily focused on the early detection of compromised kidney function, as effective practical clinical measures to prevent or treat AKI remain controversial. Prediction models or scoring systems for AKI in the field of cardiac surgery have proliferated in recent years. Additionally, clinicians and policy-makers have increasingly recommended the use of prediction models in clinical practice guidelines to make informed decisions at various AKI stages[9]. Although several scoring systems for AKI risk prediction after cardiac surgery have been published[10– 17], they carry several limitations. First, the definitions of AKI in previous studies are different. In 2012, the definition and diagnostic criteria of AKI were significantly revised by the Kidney Disease: Improving Global Outcomes (KDIGO) organization, which made scoring systems developed before 2012 imprecise, Page 2/23 Page 2/23 outdated and even likely to be eliminated[18]. Second, in previous models, dialysis induced by AKI was the most widely used endpoint. However, we cannot ignore the impact of milder forms of AKI; hence, risk models for any-stage AKI are also needed. Third, only two published scores (the Cleveland clinic score and the Mehta score) have undergone independent external validation by investigators. However, neither has demonstrated adequate discrimination and calibration in non-North American patient outdated and even likely to be eliminated[18]. Second, in previous models, dialysis induced by AKI was the most widely used endpoint. However, we cannot ignore the impact of milder forms of AKI; hence, risk models for any-stage AKI are also needed. Third, only two published scores (the Cleveland clinic score and the Mehta score) have undergone independent external validation by investigators. However, neither has demonstrated adequate discrimination and calibration in non-North American patient populations[19–22]. Background Fourth, the sample sizes of previous prediction models were relatively limited. Finally, to date, innovative AKI predictors have been widely explored, but some indicators such as hyperuricaemia have not been incorporated into these risk score systems. populations[19–22]. Fourth, the sample sizes of previous prediction models were relatively limited. Finally, to date, innovative AKI predictors have been widely explored, but some indicators such as hyperuricaemia have not been incorporated into these risk score systems. In 2013, the International Society of Nephrology launched a worldwide target of “0 by 25”—no patient deaths due to untreated acute kidney failure by 2025—to improve the diagnosis and treatment of AKI globally[23]. An important step to achieve this target is to establish an ideal risk prediction model for AKI after cardiac surgery. We sought to derive and validate scoring systems that can predict the risk of any- stage and stage-3 AKI following cardiac surgery based on the KDIGO criteria. In 2013, the International Society of Nephrology launched a worldwide target of “0 by 25”—no patient deaths due to untreated acute kidney failure by 2025—to improve the diagnosis and treatment of AKI globally[23]. An important step to achieve this target is to establish an ideal risk prediction model for AKI after cardiac surgery. We sought to derive and validate scoring systems that can predict the risk of any- stage and stage-3 AKI following cardiac surgery based on the KDIGO criteria. Material And Methods The research was approved by the Ethics Committee of Fuwai Hospital (20191308), and written informed consent from participants was waived for this retrospective analysis. Study Design A total of 58469 adult patients underwent cardiac repair at our institute from Jan 1, 2012, to Jan 1, 2019. Digital clinical data were provided by Fuwai Hospital Information Center. We monitored and checked the collected perioperative data. All data were checked twice by postgraduates and engineers who work for the Hospital Information Center. Thus, quality and accuracy were guaranteed. Patients were included if they were aged more than 18 years and had undergone open cardiac surgery (including coronary artery bypass grafting (CABG), valve surgery, great vessel operations, congenital heart disease repair, cardiac tumour surgery and combined surgery). Patients who received RRT prior to surgery were excluded (n = 70). Finally, 58399 patient charts were enrolled retrospectively. Objectives The primary purpose of this study is to derive and then internally validate scoring systems that can predict the risk of any-stage and stage-3 AKICS based on KDIGO criteria. The secondary purpose is to further elucidate the association between AKI occurrence and postoperative clinical outcomes. Variable Definitions A list of 27 candidate variables was selected based on our clinical experience and other evidence obtained by previous investigations. The definitions of risk factors were mainly referenced from the STS website (www.sts.org). The rationale for using these variables in the scoring model was based on the results of univariable analysis and clinical relevance. Statistical Analysis Descriptive statistics are expressed as frequencies (n), percentages (%) or medians, and interquartile ranges (IQRs) as appropriate. Categorical variables were analysed using Pearson’s chi-square test or Fisher’s exact test. We transformed some continuous variables into categorical variables according to clinically meaningful cut-off values or previous literature reports. A single imputation was used for variables missing less than 2% of their values (defaulted to the most common value). Missing data for the history of peripheral vascular disease (accounting for 2.2% of the data) were modelled as unknown categories. Statistical significance was considered for P values less than 0.05. The data set was randomly divided into a derivation and a validation cohort. Any-stage AKI and stage-3 AKI predictive systems were formulated after using multivariable logistic regression analysis. The final models were assessed by the Hosmer-Lemeshow goodness-of-fit test for calibration and the area under the receiver operating characteristic (ROC) curve (AUC) for discrimination. Next, the predictive utility was assessed by calculating the AUC using the validation cohort data. The final scores, obtained by a linear transformation of the regression coefficients, were calculated by dividing the regression coefficient of each risk factor by the absolute value of the sex coefficient. Statistical analysis was performed with SPSS software, version 25.0. Patient Outcomes The diagnosis of AKI was based on the KDIGO criteria, listed as follows: Stage 1: an increase in creatinine from baseline of ≥ 26.5 µmol/L (0.3 mg/dl) within 48 hours or to 1.5 to 1.9 times the baseline level within 7 days; Page 3/23 Stage 2: an increase in creatinine to 2.0 to 2.9 times the baseline level Page 3/23 Stage 2: an increase in creatinine to 2.0 to 2.9 times the baseline level Stage 3: an increase in creatinine to ≥ 3 times the baseline level or to ≥ 354 µmol/L or when the patient required RRT. Stage 3: an increase in creatinine to ≥ 3 times the baseline level or to ≥ 354 µmol/L or when the patient required RRT. The baseline creatinine level was defined as the latest obtained prior to surgery. Urine output speed, although a reliable indicator of AKI as defined by the KDIGO criteria, was not included to evaluate AKI onset due to its unavailability at our institute for the majority of patients. Characteristics of Patients A total of 58399 patients (43799 in the derivation cohort and 14600 in the validation cohort) were retrospectively analysed during the 7-year period. The most common procedure was CABG (44.6%), followed by valve surgery (22.5%), combined surgeries (18.3%), great vessel surgery (7.8%), congenital heart repair and cardiac tumour surgery (6.6%). The prevalence of any-stage AKI and stage-3 AKI in the whole cohort after cardiac surgery was 34.3% and 1.7%, respectively. Baseline clinical characteristics of Page 4/23 Page 4/23 the patients for both the derivation cohort and the validation cohort are illustrated in additional file 1 in the Supplement. the patients for both the derivation cohort and the validation cohort are illustrated in additional file 1 in the Supplement. Pre- and intraoperative information for patients in the derivation group is shown in Table 1. Patients with any-stage or stage-3 AKI were older and had a greater likelihood of previous cardiac surgery, intra-aortic balloon pump (IABP) use, atrial fibrillation, urine protein, New York Heart Association (NYHA) classification ≥ 4, blood transfusion product use,hyperuricaemia, and left ventricular ejection fraction (LVEF) damage before surgery. Emergency surgery, cardiopulmonary bypass, haemorrhage volume and greater amounts of red blood cell (RBC), platelet, or plasma transfusion during surgery were also significant characteristics of the AKI cohorts. Characteristics of Patients AKI, acute kidney injury; BMI, body mass index; COPD, chronic obstructive pulmonary disease; CABG, coronary artery bypass grafting; eGFR, estimated glomerular filtration rate, calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula; IABP, intra-aortic balloon pump; LVEF, left ventricular ejection fraction; NYHA, New York Heart Association ; RBC, red blood cell.*included congenital heart disease repair, cardiac tumor surgery. Data presented as numbers and percentages. AKI, acute kidney injury; BMI, body mass index; COPD, chronic obstructive pulmonary disease; CABG, coronary artery bypass grafting; eGFR, estimated glomerular filtration rate, calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula; IABP, intra-aortic balloon pump; LVEF, left ventricular ejection fraction; NYHA, New York Heart Association ; RBC, red blood cell.*included congenital heart disease repair, cardiac tumor surgery. Characteristics of Patients Page 5/23 Page 6/23 Table 1 Clinical Characteristics of Derivation Cohort(N = 43799)(%) Preoperative variable No any-stage AKI (n =  28659,65.4%) Any-stage AKI(n =  15140,34.6%) P Value No stage-3 AKI (n =  43046,98.3%) Stage-3 AKI (n =  753,1.7%) P Value Age(year)     <  0.001     0.080 < 60 16685(58.2) 7800(51.5)   24075(55.9) 410(54.4)   60–74 10763(37.6) 6554(43.3)   17021(39.5) 296(39.3)   ≥ 75 1211(4.2) 786(5.2)   1950(4.5) 47(6.2)   Gender     0.003     <  0.001 Female 9943(34.7) 5038(33.3)   14673(34.1) 308(40.9)   Male 18716(65.3) 10102(66.7)   28373(65.9) 445(59.1)   Obesity(BMI ≥  30 kg/m²) 1867(6.5) 1674(11.1) <  0.001 3483(8.1) 58(7.7) 0.698 Previous COPD 402(1.4) 264(1.7) 0.006 654(1.5) 12(1.6) 0.765 History of cardiac surgery 542(1.9) 507(3.3) <  0.001 1002(2.3) 47(4.5) <  0.001 History of peripheral vascular disease   0.003     0.258 Yes 3182(11.1) 1841(12.2)   4929(11.5) 94(12.5)   Unknown 626(2.2) 342(2.3)   946(2.2) 22(2.9)   IABP use 48(0.2) 49(0.3) 0.001 92(0.2) 5(0.7) 0.026 Infective endocarditis 235(0.8) 109(0.7) 0.259 338(0.8) 6(0.8) 0.971 Atrial fibrillation 3389(11.8) 3031(20.0) <  0.001 6259(14.5) 161(21.4) <  0.001 Urine protein 274(1.0) 371(2.5) <  0.001 611(1.4) 34(4.5) <  0.001 Hypertension 10193(35.6) 5409(35.7) 0.739 15335(35.6) 267(35.5) 0.925 Type 2 diabetes 5672(19.8) 3052(20.2) 0.360 8588(20.0) 136(18.1) 0.198 NYHA classification ≥ 4 648(2.3) 553(3.7) <  0.001 1150(2.7) 51(6.8) <  0.001 Page 6/23 Page 6/23 Preoperative variable No any-stage AKI (n =  28659,65.4%) Any-stage AKI(n =  15140,34.6%) P Value No stage-3 AKI (n =  43046,98.3%) Stage-3 AKI (n =  753,1.7%) P Value Blood transfusion products 26(0.1) 47(0.3) <  0.001 67(0.2) 6(0.8) 0.002 hyperuricemia 6297(22.0) 4513(29.8) <  0.001 10564(24.5) 246(32.7) <  0.001 dyslipidemia 21406(74.7) 11184(73.9) 0.061 32083(74.5) 507(67.3) <  0.001 LVEF(> 60%,reference group)   0.001     0.006 Mild damage (46%-60%) 451(1.6) 267(1.8)   699(1.6) 19(2.5)   Moderate damage (30%-45%) 112(0.4) 86(0.6)   192(0.4) 6(0.8)   Severe damage(<  30%) 9(0.0) 14(0.1)   21(0.0) 2(0.3)   Serum creatinine (umol/L)   0.062     0.346 < 70 9077(31.7) 4844(32.0)   13680(31.8) 241(32.0)   70–100 16543(57.7) 8560(56.6)   24666(57.3) 428(56.8)   101–120 2015(7.0) 1149(7.6)   3106(7.2) 58(7.7)   121–150 601(2.1) 346(2.3)   937(2.2) 10(1.3)   > 150 432(1.5) 241(1.6)   657(1.5) 16(2.1)   eGFR (mL/min/1.73 m2)     0.400     0.386 ≥ 90 13598(47.4) 7133(47.1)   20377(47.3) 354(47.0)   60-89.9 11749(41.0) 6205(41.0)   17649(41.0) 305(40.5)   30-59.9 2873(10.0) 1586(10.5)   4382(10.2) 77(10.2)   < 30 439(1.5) 216(1.4)   638(1.5) 17(2.3)   Emergency surgery 1164(4.1) 714(4.7) 0.001 1819(4.2) 59(7.8) <  0.001 The type of surgery     <  0.001     <  0.001 Page 7/23 Preoperative variable No any-stage AKI (n =  28659,65.4%) Any-stage AKI(n =  15140,34.6%) P Value No stage-3 AKI (n =  43046,98.3%) Stage-3 AKI (n =  753,1.7%) P Value CABG 14190(49.5) 5633(37.2)   19696(45.8) 127(16.9)   Valve Surgery 6141(21.4) 4167(27.5)   10165(23.6) 143(19.0)   Great vessel surgery 1764(6.2) 1699(11.2)   3187(7.4) 276(36.7)   Combined surgery 4183(14.6) 2905(19.2)   6904(16.0) 184(24.4)   Bypass time (min)     <  0.001     <  0.001 Off-pump surgery 9425(32.9) 2541(16.8) <  0.001 11910(27.7) 56(7.4) <  0.001 ≤ 120 min 14619(51.0) 7478(49.4)   21872(50.8) 225(29.9)   > 120 min 4615(16.1) 5121(33.8)   9264(21.5) 472(62.7)   Hemorrhage volume     <  0.001     <  0.001 ≤ 600 mL 24732(86.3) 12506(82.6)   36762(85.4) 476(63.2)   ༞600 mL 3927(13.7) 2634(17.4)   6284(14.6) 277(36.8)   Intraoperative RBC use 4276(14.9) 3621(23.9) <  0.001 7555(17.6) 342(45.4) <  0.001 Intraoperative platelet 1013(3.5) 1487(9.8) <  0.001 2221(5.2) 279(37.1) <  0.001 Intraoperative plasma 2646(9.2) 2626(17.3) <  0.001 5002(11.6) 270(35.9) <  0.001 Data presented as numbers and percentages. Development of Risk Prediction Score Coefficients, point scores, odds ratios (ORs) and 95% confidence intervals (CIs) are presented in Table 2 (any-stage AKI scoring system) and Table 3 (stage-3 AKI scoring system). There were 20 variables included in the any-stage AKI prediction model and 17 in the stage-3 AKI model. Next, we developed point score systems for any-stage and stage-3 AKI after cardiac surgery. Page 8/23 Page 8/23 Table 2 Table 2 P 9/23 Preoperative and intraoperative risk variables in the prediction model for postoperative any-stage AKI after cardiac surgery. Preoperative Coefficient Scores OR 95%CI P value Age(<60 years, reference group)         < 0.001 60–74 0.434 3 1.544 (1.474, 1.617) < 0.001 ≥ 75 0.670 4 1.954 (1.765,2.164) < 0.001 Male 0.165 1 1.179 (1.123, 1.238) < 0.001 Obesity(BMI ≥ 30 kg/m²) 0.687 4 1.987 (1.846, 2.139) < 0.001 History of cardiac surgery 0.371 2 1.449 (1.291, 1.626) < 0.001 History of peripheral vascular disease         < 0.001 Yes 0.169 1 1.184 (1.109,1.263) < 0.001 Unknown 0.032 0 1.032 (0.896,1.189) 0.663 IABP use 0.519 3 1.680 (1.088,2.593) 0.019 Atrial fibrillation 0.348 2 1.417 (1.331, 1.508) < 0.001 Urine protein 0.832 5 2.297 (1.994,2.714) < 0.001 Hypertension 0.094 1 1.099 (1.048,1.152) < 0.001 Type 2 diabetes 0.061 0 1.062 (1.008,1.120) 0.024 NYHA classification ≥ 4 0.150 1 1.162 (1.026,1.315) 0.018 Blood transfusion products use 0.663 4 1.940 (1.158,3.251) 0.012 hyperuricemia 0.229 1 1.257 (1.197,1.320) < 0.001 LVEF(> 60%,reference group)         0.029 Mild damage(46%-60%) 0.062 0 1.064 (0.905,1.252) 0.453 Moderate damage(30–45%) 0.321 2 1.379 (1.023,1.858) 0.035 Severe damage(< 30%) 0.913 6 2.491 (1.029,6.034) 0.043 The type of surgery(Others* ,reference group)       < 0.001 CABG 0.183 1 1.201 (1.088,1.326) < 0.001 Valve Surgery 0.354 2 1.424 (1.292,1.570) < 0.001 Great vessel surgery 0.693 4 2.000 (1.775,2.255) < 0.001 Combined surgery 0.505 3 1.656 (1.497,1.832) < 0.001 Page 9/23 Page 9/23 Preoperative Coefficient Scores OR 95%CI P value Intraoperative   Bypass time (Off-pump surgery, reference group)     < 0.001 ≤ 120 min 0.676 4 1.967 (1.853, 2.087) < 0.001 > 120 min 1.269 8 3.557 (3.322, 3.809) < 0.001 Hemorrhage volume༞600 ml 0.064 0 1.067 (1.005,1.132) 0.003 Intraoperative RBC use 0.186 1 1.204 (1.132, 1.281) < 0.001 Intraoperative platelet use 0.293 2 1.340 (1.207, 1.487) < 0.001 Intraoperative plasma use 0.355 2 1.426 (1.329, 1.529) < 0.001 Intercept -2.301       < 0.001 Minimum score = 0; maximum score = 52. Development of Risk Prediction Score Minimum score = -2; maximum score = 30. AKI, acute kidney injury; CABG, coronary artery bypass grafting; CI, confidence interval; LVEF, left ventricular ejection fraction; OR, odds ratio; RBC, red blood cell.*included congenital heart disease repair, cardiac tumor surgery. Risk Variables Associated with Postoperative AKI The details for the any-stage AKI and stage-3 AKI prediction models are shown in Table 2 and Table 3, respectively. The risk variables that contributed to any-stage AKI were age, male sex, obesity, history of cardiac surgery, history of peripheral vascular disease, IABP use, atrial fibrillation, urine protein, hypertension, type 2 diabetes, NYHA classification, blood transfusion product use, hyperuricaemia, LVEF damage, cardiopulmonary bypass, haemorrhage volume and greater amounts of RBC, platelet, or plasma transfusion during surgery. Among the variables mentioned above, obesity, hypertension, type 2 diabetes, and blood transfusion products used could not be included in the stage-3 AKI model. The remaining variables as well as dyslipidaemia were incorporated into the stage-3 AKI model. However, female rather than male sex was included as the risk factor for stage-3 AKI onset. Development of Risk Prediction Score AKI, acute kidney injury; BMI, body mass index; CABG, coronary artery bypass grafting; CI, confidence interval; LVEF, left ventricular ejection fraction; OR, odds ratio; RBC, red blood cell.*included congenital heart disease repair, cardiac tumor surgery. Minimum score = 0; maximum score = 52. AKI, acute kidney injury; BMI, body mass index; CABG, coronary artery bypass grafting; CI, confidence interval; LVEF, left ventricular ejection fraction; OR, odds ratio; RBC, red blood cell.*included congenital heart disease repair, cardiac tumor surgery. Page 10/23 Table 3 h d Table 3 Preoperative and intraoperative risk variables in the prediction model for postoperative stage-3 AKI after cardiac surgery. Preoperative Coefficient Scores OR 95%CI P value Age(<60 years, reference group)         < 0.001 60–74 0.318 1 1.374 (1.165, 1.621) < 0.001 ≥ 75 0.871 2 2.389 (1.713, 3.331) < 0.001 Male -0.367 -1 0.693 (0.585, 0.821) < 0.001 History of cardiac surgery 0.372 1 1.451 (1.117, 1.886) 0.005 History of peripheral vascular disease         0.003 Yes 0.377 1 1.458 (1.158,1.837) 0.001 Unknown 0.332 1 1.393 (0.881,2.204) 0.156 IABP use 0.977 3 2.655 (1.002,7.038) 0.050 Atrial fibrillation 0.257 1 1.293 (1.050,1.592) 0.016 Urine protein 0.844 2 2.326 (1.578, 3.427) < 0.001 NYHA classification ≥ 4 0.361 1 1.434 (1.043, 1.972) 0.026 hyperuricemia 0.224 1 1.251 (1.057,1.482) 0.009 dyslipidemia -0.232 -1 0.793 (0.674, 0.933) 0.005 LVEF(> 60%,reference group)         0.025 Mild damage(46%-60%) 0.342 1 1.408 (0.863, 2.298) 0.171 Moderate damage(30–45%) 0.472 1 1.604 (0.679, 3.790) 0.282 Severe damage(< 30%) 1.942 5 6.973 (1.560,31.166) 0.011 The type of surgery(Others* ,reference group)       < 0.001 CABG -0.040 0 0.961 (0.600,1.537) 0.867 Valve Surgery 0.061 0 1.062 (0.671,1.682) 0.796 Great vessel surgery 1.501 4 4.487 (2.840,7.088) < 0.001 Combined surgery 0.778 2 2.177 (1.389,3.411) 0.001 Intraoperative   Bypass time (Off-pump surgery, reference group)       < 0.001 ≤ 120 min 0.485 1 1.625 (1.187, 2.224) 0.002 Page 11/23 Preoperative Coefficient Scores OR 95%CI P value > 120 min 1.625 4 5.079 (3.726, 6.922) < 0.001 Hemorrhage volume > 600 ml 0.540 1 1.716 (1.451, 2.028) < 0.001 Intraoperative RBC use 0.355 1 1.427 (0.170, 1.740) < 0.001 Intraoperative platelet use 0.776 2 2.173 (1.725, 2.736) < 0.001 Intraoperative plasma use 0.308 1 1.361 (1.110, 1.668) 0.003 Intercept -5.821       < 0.001 Minimum score = -2; maximum score = 30. AKI, acute kidney injury; CABG, coronary artery bypass grafting; CI, confidence interval; LVEF, left ventricular ejection fraction; OR, odds ratio; RBC, red blood cell.*included congenital heart disease repair, cardiac tumor surgery. 3. Any-stage or stage-3 AKI model from preoperative variables 3. Any-stage or stage-3 AKI model from preoperative variables 3. Any-stage or stage-3 AKI model from preoperative variables After using only preoperative variables to construct the AKI prediction models, the AUC for the any-stage AKI model was 0.64 (95% CI, 0.64, 0.65), and that for the stage-3 AKI model was 0.79 (95% CI, 0.77, 0.80). Nevertheless, the calibration from the Hosmer-Lemshow test was poor for both models (P < 0.001, P =  0.015, respectively, Additional file 2). Secondary Outcomes AKI was associated with greater medical expenditure and a prolonged length of hospital stay (Additional file 6 ). Patients developing any degree of AKI incurred a significantly greater cost during their treatment in the hospital (P < 0.001). Patients with AKI had a significantly longer length of stay than those without AKI (P < 0.001). In the whole cohort (N = 58399), any degree AKI was associated with higher odds of agitated delirium, pulmonary complications, re-intubation, and death in the hospital (Additional file 7). AKI Score Classification Figure 2 shows the predicted and observed incidences of AKI, which were based on the risk prediction scores for the validation cohorts. According to the scores observed in the validation cohorts, the predictive risk of AKI could be grouped into three classifications: low, medium, and high (Additional file 3 in the Supplement). For the validation cohorts, the incidence of AKI onset predicted by the model was similar to that observed clinically. The risk score and its associated predictive risk charts are presented in additional file 4 and additional file 5 in the Supplement. 1. Any-stage AKI model from pre- and intraoperative variables 1. Any-stage AKI model from pre- and intraoperative variables For the derivation data set, the performance of the any-stage AKI risk prediction model, which was derived from pre- and intraoperative variables, was as follows. The AUC was 0.69 (95% CI, 0.68, 0.69; Fig. 1), indicating an acceptable discriminability. The Hosmer-Lemshow test showed that the calibration of the any-stage AKI model from the pre- and intraoperative variables was good (P = 0.95). The model sensitivity, specificity, positive predictive value, and negative predictive value were 29.3%, 90.0%, 60.7%, and 70.7%, respectively. The AUC with the validation cohort was very similar (AUC = 0.69, 95% CI, 0.68, 0.70), with a sensitivity, specificity, positive predictive value, and negative predictive value of 27.5%, 91.0%, 60.5%, 71.4%, respectively. 2. Stage-3 AKI model from pre- and intraoperative variables 2. Stage-3 AKI model from pre- and intraoperative variables Page 12/23 Page 12/23 Page 12/23 The AUC for the stage-3 AKI risk prediction model with the derivation cohort was 0.84 (95% CI, 0.83, 0.85, Fig. 1). The Hosmer-Lemshow test demonstrated good calibration for the derivation cohort (P = 0.73). The model sensitivity, specificity, positive predictive value, and negative predictive value were 0.4%, 100%, 42.9%, and 98.3%, respectively. The performance with the validation cohort also indicated good discriminability (AUC = 0.82, 95% CI, 0.80, 0.85), and the model sensitivity, specificity, positive predictive value, and negative predictive value were 1.8%, 100%, 100%, and 98.5%, respectively. The AUC for the stage-3 AKI risk prediction model with the derivation cohort was 0.84 (95% CI, 0.83, 0.85, Fig. 1). The Hosmer-Lemshow test demonstrated good calibration for the derivation cohort (P = 0.73). The model sensitivity, specificity, positive predictive value, and negative predictive value were 0.4%, 100%, 42.9%, and 98.3%, respectively. The performance with the validation cohort also indicated good discriminability (AUC = 0.82, 95% CI, 0.80, 0.85), and the model sensitivity, specificity, positive predictive value, and negative predictive value were 1.8%, 100%, 100%, and 98.5%, respectively. Discussion We developed two scoring systems for AKI risk prediction following cardiac surgery, primarily for any- stage AKI and stage-3 AKI. Scores were evolved from pre- and intraoperative risk variables with a large cohort of cardiac patients. The scoring systems were also well internally validated. The prominent strengths of our study are listed as follows. The prominent strengths of our study are listed as follows. In addition to preoperative variables, intraoperative parameters were also incorporated to investigate the risk score for AKI. Comparatively, previous predictive scores primarily and solely attached importance to preoperative associated variables without any consideration for surgery-derived physiopathology induced by cardiopulmonary bypass (CPB), transfusion product use and excessive bleeding[10–17]. Additionally, Page 13/23 Page 13/23 Page 13/23 we failed to construct a predictive model for AKI using preoperative variables alone because the calibration assessed by the Hosmer-Lemshow test was not good. Other risk scores have been derived from information collected from all phases, including pre-, intra-, and postoperation[12, 15, 16]. However, it is arduous to disentangle whether morbidities occurring in the postoperative period can truly contribute to AKI onset. For example, low cardiac output syndrome can lead to compromised renal function and vice versa. Furthermore, at present, the number of postoperative parameters investigated following cardiac surgery is apparently limited. Postoperative central venous pressure > 14 cmH2O and low cardiac output syndrome are perceived as prognostic factors in the AKICS score[12]. Ng scores[14] only include intra-aortic balloon pump use in the postoperative period. The Jiang dynamic predictive score[16] only includes low cardiac output syndrome following cardiac surgery. However, pre-, intra- and postoperative models are limited in clinical effectiveness and utility because it is not easy to distinguish the causal relationship between postoperative variables and AKI. Given the differences in AKI definition and race, no statistical comparison can be performed among the abovementioned models. Our research represents a meaningful attempt to include atrial fibrillation in the prediction models. Atrial fibrillation commonly occurs in patients with kidney disease[24]. Previous studies have indicated that by inducing myocardial fibrosis, atrial fibrillation contributes to the decline in left ventricular function and the alteration of cardiac haemodynamics and then accelerates the progression of kidney disease. Atrial fibrillation also induces renal fibrosis by downregulating neutral endopeptidase expression[25]. Our research innovatively found that atrial fibrillation could predict postoperative AKI after cardiac surgery. Discussion Additionally, there is evidence that higher uric acid levels can induce AKI through fibroblast expansion, the inflammatory cascade and an increase in endothelin-1[26, 27]. Similar to previous investigations, our research found that hyperuricaemia prior to surgery was a risk factor for postoperative AKI[28, 29]. Meanwhile, we found that sex differences can significantly contribute to postoperative AKI. Male patients had a greater likelihood of developing any-stage AKI, whereas female patients were prone to develop stage-3 AKI; the underlying mechanisms remain to be elucidated. The AKI endpoint was defined by the KDIGO criteria in our study, which is consistent with that by Birnine et al[15] and Jiang et al[16]. Compared with investigations carried out by Birnine et al[15] (n = 30854) and Jiang et al[16](n = 7233), our study is characterized by an unprecedentedly larger sample size. In fact, the criteria for defining AKI following cardiac surgery varied greatly in previous studies on risk scores[12–16]. In 2012, based on evidence review and appraisal, the KDIGO organization compiled the first international and multidisciplinary clinical practice guidelines for AKI. At present, these guidelines are recognized as a consensus on the AKI definition from all over the world[30]. However, due to differences in race, religious belief, living habits and medical level, foreign prediction models cannot be effectively applied to other individual, local patient cohorts. Previous models have demonstrated poor discrimination and calibration in non-local populations[19–22]; hence, it is necessary to build prediction models for AKI for individual populations after cardiac surgery. For the Asian population, Jiang et al. developed a dynamic predictive score for any-stage AKI after cardiac surgery. However, in this study, we not only provided a robust Page 14/23 Page 14/23 predictive scoring system for any-stage AKI after cardiac surgery in the Chinese population but also developed a separate system for postoperative stage-3 AKI. predictive scoring system for any-stage AKI after cardiac surgery in the Chinese population but also developed a separate system for postoperative stage-3 AKI. In the future, both the any-stage AKI and stage-3 AKI scoring systems will be available as online calculators, which not only will be freely available and accessible by any computer system but can also be completed in a short time. Our model can even be integrated into hospital information management systems as a module to reduce labour costs in predicting AKI. Conclusion Using pre- and intraoperative data, we developed individual scoring systems for any-stage AKI or stage-3 AKI in a cardiac surgery population in accordance with KDIGO criteria. These scoring systems are beneficial for guiding clinicians towards decision-making for AKI detection and therapy after surgery. This study will contribute to AKI-predicting software development and further explorations into effective clinical interventions for AKI. Acknowledgements We thank Yuefu Wang for providing the idea for this study. Discussion As a retrospective analysis that is pending prospective validation, our research has intrinsic limitations as a result of unmeasured confounders. Variables such as the Canadian Cardiovascular Society (CCS) class, contrast-induced nephropathy, aorta cannulation and cross-clamping time were not included as potential predictive factors because records on these variables were not completed for a majority of the recruited patients. Additionally, the AKI diagnosis currently used in the study is not strictly consistent with the KDIGO criteria because it lacks the urinary output criteria due to its unavailability at our institute for the majority of patients. This may not be a very detrimental problem, however, because urinary output criteria often perform poorly in diagnosing clinically relevant AKI. Furthermore, it is generally accepted that the sicker the patient, the longer the length of stay and the higher the cost are, so the long-term outcome is more important than the calculated administrative cost. Our study is characterized by a large sample size, but data about long-term outcomes are unavailable at our institute for the majority of patients. The AUC of the any-stage AKI risk prediction model was 0.69, which is acceptable. We will focus on updating and optimizing the existing models by exploring new biomarkers and using smarter modelling methods in future work. Finally, data were obtained from a single centre, which could lead to bias in the results; hence, there is a need to validate this scoring system with clinical data from other cardiac centres. Yu Tian1 and Wei Zhao2 contributed equally to this work. From the 1Department of Anesthesiology, Fuwai Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China; 2Information Center, Fuwai Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China From the 1Department of Anesthesiology, Fuwai Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China; 2Information Center, Fuwai Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China Funding Supported by the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2019XK320052). Supported by the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2019XK320052). Availability of data and materials The data-sets generated during and/or analyzed during the current study are not publicly available due to IRB provisions but are available from the corresponding author on reasonable request. Authors’ contributions Yuefu Wang provided the idea for this study. Xiaolin Diao and Weiwei Wang provided Digital clinical data of patients. Yuchen Gao and Sudena Wang monitored and checked the collected data. Yu Tian and Wei Page 15/23 Zhao wrote and edited the paper. Chunrong Wang was responsible for revision in this paper. The authors read and approved the final manuscript. Ethics approval and consent to participate The research was approved by the Ethics Committee of Fuwai Hospital (20191308), and written informed consent from participants was waived for this retrospective analysis. Competing interests Authors declare no conflicts of interest related to this manuscript content. Authors declare no conflicts of interest related to this manuscript content. Consent for publication Patients agreed to allow all the manuscript data to be reported. References 1. Ozrazgat-Baslanti T, Thottakkara P, Huber M, et al. 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References Palomba H, de Castro I, Neto ALC, Lage S, Yu L. Acute kidney injury prediction following elective cardiac surgery: AKICS Score. Kidney Int. 2007;72(5):624-631. 13. Berg KS, Stenseth R, Wahba A, Pleym H, Videm V. How can we best predict acute kidney injury following cardiac surgery? a prospective observational study. Eur J Anaesthesiol. 2013;30(11):704-712. 14. Birnie K, Verheyden V, Pagano D, et al. Predictive models for kidney disease: improving global outcomes (KDIGO) defined acute kidney injury in UK cardiac surgery. Crit Care. 2014;18(6):606. 15. Ng SY, Sanagou M, Wolfe R, Cochrane A, Smith JA, Reid CM. Prediction of acute kidney injury within 30 days of cardiac surgery. The Journal of Thoracic and Cardiovascular Surgery. 2014;147(6):1875-1883. 16. Jiang W, Teng J, Xu J, et al. Dynamic Predictive Scores for Cardiac Surgery-Associated Acute Kidney Injury. J Am Heart Assoc. 2016;5(8). 17. Pannu N, Graham M, Klarenbach S, et al. A new model to predict acute kidney injury requiring renal replacement therapy after cardiac surgery. Can Med Assoc J. 2016;188(15):1076-1083. Page 17/23 Page 17/23 18. Kidney Disease：Improving Global Outcomes(KDIGO)Acute Kidney Injury Work Group. KDIGO Clinical Practice Guideline for Acute Kidney Injury(J).Kidney Inter,2012,2：1-138． 19. Rao S N, Shenoy M P, Gopalakrishnan M, et al. Applicability of the Cleveland clinic scoring system for the risk prediction of acute kidney injury after cardiac surgery in a South Asian cohort(J). Indian Heart J, 2018,70(4):533-537. 20. Ranucci M, Aloisio T, Cazzaniga A, et al. Validation of renal-risk models for the prediction of non-renal replacement therapy cardiac surgery-associated acute kidney injury(J). International Journal of Cardiology, 2018,272:49-53. 21. Jiang W, Xu J, Shen B, et al. Validation of Four Prediction Scores for Cardiac Surgery-Associated Acute Kidney Injury in Chinese Patients(J). Brazilian Journal of Cardiovascular Surgery, 2017,32(6):481-486. 22. Chew S T, Mar W M, Ti L K. Association of ethnicity and acute kidney injury after cardiac surgery in a South East Asian population(J).Br J Anaesth, 2013,110(3):397-401. 23. Remuzzi G, Horton R. Acute renal failure: an unacceptable death sentence globally. Lancet. 2013;382(9910):2041-2042. 24. Guo Y, Gao J, Ye P, et al. Comparison of atrial fibrillation in CKD and non-CKD populations: A cross- sectional analysis from the Kailuan study. Int J Cardiol. 2019;277:125-129. 25. Tapoi L, Ureche C, Sascau R, Badarau S, Covic A. Atrial fibrillation and chronic kidney disease conundrum: an update. J Nephrol. 2019. 26. Romi MM, Arfian N, Tranggono U, Setyaningsih W, Sari D. References Uric acid causes kidney injury through inducing fibroblast expansion, Endothelin-1 expression, and inflammation. Bmc Nephrol. 2017;18(1):326. 27. Kanbay M, Solak Y, Afsar B, et al. Serum Uric Acid and Risk for Acute Kidney Injury Following Contrast. Angiology. 2017;68(2):132-144. 28. Ejaz AA, Johnson RJ, Shimada M, et al. The Role of Uric Acid in Acute Kidney Injury[J]. Nephron. 2019, 142(4): 275-283. 29.Kang MW, Chin HJ, Joo KW, et al. Hyperuricemia is associated with acute kidney injury and all-cause mortality in hospitalized patients[J]. Nephrology. 2019, 24(7): 718-724. 30. Kellum JA, Lameire N, KDIGO AGWG, For TKAG. Diagnosis, evaluation, and management of acute kidney injury: a KDIGO summary (Part 1). Critical care. 2013;17(1):204. Figures Figures Page 18/23 Receiver operating characteristic curves (ROC) for any-stage AKI and stage-3 AKI prediction models in the derivation cohort and validation cohort. AKI: acute kidney injury Figure 1 Figure 1 Receiver operating characteristic curves (ROC) for any-stage AKI and stage-3 AKI prediction models in the derivation cohort and validation cohort. AKI: acute kidney injury Receiver operating characteristic curves (ROC) for any-stage AKI and stage-3 AKI prediction models in the derivation cohort and validation cohort. AKI: acute kidney injury Page 19/23 Receiver operating characteristic curves (ROC) for any-stage AKI and stage-3 AKI prediction models in the derivation cohort and validation cohort. AKI: acute kidney injury Figure 1 Figure 1 Receiver operating characteristic curves (ROC) for any-stage AKI and stage-3 AKI prediction models in the derivation cohort and validation cohort. AKI: acute kidney injury Receiver operating characteristic curves (ROC) for any-stage AKI and stage-3 AKI prediction models in the derivation cohort and validation cohort. AKI: acute kidney injury Page 20/23 d predicted risk of any-stage AKI (A) and stage-3 AKI (B) in the validation cohorts Figure 2 Observed and predicted risk of any-stage AKI (A) and stage-3 AKI (B) in the validation cohorts. AKI: acute kidney injury Figure 2 Observed and predicted risk of any-stage AKI (A) and stage-3 AKI (B) in the validation cohorts. AKI: acute kidney injury Observed and predicted risk of any-stage AKI (A) and stage-3 AKI (B) in the validation cohorts. AKI: acute kidney injury Observed and predicted risk of any-stage AKI (A) and stage-3 AKI (B) in the validation cohorts. AKI: acute kidney injury Page 21/23 Page 21/23 d predicted risk of any-stage AKI (A) and stage-3 AKI (B) in the validation cohorts re 2 erved and predicted risk of any-stage AKI (A) and stage-3 AKI (B) in the validation cohorts. AKI: acute ey injury pplementary Files Figure 2 Observed and predicted risk of any-stage AKI (A) and stage-3 AKI (B) in the validation cohorts. AKI: acute kidney injury Observed and predicted risk of any-stage AKI (A) and stage-3 AKI (B) in the validation cohorts. AKI: acute kidney injury Observed and predicted risk of any-stage AKI (A) and stage-3 AKI (B) in the validation cohorts. AKI: acute kidney injury Supplementary Files Page 22/23 This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. AdditionalfileSupplements.docx AdditionalfileSupplements.docx Page 23/23
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Isotretinoin Use in Transmasculine Patients and Its Implication on Chest Masculinization Surgery: Scoping Review of the Literature (Preprint)
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Corresponding Author: Corresponding Author: Robert P Dellavalle, MD, MSPH, PhD Dermatology Service Rocky Mountain Regional Veterans Affairs Medical Center 1700 N Wheeling St Rm E1-342 Aurora, CO, 80045 United States Phone: 1 720 857 5562 Fax: 1 720 723 7876 Email: robert.dellavalle@cuanschutz.edu Abstract Background: Acne often worsens in transmasculine patients who are on prolonged testosterone therapy. Isotretinoin is an oral retinoid used in the treatment of severe or refractory cases of acne, but it has the potential to cause delayed wound healing. Transmasculine patients may potentially be prescribed treatment for acne with isotretinoin while also planning to undergo chest masculinization surgery. Objective: This scoping review aims to determine whether isotretinoin has a negative impact on postoperative healing in transmasculine patients undergoing chest masculinization surgery. Methods: A scoping review was performed using the PubMed and Ovid databases. A total of 16 publications were selected for inclusion. Results: Acne tends to peak in transmasculine patients 6 months after initiation of testosterone treatment. Severe cases can be treated with isotretinoin; however, acne may recur once treatment is discontinued, given ongoing hormone therapy. There is little to no evidence in the medical literature regarding perioperative use of isotretinoin specifically among transmasculine patients undergoing chest masculinization surgery. In general, however, recent studies have found no evidence of increased hypertrophic scars or keloids in patients taking isotretinoin. Conclusions: Further studies are required to strengthen the current evidence that suggests that isotretinoin does not need to be discontinued before or after incisional or excisional surgeries, including chest masculinization surgery in transmasculine patients. JMIR DERMATOLOGY JMIR DERMATOLOGY Strock et al (JMIR Dermatol 2023;6:e45351) doi: 10.2196/45351 (JMIR Dermatol 2023;6:e45351) doi: 10.2196/45351 https://derma.jmir.org/2023/1/e45351 Daniel Strock1, BSc; Torunn E Sivesind2, MD; Robert P Dellavalle2,3,4, MD, MSPH, PhD; Gerhard S Mundinger5, MD, MBA 1School of Medicine, Eastern Virginia Medical School, Norfolk, VA, United States 2Department of Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States 3Department of Epidemiology, Colorado School of Public Health, University of Colorado Anschutz Medical Ca 4Dermatology Service, Rocky Mountain Regional Veterans Affairs Medical Center, Aurora, CO, United States 5Crane Center for Transgender Surgery, Austin, TX, United States 1School of Medicine, Eastern Virginia Medical School, Norfolk, VA, United States 2Department of Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States 3Department of Epidemiology, Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO, United States 4Dermatology Service, Rocky Mountain Regional Veterans Affairs Medical Center, Aurora, CO, United States 5Crane Center for Transgender Surgery, Austin, TX, United States Isotretinoin Use in Transmasculine Patients and Its Implication on Chest Masculinization Surgery: Scoping Review of the Literature Daniel Strock1, BSc; Torunn E Sivesind2, MD; Robert P Dellavalle2,3,4, MD, MSPH, PhD; Gerhard S Mundinger5, MD MBA JMIR Dermatol 2023 | vol. 6 | e45351 | p. 1 (page number not for citation purposes) KEYWORDS acne; isotretinoin; transsexual; scoping; post operative; surgical; pharmacology; pharmaceutic; retinoid; testosterone therapy; sex reassignment; gender affirmation surgery; masculinization surgery; gender reassignment; derm; dermatology; dermis; oily skin; hormone therapy; perioperative; operation; scar; keloid; surgery; surgeries; trans; transmen; transgender; transex; top surgery; chest masculinization; accutane; roaccutane JMIR Dermatol 2023 | vol. 6 | e45351 | p. 1 (page number not for citation purposes) XSL•FO RenderX JMIR DERMATOLOGY Strock et al AND (isotretinoin OR accutane OR roaccutane)” yielded zero results. A search using the key terms “(transmen OR “trans men” OR trans-men OR transgender* OR transex* OR transmasc*) AND (isotretinoin OR accutane OR roaccutane)” yielded 17 results, though no articles were new in respect to the previously conducted PubMed search. The key terms “(surg*) AND (heal* OR scar*) AND (isotretinoin OR accutane OR roaccutane),” limited to publications within 10 years, yielded 33 results, all of which were similarly included in the previous PubMed search. Results Acne and Isotretinoin Use in Transmasculine Patients Masculinizing hormone therapy used in transmasculine patients has the potential to worsen acne, mediated largely by testosterone’s effect on the pilosebaceous unit [4]. In a prospective study of 20 transmasculine patients undergoing exogenous testosterone therapy, rates of facial acne increased from 35% to 82.4% following 6 months of treatment, whereas rates of chest and back acne increased from 15% to 88.2% within the same time frame [4]. Another prospective cohort study of 193 transmasculine patients demonstrated that self-reported scores of acne severity peaked 6 months after initiating testosterone therapy, and were still significantly higher than those at baseline after 12 months of therapy [5]. A retrospective review of medical records of 55 transmasculine patients also demonstrated a significant association between acne and a serum testosterone level greater than 630 ng/dL [6]. No evidence-based guidelines currently exist for treating acne in the context of testosterone therapy [7]. Though acne caused by exogenous testosterone is often mild to moderate in severity [8], more persistent or extensive cases may necessitate treatment with isotretinoin. While isotretinoin can be an appropriate treatment for acne in the setting of prolonged testosterone therapy, there are many special considerations when prescribing isotretinoin for transmasculine patients [9]. Given the teratogenicity of isotretinoin, clinicians should be prepared to engage their patients in thorough but sensitive conversations regarding their risk of pregnancy. Additionally, though the relationship between isotretinoin use and idiosyncratic depression is largely controversial, it should be noted that depressive symptoms, suicidal ideation, and self-harm are more common in transgender individuals. These comorbidities are posited to be largely the result of social stresses including bullying, harassment, social stigma, and decreased support from family members [10]. It also unclear whether long-term remission can be achieved with one “cycle” of isotretinoin in the context of ongoing testosterone therapy. No large-scale randomized studies have examined isotretinoin use in this specific population, but several case studies have demonstrated the efficacy of isotretinoin therapy in the treatment of testosterone-dependent acne; these results are presented in Table 1 [11,12]. Introduction Acne is an inflammatory condition of the skin commonly treated by dermatologists. While the pathogenesis of acne is multifactorial, recent research has emphasized the importance of hormones in the function of the pilosebaceous unit [1]. In sebaceous glands, testosterone is converted to dihydrotestosterone and binds to cytoplasmic androgen receptors, promoting epidermal growth and sebocyte proliferation. This increased sebum production plays a principal role in the development of acne. This is of particular relevance to female-to-male transgender patients (herein referred to as “transmasculine patients”) who are often prescribed masculinizing doses of exogenous testosterone as part of gender-affirming hormone therapy. Isotretinoin may be prescribed for more severe or refractory cases of acne, but it has the potential to delay wound healing due to its effect on the stem cells responsible for repopulating the epidermis. Studies in animal models have demonstrated isotretinoin’s effect on the pilosebaceous unit, whereby the sebaceous glands were greatly reduced in volume [2]. Given that transmasculine patients may pursue bilateral mastectomy and chest masculinization surgery (often referred to as “top surgery”) as a means of gender affirmation, it is vital for dermatologists to be aware of how isotretinoin use may impact chest masculinization surgery in this population. A scoping review [3] of relevant literature available via a web-based database search was performed in order to assist clinicians in making informed, evidence-based decisions with their patients and to identify any existing knowledge gaps regarding isotretinoin treatment and chest masculinization surgery. JMIR Dermatol 2023 | vol. 6 | e45351 | p. 2 (page number not for citation purposes) https://derma.jmir.org/2023/1/e45351 Discussion Acne is common in transmasculine patients undergoing masculinizing hormone therapy, and some individuals with testosterone-induced acne may benefit from isotretinoin therapy. In a survey of transgender individuals, those who self-identified as men were more likely to prioritize treatment of the chest over that of their face or genitals [19]. In a cross-sectional analysis of 90 transgender men, exogenous testosterone demonstrated a significant effect on the presence of chest acne, which was present in 52% of transmasculine patients on testosterone therapy, compared to 9% of those who were not on testosterone therapy. Given that exogenous testosterone therapy has a prominent effect on the chest [20], it is vital for clinicians to recognize the impact that acne treatment has on patients’ body image–related quality of life. The ASDS guidelines (published in 2017) for following comprehensive literature review and task force consensus state, “The data for incisional and excisional cutaneous surgery on isotretinoin is insufficient to make any recommendations. In particular cases, incisional or excisional surgery may be medically necessary in patients receiving isotretinoin” [15]. Quality of Evidence was assigned a “D” score by task force members, using the GRADE (Grading of Recommendations, Assessment, Development, and Evaluation) system, and representing “very low” confidence in their final recommendation [15]. Another systematic review with consensus recommendations, also published in 2017, determined that there is “insufficient evidence to delay cutaneous surgery for patients currently taking or having recently completed isotretinoin therapy” [16]. When initiating isotretinoin therapy in transgender male patients, it should be standard practice for dermatologists to discuss plans for any upcoming surgery, including chest masculinization surgery, with their patients. While current literature does not suggest any effect of isotretinoin on postsurgical wound healing, risks and benefits of perioperative treatment should be discussed with patients, and a treatment plan should be formulated on the basis of their individual goals of care and severity of acne. Given the lack of specific evidence in this patient population, surgery would ideally be performed before initiation of isotretinoin therapy in order to minimize any interactions. However, in cases of severe acne with a risk of scarring, it is not recommended to delay treatment with isotretinoin or to discontinue isotretinoin before chest masculinization surgery. More recently, a randomized controlled trial of 303 patients, published in 2020, examined the effects of perioperative isotretinoin therapy on rhinoplasty outcomes [17]. Methods A search of the available biomedical literature was performed using the PubMed database on September 1, 2022. An initial query using the search string, “(transmen OR “trans men” OR trans-men OR transgender* OR transex* OR transmasc*) AND (“top surgery” OR “Chest masculinization”) AND (isotretinoin OR accutane OR roaccutane),” yielded zero results. A more focused search used the string, “(transmen OR “trans men” OR trans-men OR transgender* OR transex* OR transmasc*) AND (isotretinoin OR accutane OR roaccutane),” yielded 19 results, all of which were published between 2015 and 2022. Another search using the string, “(surg*) AND (heal* OR scar*) AND (isotretinoin OR accutane OR roaccutane),” limited to publications within 10 years, yielded 109 results, for a combined total of 128 articles selected for title and abstract screening. This was performed by 2 independent reviewers (DS and TS), with 40 articles selected for subsequent full-text review. After full-text review, a total of 11 studies were selected for inclusion. Surveying the references of these 11 articles revealed an additional 5 relevant studies, which were also chosen for inclusion. testosterone 1 [11,12]. An additional search was later conducted using the Ovid database on June 18, 2023. The search terms “(transmen OR “trans men” OR trans-men OR transgender* OR transex* OR transmasc*) AND (“top surgery” OR “Chest masculinization”) All 4 patients presented in these case studies had a positive response to isotretinoin therapy. Patients 1 and 2 achieved resolution of their acne, but ultimately presented with recurrence necessitating ongoing isotretinoin therapy. Patient 3 achieved JMIR Dermatol 2023 | vol. 6 | e45351 | p. 2 (page number not for citation purposes) https://derma.jmir.org/2023/1/e45351 XSL•FO RenderX XSL•FO RenderX JMIR DERMATOLOGY Strock et al remission and decided to discontinue treatment at 4 months. Patient 4 demonstrated partial clearance of his acne, but associated therapy with a depressive episode and discontinued at 3.5 months of treatment. Table 1. Case series of transgender males treated with isotretinoin [11,12]. Methods Isotretinoin dose Length of testosterone treat- ment before starting isotretinoin Testosterone dose Patient age (years) Patient number 30 mg/day for 9 months, discontinue for 3 months, ongoing at 20 mg/day 6 months Testosterone undecanoate, 1000 mg every 3 months 20-29a 1 20 mg/d for 8 months, discontinue for 6 months, ongoing at 20 mg 3 times per week 6 months Testosterone undecanoate, 1000 mg every 3 months 20-29a 2 20 mg/day for 3.5 months 6 months Testosterone (formulation unspeci- fied), 250 mg every 21 days 17 3 20 mg/day for 4 months 5 months Testosterone (formulation unspeci- fied), 250 mg every 21 days 17 4 aThe patient in the original case report was referred to as being “in his 20s.” associated therapy with a depressive episode and discontinued at 3.5 months of treatment. aThe patient in the original case report was referred to as being “in his 20s.” transmasculine patients on isotretinoin therapy undergoing chest masculinization surgery. Isotretinoin and Surgical Healing It has traditionally been taught that surgical procedures should not be performed while a patient is taking oral isotretinoin, given concerns that retinoids may alter connective tissue healing or lead to keloids or hypertrophic scarring. These recommendations are based largely on 3 case series published in the 1980s, which reported the formation of keloids in 9 patients on isotretinoin therapy, who subsequently underwent dermabrasion or argon laser phototherapy [13,14]. However, recent literature on the subject contradicts these earlier reports, as noted by the American Society for Dermatologic Surgery (ASDS). https://derma.jmir.org/2023/1/e45351 JMIR Dermatol 2023 | vol. 6 | e45351 | p. 3 (page number not for citation purposes) Discussion This study suggests that isotretinoin caused no significant disturbances in postoperative healing, with no hypertrophic scarring or cartilaginous deformities. A case study published in 2022 demonstrated normal scar tissue healing in 2 cisgender females who underwent bilateral reduction mammoplasty while on isotretinoin therapy [18]. Neither patient had a family history of keloids or hypertrophic scarring. Our review did not reveal any studies that specifically examined postoperative healing in Further research is needed on this particular subject matter and patient population in order to better to characterize the exact relationship between isotretinoin use and chest masculinization surgery, so that dermatologists may better coordinate care for their transgender patients. JMIR Dermatol 2023 | vol. 6 | e45351 | p. 3 (page number not for citation purposes) XSL•FO RenderX JMIR DERMATOLOGY Strock et al Conflicts of Interest RPD is the Editor-in-Chief of JMIR Dermatology, joint coordinating editor for Cochrane Skin, a dermatology section editor for UpToDate, and a coordinating editor representative and cochair on the Cochrane Council. TES is an editorial board member–at-large for JMIR Dermatology and a member of the Cochrane Collaboration. DS is a social media editor for JMIR Dermatology. RPD receives editorial stipends (Journal of the American Academy of Dermatology and Journal of Investigative Dermatology), royalties (UpToDate), and expense reimbursement from Cochrane Skin. TES receives fellowship funding from Pfizer (grant 25B1519; principal investigator, Stanca Birlea) and the National Institutes of Health (grant 2T32AR00741136A1; principal investigator, Dennis Roop). References Explorative prospective evaluation of short-term subjective effects of hormonal treatment in trans people-results from the European network for the investigation of gender incongruence. J Sex Med 2019 Aug;16(8):1297-1309 [doi: 10.1016/j.jsxm.2019.05.009] [Medline: 31230940] 6. Park JA, Carter EE, Larson AR. Risk factors for acne development in the first 2 years after initiating masculinizing testosterone therapy among transgender men. J Am Acad Dermatol 2019 Aug;81(2):617-618 [doi: 10.1016/j.jaad.2018.12.040] [Medline: 30711356] 7. Ragmanauskaite L, Kahn B, Ly B, Yeung H. Acne and the lesbian, gay, bisexual, or transgender teenager. Dermatol Clin 2020 Apr;38(2):219-226 [FREE Full text] [doi: 10.1016/j.det.2019.10.006] [Medline: 32115131] 7. Ragmanauskaite L, Kahn B, Ly B, Yeung H. Acne and the lesbian, gay, bisexual, or transgender teenager. Dermatol Clin 2020 Apr;38(2):219-226 [FREE Full text] [doi: 10.1016/j.det.2019.10.006] [Medline: 32115131] 8. Wierckx K, Van de Peer F, Verhaeghe E, Dedecker D, Van Caenegem E, Toye K, et al. Short- and long-term clinical skin effects of testosterone treatment in trans men. J Sex Med 2014 Jan;11(1):222-229 [doi: 10.1111/jsm.12366] [Medline: 24344810] 8. Wierckx K, Van de Peer F, Verhaeghe E, Dedecker D, Van Caenegem E, Toye K, et al. Short- and long-term clinical skin effects of testosterone treatment in trans men. J Sex Med 2014 Jan;11(1):222-229 [doi: 10.1111/jsm.12366] [Medline: 24344810] 9. Richer V, Kuritzky LA. Considerations in treating severe acne with isotretinoin in transgender men. J Cutan Med Surg 2020 May 25;24(5):529-530 [doi: 10.1177/1203475420930224] [Medline: 32449627] 9. Richer V, Kuritzky LA. Considerations in treating severe acne with isotretinoin in transgender men. J Cutan Med Surg 2020 May 25;24(5):529-530 [doi: 10.1177/1203475420930224] [Medline: 32449627] 10. Reisner SL, Greytak EA, Parsons JT, Ybarra ML. Gender minority social stress in adolescence: disparities in adolescent bullying and substance use by gender identity. J Sex Res 2015 Apr 17;52(3):243-256 [FREE Full text] [doi: 10. Reisner SL, Greytak EA, Parsons JT, Ybarra ML. Gender minority social stress in adolescence: disparities in adolescent bullying and substance use by gender identity. J Sex Res 2015 Apr 17;52(3):243-256 [FREE Full text] [doi: 10.1080/00224499.2014.886321] [Medline: 24742006] 11. Turrion-Merino L, Urech-García-de-la-Vega M, Miguel-Gomez L, Harto-Castaño A, Jaen-Olasolo P. Severe acne in female-to-male transgender patients. JAMA Dermatol 2015 Nov 01;151(11):1260-1261 [doi: 10 1001/j d l 2015 0761] [M dli 25992965] 11. Turrion-Merino L, Urech-García-de-la-Vega M, Miguel-Gomez L, Harto-Castaño A, Jaen-Olasolo P. Severe acne in female-to-male transgender patients. JAMA Dermatol 2015 Nov 01;151(11):1260-1261 [doi: 10.1001/jamadermatol.2015.0761] [Medline: 25992965] 12. Campos-Muñoz L, López-De Lara D, Rodríguez-Rojo ML, Conde-Taboada A, López-Bran E. References Transgender adolescents and acne: a cases series. Pediatr Dermatol 2018 May 25;35(3):e155-e158 [doi: 10.1111/pde.13448] [Medline: 29575091] 12. Campos-Muñoz L, López-De Lara D, Rodríguez-Rojo ML, Conde-Taboada A, López-Bran E. Transgender adolescents and acne: a cases series. Pediatr Dermatol 2018 May 25;35(3):e155-e158 [doi: 10.1111/pde.13448] [Medline: 29575091] 12. Campos-Muñoz L, López-De Lara D, Rodríguez-Rojo ML, Conde-Taboada A, López-Bran E. Transgender adolescents and acne: a cases series. Pediatr Dermatol 2018 May 25;35(3):e155-e158 [doi: 10.1111/pde.13448] [Medline: 29575091] 13. Zachariae H. Delayed wound healing and keloid formation following argon laser treatment or dermabrasion during isotretinoin treatment. Br J Dermatol 1988 May;118(5):703-706 [doi: 10.1111/j.1365-2133.1988.tb02574.x] [Medline: 2969261] 14. Rubenstein R, Roenigk HH, Stegman SJ, Hanke CW. Atypical keloids after dermabrasion of patients taking isotretinoin. J Am Acad Dermatol 1986 Aug;15(2 Pt 1):280-285 [doi: 10.1016/s0190-9622(86)70167-9] [Medline: 3018052] 15. Waldman A, Bolotin D, Arndt KA, Dover JS, Geronemus RG, Chapas A, et al. ASDS Guidelines Task Force: consensus recommendations regarding the safety of lasers, dermabrasion, chemical peels, energy devices, and skin surgery during and after isotretinoin use. Dermatol Surg 2017;43(10):1249-1262 [doi: 10.1097/dss.0000000000001166] 16. Spring LK, Krakowski AC, Alam M, Bhatia A, Brauer J, Cohen J, et al. Isotretinoin and timing of procedural interventions: a systematic review with consensus recommendations JAMA Dermatol 2017 Aug 01;153(8):802 809 [doi: 12. Campos-Muñoz L, López-De Lara D, Rodríguez-Rojo ML, Conde-Taboada A, López-Bran E. Transgender adolescents and acne: a cases series. Pediatr Dermatol 2018 May 25;35(3):e155-e158 [doi: 10.1111/pde.13448] [Medline: 29575091] 13. Zachariae H. Delayed wound healing and keloid formation following argon laser treatment or dermabrasion during isotretinoin treatment. Br J Dermatol 1988 May;118(5):703-706 [doi: 10.1111/j.1365-2133.1988.tb02574.x] [Medline: 2969261] 14. Rubenstein R, Roenigk HH, Stegman SJ, Hanke CW. Atypical keloids after dermabrasion of patients taking isotretinoin. J Am Acad Dermatol 1986 Aug;15(2 Pt 1):280-285 [doi: 10 1016/s0190-9622(86)70167-9] [Medline: 3018052] and acne: a cases series. Pediatr Dermatol 2018 May 25;35(3):e155-e158 [doi: 10.1111/pde.13448] [Medline: 29575091] 13. Zachariae H. Delayed wound healing and keloid formation following argon laser treatment or dermabrasion during isotretinoin treatment. Br J Dermatol 1988 May;118(5):703-706 [doi: 10.1111/j.1365-2133.1988.tb02574.x] [Medline: 2969261] 14 R b t i R R i k HH St SJ H k CW At i l k l id ft d b i f ti t t ki i t ti i y ( ) p 13. Zachariae H. Delayed wound healing and keloid formation following argon laser treatment or dermabrasion during isotretinoin treatment. Br J Dermatol 1988 May;118(5):703-706 [doi: 10.1111/j.1365-2133.1988.tb02574.x] [Medline: 2969261] 14. References 1. Hazarika N. Acne vulgaris: new evidence in pathogenesis and future modalities of treatment. J Dermatolog Treat 2021 May 29;32(3):277-285 [doi: 10.1080/09546634.2019.1654075] [Medline: 31393195] 2. Gomez EC. Actions of isotretinoin and etretinate on the pilosebaceous unit. J Am Acad Dermatol 1982 Apr;6(4 Pt 2 Suppl):746-750 [doi: 10.1016/s0190-9622(82)70064-7] [Medline: 6950956] 3. Munn Z, Peters MDJ, Stern C, Tufanaru C, McArthur A, Aromataris E. Systematic review or scoping review? Guidance for authors when choosing between a systematic or scoping review approach. BMC Med Res Methodol 2018 Nov 19;18(1):143 [FREE Full text] [doi: 10.1186/s12874-018-0611-x] [Medline: 30453902] 4. Motosko C, Zakhem G, Pomeranz M, Hazen A. Acne: a side-effect of masculinizing hormonal therapy in transgender patients. Br J Dermatol 2019 Jan 14;180(1):26-30 [doi: 10.1111/bjd.17083] [Medline: 30101531] 5. van Dijk D, Dekker MJ, Conemans EB, Wiepjes CM, de Goeij EG, Overbeek KA, et al. Explorative prospective evaluation of short-term subjective effects of hormonal treatment in trans people-results from the European network for the investigation of gender incongruence. J Sex Med 2019 Aug;16(8):1297-1309 [doi: 10.1016/j.jsxm.2019.05.009] [Medline: 31230940] 6. Park JA, Carter EE, Larson AR. Risk factors for acne development in the first 2 years after initiating masculinizing testosterone therapy among transgender men. J Am Acad Dermatol 2019 Aug;81(2):617-618 [doi: 10.1016/j.jaad.2018.12.040] [Medline: 30711356] 1. Hazarika N. Acne vulgaris: new evidence in pathogenesis and future modalities of treatment. J Dermatolog Treat 2021 May 29;32(3):277-285 [doi: 10.1080/09546634.2019.1654075] [Medline: 31393195] 1. Hazarika N. Acne vulgaris: new evidence in pathogenesis and future modalities of treatment. J Dermatolog Treat 2021 May 29;32(3):277-285 [doi: 10.1080/09546634.2019.1654075] [Medline: 31393195] y 2. Gomez EC. Actions of isotretinoin and etretinate on the pilosebaceous unit. J Am Acad Dermatol 1982 Apr;6(4 Pt 2 Suppl):746-750 [doi: 10.1016/s0190-9622(82)70064-7] [Medline: 6950956] 3. Munn Z, Peters MDJ, Stern C, Tufanaru C, McArthur A, Aromataris E. Systematic review or scoping review? Guidance for authors when choosing between a systematic or scoping review approach. BMC Med Res Methodol 2018 Nov 19;18(1):143 [FREE Full text] [doi: 10.1186/s12874-018-0611-x] [Medline: 30453902] 4. Motosko C, Zakhem G, Pomeranz M, Hazen A. Acne: a side-effect of masculinizing hormonal therapy in transgender patients. Br J Dermatol 2019 Jan 14;180(1):26-30 [doi: 10.1111/bjd.17083] [Medline: 30101531] 4. Motosko C, Zakhem G, Pomeranz M, Hazen A. Acne: a side-effect of masculinizing hormonal the patients. Br J Dermatol 2019 Jan 14;180(1):26-30 [doi: 10.1111/bjd.17083] [Medline: 30101531] 5. van Dijk D, Dekker MJ, Conemans EB, Wiepjes CM, de Goeij EG, Overbeek KA, et al. References Rubenstein R, Roenigk HH, Stegman SJ, Hanke CW. Atypical keloids after dermabrasion of patients taking isotretinoin. J Am Acad Dermatol 1986 Aug;15(2 Pt 1):280-285 [doi: 10.1016/s0190-9622(86)70167-9] [Medline: 3018052] 15. Waldman A, Bolotin D, Arndt KA, Dover JS, Geronemus RG, Chapas A, et al. ASDS Guidelines Task Force: consensus recommendations regarding the safety of lasers, dermabrasion, chemical peels, energy devices, and skin surgery during y p 13. Zachariae H. Delayed wound healing and keloid formation following argon laser treatment or dermabrasion during isotretinoin treatment. Br J Dermatol 1988 May;118(5):703-706 [doi: 10.1111/j.1365-2133.1988.tb02574.x] [Medline: 2969261] 14. Rubenstein R, Roenigk HH, Stegman SJ, Hanke CW. Atypical keloids after dermabrasion of patients taking isotretinoin. J Am Acad Dermatol 1986 Aug;15(2 Pt 1):280-285 [doi: 10.1016/s0190-9622(86)70167-9] [Medline: 3018052] 14. Rubenstein R, Roenigk HH, Stegman SJ, Hanke CW. Atypical keloids after dermabrasion of patients taking isotretinoin. J Am Acad Dermatol 1986 Aug;15(2 Pt 1):280-285 [doi: 10.1016/s0190-9622(86)70167-9] [Medline: 3018052] J Am Acad Dermatol 1986 Aug;15(2 Pt 1):280 285 [doi: 10.1016/s0190 9622(86)70167 9] [Medline: 3018052] 15. Waldman A, Bolotin D, Arndt KA, Dover JS, Geronemus RG, Chapas A, et al. ASDS Guidelines Task Force: consensus recommendations regarding the safety of lasers, dermabrasion, chemical peels, energy devices, and skin surgery during and after isotretinoin use. Dermatol Surg 2017;43(10):1249-1262 [doi: 10.1097/dss.0000000000001166] 15. Waldman A, Bolotin D, Arndt KA, Dover JS, Geronemus RG, Chapas A, et al. ASDS Guidelines Task Force: consensus recommendations regarding the safety of lasers, dermabrasion, chemical peels, energy devices, and skin surgery during and after isotretinoin use. Dermatol Surg 2017;43(10):1249-1262 [doi: 10.1097/dss.0000000000001166] g 16. Spring LK, Krakowski AC, Alam M, Bhatia A, Brauer J, Cohen J, et al. Isotretinoin and timing of procedural interventions: a systematic review with consensus recommendations. JAMA Dermatol 2017 Aug 01;153(8):802-809 [doi: 10.1001/jamadermatol.2017.2077] [Medline: 28658462] g 16. Spring LK, Krakowski AC, Alam M, Bhatia A, Brauer J, Cohen J, et al. Isotretinoin and timing of procedural interventions: a systematic review with consensus recommendations. JAMA Dermatol 2017 Aug 01;153(8):802-809 [doi: 10.1001/jamadermatol.2017.2077] [Medline: 28658462] 17. Yahyavi S, Jahandideh H, Izadi M, Paknejad H, Kordbache N, Taherzade S. Analysis of the effects rhinoplasty patients. Aesthet Surg J 2020 Nov 19;40(12):NP657-NP665 [doi: 10.1093/asj/sjaa219] 17. Yahyavi S, Jahandideh H, Izadi M, Paknejad H, Kordbache N, Taherzade S. Analysis of the effects of isotretinoin on rhinoplasty patients. Aesthet Surg J 2020 Nov 19;40(12):NP657-NP665 [doi: 10.1093/asj/sjaa219] [Medline: 32756944] 18. Davies MJ, Perkins D. References Oral isotretinoin (Roaccutane) use during incisional surgery: safe or risky? Case Reports Plast Surg Hand Surg 2022 May 13;9(1):131-134 [FREE Full text] [doi: 10.1080/23320885.2022.2071275] [Medline: 35601983] 19. Ginsberg BA, Calderon M, Seminara NM, Day D. A potential role for the dermatologist in the physical transformation of transgender people: a survey of attitudes and practices within the transgender community. J Am Acad Dermatol 2016 Feb;74(2):303-308 [doi: 10.1016/j.jaad.2015.10.013] [Medline: 26669479] rhinoplasty patients. Aesthet Surg J 2020 Nov 19;40(12):NP657-NP665 [doi: 10.1093/asj/sjaa219] [Medline: 32756944] 18. Davies MJ, Perkins D. Oral isotretinoin (Roaccutane) use during incisional surgery: safe or risky? Case Reports Plast Surg Hand Surg 2022 May 13;9(1):131-134 [FREE Full text] [doi: 10.1080/23320885.2022.2071275] [Medline: 35601983] 19. Ginsberg BA, Calderon M, Seminara NM, Day D. A potential role for the dermatologist in the physical transformation of transgender people: a survey of attitudes and practices within the transgender community. J Am Acad Dermatol 2016 F b 74(2) 303 308 [d i 10 1016/j j d 2015 10 013] [M dli 26669479] p y p g ( ) j j 18. Davies MJ, Perkins D. Oral isotretinoin (Roaccutane) use during incisional surgery: safe or risky? Case Reports Plast Surg Hand Surg 2022 May 13;9(1):131-134 [FREE Full text] [doi: 10.1080/23320885.2022.2071275] [Medline: 35601983] 18. Davies MJ, Perkins D. Oral isotretinoin (Roaccutane) use during incisional surgery: safe or risky? Case Reports Plast Surg Hand Surg 2022 May 13;9(1):131-134 [FREE Full text] [doi: 10.1080/23320885.2022.2071275] [Medline: 35601983] 19. Ginsberg BA, Calderon M, Seminara NM, Day D. A potential role for the dermatologist in the physical transformation of transgender people: a survey of attitudes and practices within the transgender community. J Am Acad Dermatol 2016 Feb;74(2):303-308 [doi: 10.1016/j.jaad.2015.10.013] [Medline: 26669479] 19. Ginsberg BA, Calderon M, Seminara NM, Day D. A potential role for the dermatologist in the physical transformation of transgender people: a survey of attitudes and practices within the transgender community. J Am Acad Dermatol 2016 Feb;74(2):303-308 [doi: 10.1016/j.jaad.2015.10.013] [Medline: 26669479] JMIR Dermatol 2023 | vol. 6 | e45351 | p. 4 (page number not for citation purposes) https://derma.jmir.org/2023/1/e45351 XSL•FO RenderX JMIR DERMATOLOGY JMIR DERMATOLOGY Strock et al 20. Motosko CC, Zakhem GA, Pomeranz MK, Pomerantz R, Saadeh PB, Gothard MD, et al. Effect of testosterone on chests and abdomens of transgender men. J Am Acad Dermatol 2019 Aug;81(2):634-636 [doi: 10.1016/j.jaad.2019.01.030] [Medline: 30685270] JMIR Dermatol 2023 | vol. 6 | e45351 | p. 5 (page number not for citation purposes) https://derma.jmir.org/2023/1/e45351 Abbreviations ASDS: American Society for Dermatologic Surgery GRADE: Grading of Recommendations, Assessment, Development, and Evaluation by R Alhusayen; submitted 26.12.22; peer-reviewed by T Simonart, OP Hamnvik, S Hidig; comments to author 16.04.2 d version received 29.06.23; accepted 23.07.23; published 10.08.23 Please cite as: Strock D, Sivesind TE, Dellavalle RP, Mundinger GS Isotretinoin Use in Transmasculine Patients and Its Implication on Chest Masculinization Surgery: Scoping Review of the Literature JMIR Dermatol 2023;6:e45351 URL: https://derma.jmir.org/2023/1/e45351 doi: 10.2196/45351 PMID: ©Daniel Strock, Torunn E Sivesind, Robert P Dellavalle, Gerhard S Mundinger. Originally published in JMIR Dermatology (http://derma.jmir.org), 10.08.2023. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Dermatology, is properly cited. The complete bibliographic information, a link to the original publication on http://derma.jmir.org, as well as this copyright and license information must be included. https://derma.jmir.org/2023/1/e45351 XSL•FO RenderX XSL•FO RenderX
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Application of Bloom’s Taxonomy in Categorization of Cognitive Process Development in Colleges
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Abstract Bloom’s Taxonomy is crucial in any teaching and learning environment because it determines direction for a course of study. This study assessed application of Bloom’s Taxonomy in courses of study offered in colleges in Lusaka District. Objectives were: Assess the application of Bloom’s Taxonomy in setting learning outcomes for the courses; Assess reflection of Bloom’s Taxonomy in activities set for course participants; Ascertain significance attached to Bloom’s Taxonomy in courses of study designed for participants. The study employed a mixed methodology, in particular employing a descriptive research design to assess application of the Taxonomy in course outcomes/objective. The population comprised college lecturers in Lusaka District. Sample size was 36 lecturers who were conveniently sampled. Data were collected using a questionnaire and analyzed using frequencies and Spearman correlation coefficient. Findings revealed that the majority of the lecturers did not illustrate how to use Bloom’s Taxonomy in planning course of study, setting objectives/outcomes, creating learning activities or create assessment tasks for course participants. However, most of the lecturers demonstrated use of active verbs which they use in objectives/outcomes. Findings revealed that lecturers hardly used Bloom’s Taxonomy. There is no relationship between lecturers’ use of learning outcomes as a basis for preparing class tasks and their assertions that they compared objectives with tasks prepared for students. It is recommended that lecturers should be oriented on use of the learning Taxonomy. g y Keywords: Bloom’s Taxonomy, Objectives, Outcomes, Lecturers DOI: 10.7176/JEP/14-4-02 g y Keywords: Bloom’s Taxonomy, Objectives, Outcomes, Lecturers DOI: 10 7176/JEP/14-4-02 g y Keywords: Bloom’s Taxonomy, Objectives, Outcomes, Lecturers Keywords: Bloom’s Taxonomy, Objectives, Outcomes, Lecturers Publication date: February 28th 2023 INTRODUCTION Bloom’s Taxonomy is an indispensable component in any teaching and learning activity. It classifies levels of cognitive development expected to be attained in various learning stages. Bloom’s Taxonomy provides an understanding of how to formulate appropriate objectives in the teaching and learning process. This model also guides instructors to change complexity of the questions and help learners to achieve higher levels of hierarchy. Stanny (2016) explains the effectiveness of Bloom Taxonomy in classifying thinking skills in a hierarchy that ranges from lower level cognitive skills through higher order cognitive skills. Further, it helps in the development of critical thinking among teachers as they set assessment tasks. However, this taxonomy is hardly used in some of the teaching and learning processes. Attributed to this lack or partial application of the taxonomy is twofold. Firstly, lack of proper understanding of the taxonomy in using it to set objectives, questions, learning outcomes as well as corresponding assessments. Secondly, it is none exposure of teachers to application of taxonomy in the teaching and learning process. Akinboboye and Ayanwale (2021) affirm this disparity in the use of the taxonomy by some of the teachers who face challenges in using this taxonomy because of lack of complete understanding of how it is supposed to be applied. While some of the teachers have ideas in using the taxonomy, others have not been exposed to it at all. Such are problems associated to use of Bloom’s Taxonomy among teachers. Earlier on, Gluga, Kay, Lister, Simon, and Kleitman (2013) confirmed that educators often need support from educational experts, which are not easily available in many departments in higher education. It was against this background that this study was conducted to assess the application of the Taxonomy among lecturers in colleges. Application of Bloom’s Taxonomy in Categorization of Cognitive Process Development in Colleges Selina Banda1 , Florence Phiri2, Jack Kaale1, Agnes M. Banda3, Daniel L. Mpolomoka4 Rose Chikopela5 and Christine Mushibwe4 1. Copperbelt University, School of Humanities and Social Sciences, Department of Social Studies 3. Zambian Open University, School of Education, Department of Primary and Early Childhood Education 4. Unicaf University Zambia, School of Education, Humanities and Social Sciences 5. Chalimbana University, School of Education, Department of Special Education-Guidance and Counselling bandaseli67@gmail.com www.iiste.org www.iiste.org Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.4, 2023 Journal of Education and Practice www.iiste.org Application of Bloom’s Taxonomy in Categorization of Cognitive Process Development in Colleges Selina Banda1*, Florence Phiri2, Jack Kaale1, Agnes M. Banda3, Daniel L. Mpolomoka4 Rose Chikopela5 and Christine Mushibwe4 1. Copperbelt University, School of Humanities and Social Sciences, Department of Social Studies 3. Zambian Open University, School of Education, Department of Primary and Early Childhood Education 4. Unicaf University Zambia, School of Education, Humanities and Social Sciences 5. Chalimbana University, School of Education, Department of Special Education-Guidance and Counselling bandaseli67@gmail.com LITERATURE REVIEW The Bloom’s Taxonomy Model is helpful to the teachers for thinking and analyzing their teaching and student’s learning. The framework is used to state clear objectives which can help the teachers to plan lessons accordingly. It also provides a framework for cognitive behaviors which can be applied to understand difficulty of tasks, conduct an assessment, and simplify or complicate the activities for students (Armstrong, 2010). However, not all teachers know how to use Bloom’s Taxonomy in preparing activities for their learners. Larson & Lockee (2019) explain that educators often find it challenging and tedious to develop learning objectives to describe cognitive skills at different levels of Bloom’s taxonomy. 6 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.4, 2023 ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.4, 2023 Li et. al. (2022) observed that some educators have difficulties in creating learning objectives applicable to the levels in Bloom’s taxonomy. They further indicated the need to reflect objectives in the advancement of learners’ skills with learning content as well as dependencies the concerning set intents. Similarly, Masapanta-Carri´on & Vel´azquez-Iturbide (2018), Mpolomoka, Banda & Dube (2017) and Mpolomoka, Muyangana, Banda, Dube, Mabenga, Kangwa & Muyoba (2016) observe difficulties that educators have in developing learning objectives appropriate to the levels specified in Bloom’s taxonomy. Teachers mostly make the mistake of assuming that learners who can remember facts can then also complete those more important higher order tasks. This partial understanding of the taxonomy limits cognitive development of learners because of lack of exposure to use of higher order thinking skills. Masapanta-Carrión, Velázquez-Iturbide & Ángel (2018) note another dimension of difficulties that teacher experience which relate to challenges faced in classifying content into specific levels of the taxonomy. Over dependance on acquisition of low level cognitive development knowledge and skills at the expense of higher level ones in teaching is problematic. The taxonomy is partially utilized which adversely affects development, intellectually. Some teachers were seen to have limited understanding of terminologies applicable in each level of the cognitive taxonomy. Masapanta-Carrión, Velázquez-Iturbide & Ángel (2018) propose to offer training to instructors in how to use it was proposed as a way of solving the problem of deficient use of the taxonomy. Furthermore, Akinboboye & Ayanwale (2021) urge teachers to set questions that based on Bloom’s taxonomy to ensure balancing cognitive skill development in learners. LITERATURE REVIEW g g p Bloom’s Taxonomy classifies learning into stages which range from the lowest to the highest levels. Adam (2015) explains that the taxonomy contains six categories of cognitive skills ranging from lower-order skills that require less cognitive processing to higher-order skills that require deeper learning and a greater degree of cognitive processing. It indicates these consecutive learning levels which are planned for learners to experience. A set of three hierarchical models is used to classify educational learning objectives into levels of complexity and specificity. The three lists cover the learning objectives in cognitive, affective and sensory domains. The cognitive domain list has been the primary focus of most traditional education and is frequently used to structure curriculum learning objectives, assessments and activities (Anderson et al, 2001). These levels can be helpful in developing learning outcomes because certain verbs are particularly appropriate at each level and not appropriate at other levels (though some verbs are useful at multiple levels). Figure 1: Bloom’s Taxonomy Cognitive Domain Source: Anderson (2001). Bloom’s Taxonomy leads to deeper learning and transfer of knowledge and skills to a greater variety of tasks and contexts (Adam, 2015). Levels of development in the cognitive domain are ordered from simple to complex in terms of thinking skills. Each level is defined and suitable verbs for learning outcomes are illustrated. Figure 1: Bloom’s Taxonomy Cognitive Domain Source: Anderson (2001). Source: Anderson (2001). Bloom’s Taxonomy leads to deeper learning and transfer of knowledge and skills to a greater variety of tasks and contexts (Adam, 2015). Levels of development in the cognitive domain are ordered from simple to complex in terms of thinking skills. Each level is defined and suitable verbs for learning outcomes are illustrated. Source: Anderson (2001). Bloom’s Taxonomy leads to deeper learning and transfer of knowledge and skills to a greater variety of tasks and contexts (Adam, 2015). Levels of development in the cognitive domain are ordered from simple to complex in terms of thinking skills. Each level is defined and suitable verbs for learning outcomes are illustrated. 7 Journal of Education and Practice www.iiste.org ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.4, 2023 Table 1: Bloom’s Taxonomy Cognitive Domain Level Definition Suitable Outcome Verbs Remember Retrieve, recall, recognize Cite, define Cite, define, describe, identify, label, list, match, name, outline, quote, recall, report, reproduce, retrieve, show, state, tabulate, and tell. Understand Demonstrating comprehension through different forms of explanation. LITERATURE REVIEW abstract, arrange, articulate, associate, categorize, clarify, classify, compare, compute, conclude, contrast, defend, diagram, differentiate, discuss, distinguish, estimate, exemplify, explain, extend, extrapolate, generalize, give examples of, illustrate, infer, interpolate, interpret, match, outline, paraphrase, predict, rearrange, reorder, rephrase, represent, restate, summarize, transform, and translate. Apply Use information or a skill in a new situation Apply, calculate, carry out, classify, complete, compute, demonstrate, dramatize, employ, examine, execute, experiment, generalize, illustrate, implement, infer, interpret, manipulate, modify, operate, organize, outline, predict, solve, transfer, translate, and use. Analyze Break material into its constituent parts and determine how the parts relate to one another and/or to an overall structure or purpose Analyze, arrange, break down, categorize, classify, compare, connect, contrast, deconstruct, detect, diagram, differentiate, discriminate, distinguish, divide, explain, identify, integrate, inventory, order, organize, relate, separate, and structure. Evaluate Make judgments based on criteria and standards Appraise, apprise, argue, assess, compare, conclude, consider, contrast, convince, criticize, critique, decide, determine, discriminate, evaluate, grade, judge, justify, measure, rank, rate, recommend, review, score, select, standardize, support, test, and validate. Create Put elements together to form a new coherent or functional whole; reorganize elements into a new pattern or structure Arrange, assemble, build, collect, combine, compile, compose, constitute, construct, create, design, develop, devise, formulate, generate, hypothesize, integrate, invent, make, manage, modify, organize, perform, plan, prepare, produce, propose, rearrange, reconstruct, reorganize, revise, rewrite, specify, synthesize, and write. T bl 1 b i th iti d i it l l d li bl b h i l b Ill t t d i th Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.4, 2023 www.iiste.org Table 1 above summarizes the cognitive domain, its levels and applicable behavioural verbs. Illustrated in the table, is the incremental learning that builds intellectual capacity of learners. Blooms taxonomy produced according to Nkhoma (2016) desired effects of incremental learning. Thus, Bloom’s taxonomy highlights the need for including learning objectives that require higher levels of cognitive skills that lead to deeper learning and transfer of knowledge and skills to a greater variety of tasks and contexts (Adam, 2015). Blooms taxonomy is indispensable to teaching and learning. It provides guidance on segmenting learning abilities expected to be achieved in each level. It is incremental because the levels are progressive from low to high in terms of cognitive development. LITERATURE REVIEW For this reason, teachers at all levels are supposed to be acquainted with knowledge and skills in how to apply the cognitive domain in their teaching. Reviewed literature shows that there some knowledge gaps in the use of the taxonomy. While some teachers partially know how to use the taxonomy, others are bereft of the required knowledge. Hypotheses Testing Hypotheses Testing HO1 - There is no relationship between use of objectives and learning outcomes in tasks prepared. HO2 - Use of Bloom’s Taxonomy in designing study courses was not associated to grouping of objectives when i l t t theses Testing There is no relationship between use of objectives and learning outcomes in tasks prepared. yp g HO1 - There is no relationship between use of objectives and learning outcomes in tasks prepared. HO2 - Use of Bloom’s Taxonomy in designing study courses was not associated to grouping of objectives when preparing lecture notes. p j g p p HO2 - Use of Bloom’s Taxonomy in designing study courses was not associated to grouping of objectives when preparing lecture notes. Theoretical Framework This study used Objectives-Oriented Evaluation Approach which focuses on determining the extent to which the purposes of a program are achieved. The objectives-oriented approach to evaluation is attributed to Ralph W. Tyler (1942, 1950) who conceptualized and popularized the focus on objectives in education. According to Tyler (1942) the goals and the objectives of a program must be defined as a prerequisite to evaluation. The objectives-oriented evaluation determines whether some or all of the program objectives are achieved and, if so, how well they are achieved. In education, the objectives are concerned with the purposes of a single lesson or training program or the knowledge students should attain during an entire year. Worthen and Sanders (1987) assert that objective- oriented approach is systematic, logical, scientifically acceptable and ready to use by evaluator. This study adopted objectives-oriented approach based on the assumption that objectives provide accurate and reliable information about the insights of the programme. The focus of this study was confined to objectives/questions and learning outcome statements of the program in relation to cognitive domain in Bloom’s Taxonomy. Focus was on the application of goals, objectives and intended outcomes of a program. The major question addressed in this kind of evaluation is. Statement of the problem Preparation of education activities is guided by models. Bloom’s Taxonomy is one of the models which underly creation of courses of study in terms of setting learning outcomes, objectives, topical activities and suitable tasks. Not all course instructors/lecturers are conversant with the use of Bloom’s Taxonomy. Absence of Taxonomy result in, course, outcomes/objectives, activities and materials being mismatched and in worse cases, underteaching course participants. It was not known how much lecturers acquainted themselves with the right Taxonomy for developing appropriate cognitive processes for the learners in the colleges. This study therefore investigated the application of bloom’s Taxonomy in categorization of cognitive process development in colleges in Zambia. mbia. ) Assess the application of Bloom’s Taxonomy in setting learning outcomes for the courses; ) pp y g g 2) Assess reflection of Bloom’s Taxonomy in activities set for course participants; 3) Ascertain significance attached to Bloom’s Taxonomy in courses of study designed for participant 8 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.4, 2023 ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.4, 2023 METHODOLOGY The study employed a mixed methodology in which a concurrent design was used to allow for collection of two types of data at the same time. A descriptive research design was used to assess application of the taxonomy in course outcomes/objective. Population targeted comprised college lecturers in Lusaka District. Convenience sampling was used to get the required sample of 36 respondents. p g g q p p Data were collected using a self-administered questionnaire containing closed and open-ended statements. The closed-ended part constituted the quantitative aspect of the study and it had 14 statements. Six statements dichotomous with a ‘yes’ or ‘no’ response. The remaining eight had Likert scale which was used to measure lecturers’ opinion towards use of objective and Bloom’s Taxonomy in their courses of study. Qualitatively, the other segment comprised open-ended statements. They are textual responses and generally used for qualitative analysis (Banda, Mpolomoka, Mbono & Sampa, 2017). The first part of the questionnaire comprised question items that assessed use of objectives. The second part assessed application of objectives and Bloom’s Taxonomy in implementation of course studies. Bloom’s Taxonomy underlies determination of objectives or learning outcomes for any lesson prepared for students. Close-ended part was quantifiable, analyzed using Spearman coefficient whereas the open-ended was thematically analyzed. Data were analyzed using frequencies for computing statistics for responses obtained from lecturers. Spearman rank correlation coefficient was applicable in analyzing relationship between variables concerning use of Bloom’s Taxonomy in preparing courses of study among lecturers. Therefore, we believe that the study would generate reliable data since it employed standardized and scientifically-accepted data collection tools. FINDINGS ngs are presented following key thematic areas derived from the objectives and the emerging issues. g p g y j g g The majority of the lecturers assessed showed that they hardly applied the sub-domains for ascertaining the type of suitable objective. Spearman coefficient correlation was used to analyze association of variables reflecting application of objectives, learning outcomes and tasks inherent in the courses of study lecturers offered in colleges. The findings showed weak correlations between the tested variables of interest. Application of Bloom’s Taxonomy in Setting Learning Outcomes Lecturers did not always apply Bloom’s Taxonomy when setting objectives for their teaching and learning notes. Notably, objectives were set according to the order of grasping knowledge and skills, cognitively. Evidently, verbs used in the objectives were sourced from the same level of the cognitive domain. Responses from participants in the study indicated non-compliant to application of Bloom’s Taxonomy in grouping objectives. Domains were not stated in the expression of their understanding of grouping objectives. Application of Bloom’s Taxonomy in Setting Objectives Application of Bloom’s Taxonomy in Setting Objectives pp y g j Course lecturers indicated that they always grouped objectives when preparing lecture notes. Lecturers agreed that they used Bloom’s Taxonomy in setting objectives/outcomes. They agreed that they grouped objectives from simple to complex. Most of the lecturers demonstrated use of active verbs when setting learning outcomes. They applied active 9 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.4, 2023 ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.4, 2023 verbs in objectives which they set for their lecture notes. They indicated that they always grouped objectives according to the applicable verbs in the given lessons. verbs in objectives which they set for their lecture notes. They indicated that they always grouped objectives according to the applicable verbs in the given lessons. Application of Bloom’s Taxonomy in Designing Courses Application of Bloom s Taxonomy in Designing Courses The majority of the lecturers showed that they used theoretical framework to guide them in grouping of learning outcomes for their courses. Responses from lecturers indicated conflicting views on the use of Blooms’ Taxonomy when setting learning outcomes. The majority of the lecturers indicated that they always used the Taxonomy to design study courses. However, when asked to show the domains they used in the Taxonomy, not all of them demonstrated the required cognitive levels. Out of the 36 lecturers, seven expressed all the correct levels, 10 partially did whereas the majority, 19 of them exhibited ignorance of the sought Bloom’s Taxonomy. The majority of the lecturers did not illustrate how to use Bloom’s Taxonomy in planning course of study, setting goals/objectives, creating learning activities or create assessment tasks for course participants. Of the 36 lecturers, three showed how to apply the cognitive levels in one of the given areas. The rest, 33 of them did not. This corroborates with what empirical literature reveals regarding cognitive-reflective engagements and the recommendation to enhance learning outcomes to mirror the revised Bloom and Solo’s Taxonomies (Simui, Mpolomoka, Sakakombe & Mhango, 2020; Garrison, Anderson & Archer, 2001). p g ) The lecturers indicated that they used Bloom’s Taxonomy partially in their handling of course of study delivery. While many of the lecturers found it useful in setting objectives, others used the Taxonomy to assess the teaching and learning process. Hypotheses Testing Hypotheses Testing Table 2: Relationship Between Objectives and Learning Outcomes in Tasks There is no relationship between use of objectives and learning outcomes in tasks prepared. Correlations I use learning outcomes as a basis for preparing tasks for the class I compare objectives with tasks prepared for students Spearman's rho I use learning outcomes as a basis for preparing tasks for the class Correlation Coefficient 1.000 .187 Sig. (2-tailed) . .274 N 36 36 I compare objectives with tasks prepared for students Correlation Coefficient .187 1.000 Sig. (2-tailed) .274 . N 36 36 Table above on Spearman rho r. .187 which shows the relationship between use of learning outcomes as a basis for preparing tasks and objectives prepared for students. The result of the analysis, r. .187 is indicative of a very weak relation between the variables. This means that lecturers’ use of learning outcomes as a basis for preparing learning tasks relate to their assertions of comparing objectives with tasks prepared for students was indicative of a weak relationship. The p- value less than the significance level (α = 0.01) for any correlation coefficients can reject the null hypothesis, and the correlation coefficients are considered statistically significant with 99% confidence level. The p-value of .274 shows that the variables are uncorrelated because it is more than .01. We therefore fail to reject the null hypothesis that there is no relationship between lecturers’ use of learning outcomes as a basis for preparing tasks for the class and their assertions of comparing objectives with tasks prepared for students. Hypotheses Testing Table 2: Relationship Between Objectives and Learning Outcomes in Tasks There is no relationship between use of objectives and learning outcomes in tasks prepared. Correlations I use learning outcomes as a basis for preparing tasks for the class I compare objectives with tasks prepared for students Spearman's rho I use learning outcomes as a basis for preparing tasks for the class Correlation Coefficient 1.000 .187 Sig. (2-tailed) . .274 N 36 36 I compare objectives with tasks prepared for students Correlation Coefficient .187 1.000 Sig. (2-tailed) .274 . N 36 36 yp g Table 2: Relationship Between Objectives and Learning Outcomes in Tasks There is no relationship between use of objectives and learning outcomes in tasks prepared. Correlations Table above on Spearman rho r. Reflection of Objectives Lecturers varied in the way they grouped objectives for their notes. The majority of the lecturers showed that they did not group the objectives in any way. Objectives were set without following any format. A few who grouped their objectives indicated that they used the order of simple to complex or vice versa. Out of 36 lecturers, two showed that they grouped objectives using Bloom’s Taxonomy through application of sub cognitive domains which are: Remembering, understanding, applying, analyzing, evaluating, creating. Grouping of objectives into lower and higher thinking skills varied among lecturers. While some of them followed the given order, others did not. Notably, out of 36 lecturers, 20 showed how to group objectives into lower to higher order thinking skills. Sixteen were not sure of how to group the objectives. Hypotheses Testing This revelation is contrary to the finding by Nkhoma, et al., (2017) which report on application of Bloom’s Taxonomy in music education as a framework for aligning learning bjectives, curriculum and assessment. ISCUSSIONS Journal of Education and Practice www.iiste.org ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.4, 2023 Table 3 Objectives and Learning Outcomes Correlations I group objectives when preparing lecture notes I use learning outcomes as a basis for preparing tasks for the class Spearman's rho I group objectives when preparing lecture notes Correlation Coefficient 1.000 .305 Sig. (2-tailed) . .071 N 36 36 I use learning outcomes as a basis for preparing tasks for the class Correlation Coefficient .305 1.000 Sig. (2-tailed) .071 . N 36 36 Table 3 above on Spearman rho r. .305 shows the relationship between grouping objectives when preparing lecture notes and use of learning outcomes as a basis for preparing tasks for the class. The result of the analysis indicates that r. .305 falls between .20 and .39 which is verbally interpreted as weak. This means that there was a weak association of lecturers’ grouping of objectives when preparing lecture notes and use of learning outcomes as a basis for preparing tasks for the class. The p- value of less than the significance level (α = 0.01) for any correlation coefficients can reject the null hypothesis and the correlation coefficients are considered statistically significant with 99% confidence level The Table 3 above on Spearman rho r. .305 shows the relationship between grouping objectives when preparing lecture notes and use of learning outcomes as a basis for preparing tasks for the class. The result of the analysis indicates that r. .305 falls between .20 and .39 which is verbally interpreted as weak. This means that there was a weak association of lecturers’ grouping of objectives when preparing lecture notes and use of learning outcomes as a basis for preparing tasks for the class. The p- value of less than the significance level (α = 0.01) for any correlation coefficients can reject the null hypothesis, and the correlation coefficients are considered statistically significant with 99% confidence level. The p-value of .071 shows that the variables are uncorrelated because the p-value of.187 is more than .01. Hypotheses Testing .095 N 36 36 I group objectives when preparing lecture notes Correlation Coefficient .282 1.000 Sig. (2-tailed) .095 . N 36 36 Table above shows Spearman rho r. .282 which is about the relationship between use Bloom's Taxonomy in designing study courses and grouping objectives when preparing lecture notes. The result of the analysis, r. .282 falls between .20 and .39 which is verbally interpreted as weak. This means that there was a weak correlation between lecturers’ use Bloom's Taxonomy in designing study courses and grouping objectives when preparing lecture notes. The p- value of more than the significance level (α = 0.01) for any correlation coefficients is considered statistically insignificant with 99% confidence level. The p-value of .095 shows that the variables are uncorrelated because it is more than .01. We therefore fail to reject the null hypothesis that there is no relationship between use Bloom's Taxonomy designing study courses and grouping objectives when preparing lecture notes among course lecturers. The p- value of more than the significance level (α = 0.01) for any correlation coefficients is considered statistically insignificant with 99% confidence level. The p-value of .095 shows that the variables are uncorrelated because it is more than .01. We therefore fail to reject the null hypothesis that there is no relationship between use Bloom's Taxonomy designing study courses and grouping objectives when preparing lecture notes among course lecturers. The hypothesis tests conducted showed results indicative of weak relationships among variables. Lecturers’ application of Bloom’s Taxonomy in their preparation of teaching and learning activities was insignificant manifested. The cognitive Taxonomy was not used as a basis for determining content of study courses neither were objectives used for ascertaining lecture notes. This revelation is contrary to the finding by Nkhoma, et al., (2017) which report on application of Bloom’s Taxonomy in music education as a framework for aligning learning objectives, curriculum and assessment. Lecturers’ responses were inconsistent in their use of Bloom’s Taxonomy depending on the nature of the Hypotheses Testing We therefore fail to reject the null hypothesis that there is no relationship between grouping objectives when preparing lecture notes and use learning outcomes as a basis for preparing tasks for the class among lecturers. g p p g g Use of Bloom’s Taxonomy in designing study courses was not associated to grouping of objectives when preparing lecture notes. p p g Table 4: Bloom’s Taxonomy and Objectives Correlations I use Bloom's Taxonomy to design study courses I group objectives when preparing lecture notes Spearman's rho I use Bloom's Taxonomy to design study courses Correlation Coefficient 1.000 .282 Sig. (2-tailed) . .095 N 36 36 I group objectives when preparing lecture notes Correlation Coefficient .282 1.000 Sig. (2-tailed) .095 . N 36 36 Table above shows Spearman rho r. .282 which is about the relationship between use Bloom's Taxonomy in designing study courses and grouping objectives when preparing lecture notes. The result of the analysis, r. .282 falls between .20 and .39 which is verbally interpreted as weak. This means that there was a weak correlation between lecturers’ use Bloom's Taxonomy in designing study courses and grouping objectives when preparing lecture notes. The p- value of more than the significance level (α = 0.01) for any correlation coefficients is considered statistically insignificant with 99% confidence level. The p-value of .095 shows that the variables are uncorrelated because it is more than .01. We therefore fail to reject the null hypothesis that there is no relationship between use Bloom's Taxonomy designing study courses and grouping objectives when preparing lecture notes among course lecturers. The hypothesis tests conducted showed results indicative of weak relationships among variables. Lecturers’ application of Bloom’s Taxonomy in their preparation of teaching and learning activities was insignificant manifested. The cognitive Taxonomy was not used as a basis for determining content of study courses neither were objectives used for ascertaining lecture notes. This revelation is contrary to the finding by Nkhoma, et al., (2017) which report on application of Bloom’s Taxonomy in music education as a framework for aligning learning objectives, curriculum and assessment. Table 4: Bloom’s Taxonomy and Objectives Table 4: Bloom’s Taxonomy and Objectives Correlations I use Bloom's Taxonomy to design study courses I group objectives when preparing lecture notes Spearman's rho I use Bloom's Taxonomy to design study courses Correlation Coefficient 1.000 .282 Sig. (2-tailed) . Hypotheses Testing .187 which shows the relationship between use of learning outcomes as a basis for preparing tasks and objectives prepared for students. The result of the analysis, r. .187 is indicative of a very weak relation between the variables. This means that lecturers’ use of learning outcomes as a basis for preparing learning tasks relate to their assertions of comparing objectives with tasks prepared for students was indicative of a weak relationship. p The p- value less than the significance level (α = 0.01) for any correlation coefficients can reject the null hypothesis, and the correlation coefficients are considered statistically significant with 99% confidence level. The p-value of .274 shows that the variables are uncorrelated because it is more than .01. We therefore fail to reject the null hypothesis that there is no relationship between lecturers’ use of learning outcomes as a basis for preparing tasks for the class and their assertions of comparing objectives with tasks prepared for students. The p- value less than the significance level (α = 0.01) for any correlation coefficients can reject the null hypothesis, and the correlation coefficients are considered statistically significant with 99% confidence level. The p-value of .274 shows that the variables are uncorrelated because it is more than .01. We therefore fail to reject the null hypothesis that there is no relationship between lecturers’ use of learning outcomes as a basis for preparing tasks for the class and their assertions of comparing objectives with tasks prepared for students. 10 urnal of Education and Practice www.iiste.org SN 2222-1735 (Paper) ISSN 2222-288X (Online) ol.14, No.4, 2023 able 3 Objectives and Learning Outcomes Correlations I group objectives when preparing lecture notes I use learning outcomes as a basis for preparing tasks for the class Spearman's rho I group objectives when preparing lecture notes Correlation Coefficient 1.000 .305 Sig. (2-tailed) . .071 N 36 36 I use learning outcomes as a basis for preparing tasks for the class Correlation Coefficient .305 1.000 Sig. (2-tailed) .071 . N 36 36 Table 3 above on Spearman rho r. .305 shows the relationship between grouping objectives when preparing cture notes and use of learning outcomes as a basis for preparing tasks for the class. The result of the analysis ndicates that r. .305 falls between .20 and .39 which is verbally interpreted as weak. Hypotheses Testing This means that there was a weak association of lecturers’ grouping of objectives when preparing lecture notes and use of learning outcomes s a basis for preparing tasks for the class. The p- value of less than the significance level (α = 0.01) for any correlation coefficients can reject the null ypothesis, and the correlation coefficients are considered statistically significant with 99% confidence level. The -value of .071 shows that the variables are uncorrelated because the p-value of.187 is more than .01. We therefore ail to reject the null hypothesis that there is no relationship between grouping objectives when preparing lecture otes and use learning outcomes as a basis for preparing tasks for the class among lecturers. Use of Bloom’s Taxonomy in designing study courses was not associated to grouping of objectives when reparing lecture notes. able 4: Bloom’s Taxonomy and Objectives Correlations I use Bloom's Taxonomy to design study courses I group objectives when preparing lecture notes Spearman's rho I use Bloom's Taxonomy to design study courses Correlation Coefficient 1.000 .282 Sig. (2-tailed) . .095 N 36 36 I group objectives when preparing lecture notes Correlation Coefficient .282 1.000 Sig. (2-tailed) .095 . N 36 36 Table above shows Spearman rho r. .282 which is about the relationship between use Bloom's Taxonomy in esigning study courses and grouping objectives when preparing lecture notes. The result of the analysis, r. .282 alls between .20 and .39 which is verbally interpreted as weak. This means that there was a weak correlation etween lecturers’ use Bloom's Taxonomy in designing study courses and grouping objectives when preparing cture notes. The p- value of more than the significance level (α = 0.01) for any correlation coefficients is considered atistically insignificant with 99% confidence level. The p-value of .095 shows that the variables are uncorrelated ecause it is more than .01. We therefore fail to reject the null hypothesis that there is no relationship between use loom's Taxonomy designing study courses and grouping objectives when preparing lecture notes among course cturers. The hypothesis tests conducted showed results indicative of weak relationships among variables. Lecturers’ pplication of Bloom’s Taxonomy in their preparation of teaching and learning activities was insignificant manifested. The cognitive Taxonomy was not used as a basis for determining content of study courses neither were bjectives used for ascertaining lecture notes. ecturers’ responses were inconsistent in their use of Bloom’s Taxonomy depending on the nature o DISCUSSIONS Lecturers’ responses were inconsistent in their use of Bloom’s Taxonomy depending on the nature of the 11 Journal of Education and Practice www.iiste.org Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.4, 2023 ste.org ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.4, 2023 assessment given. They showed agreement in most parts of the questionnaire which needed a yes/no response. However, when asked to show the domains if they used the Taxonomy, not all of them demonstrated the required cognitive levels. Most of the lecturers did not show the application of the Taxonomy when asked to illustrate. Agreeing with the use of the Taxonomy and showing its applicability, did not correlate. The lecturers agreed to what they did not know which means they did not know the Taxonomy and its application in their work. assessment given. They showed agreement in most parts of the questionnaire which needed a yes/no response. However, when asked to show the domains if they used the Taxonomy, not all of them demonstrated the required cognitive levels. Most of the lecturers did not show the application of the Taxonomy when asked to illustrate. Agreeing with the use of the Taxonomy and showing its applicability, did not correlate. The lecturers agreed to what they did not know which means they did not know the Taxonomy and its application in their work. Despite agreeing that they used Bloom’s Taxonomy in their teaching and learning activities, lecturers failed to illustrate how they applied it in the preparation of courses and lecture notes. Lecturers varied in the way they grouped objectives for their notes. While some of them followed a format from simple to complex, others grouped them without any format followed. The majority of the lecturers showed that they did not group the objectives in any way. Although lecturers agreed that they used the Taxonomy, most of them failed to demonstrate the application of the required cognitive levels. The majority of the lecturers assessed showed that they hardly applied the sub- domains for ascertaining the type of suitable objectives. Responses from many of the participants in the study indicated non-compliant to application of Bloom’s Taxonomy in grouping objectives. Domains were not stated in the expression of their understanding of grouping objectives. Suggestions for further Study gg y There is need for a research in determining application of verbs used in cognitive domain in programmes offered in colleges. RECOMMENDATION There is need to orient college lecturers on the importance of use of learning objective and outcomes based on known models of which Bloom’s Taxonomy is one. This can be done through in-house trainings and other staff development processes. CONCLUSION Lecturer’s affirmation that they used objectives and Bloom’s Taxonomy in preparatory activities of their courses was inconsistent with textural responses and tested hypotheses. There was no relationship in the lecturer’s use of Bloom’s Taxonomy when preparing study courses and grouping of objectives for lecture notes. There is no relationship between lecturers’ use of learning outcomes as a basis for preparing class tasks and their assertions that they compared objectives with tasks prepared for students. Lecturers should prepare courses of study and learning activities that include all cognitive levels in the domain. This is because Bloom’s Taxonomy should serve as a basis for setting objectives and related activities. DISCUSSIONS The majority of the lecturers did not illustrate how to use Bloom’s Taxonomy in planning course of study, setting goals/objectives, creating learning activities or create assessment tasks for course participants. Chandio, Pandhaini & Igbal (2016) attest to failure to apply Bloom’s Taxonomy to lack of training of people involved in handling the cognitive domains levels. Some of the lecturers indicated that they used Bloom’s Taxonomy partially in their handling of course of study delivery. Lecturers’ use of learning outcomes as a basis for preparing did not relate to their assertions of comparing objectives with tasks prepared for students. The researchers failed to reject the null hypothesis that there is no relationship between lecturers’ use of learning outcomes as a basis for preparing class tasks and their assertions that they compared objectives with tasks prepared for students. This means that lecturers did not use Bloom’s Taxonomy to design study courses and grouping objectives when preparing lecture notes. This study contributes to strengthening the use of taxonomies as bases for preparing courses and lecture notes. This is because Bloom’s Taxonomy is termed as a pathway that guides the learning process. p g p g y Banda, S., Mpolomoka, D.L., Mbono, D. & Sampa, R.L. (2017). Use of questions in qualitative research: How questions guided our study. International Journal of Development Research, 7, (12). ; Akinboboye, J. & Musa, Ayanwale, Musa. (2021). Bloom Taxonomy Usage and Psychometric Analysis of Classroom Teacher-Made Test. Anderson, Lorin W. & David, R. Krathwohl, (eds.) (2001). A Taxonomy for Learning, Teaching, and Assessing: A Revision of Bloom's Taxonomy of Educational Objectives. New York: Addison Wesley Longman, Inc Armstrong, P. (2010) Bloom’s Taxonomy. Vanderbilt University Center for Teaching, Nashville. https://cft.vanderbilt.edu/guides-sub-pages/blooms-taxonomy Adams, N.E. (2015, July). Bloom's taxonomy of cognitive learning objectives. J Med Libr Assoc. 103(3):152-3. doi: 10.3163/1536-5050.103.3.010. PMID: 26213509; PMCID: PMC4511057. T.M., Pandhaini, M.S. & Igbal, R. (2016). Bloom’s Taxonomy: Improving Assessment and Teaching- REFERENCES Adams, N.E. (2015, July). Bloom's taxonomy of cognitive learning objectives. J Med Libr Assoc. 103(3):152-3. doi: 10.3163/1536-5050.103.3.010. PMID: 26213509; PMCID: PMC4511057. Akinboboye, J. & Musa, Ayanwale, Musa. (2021). Bloom Taxonomy Usage and Psychometric Analysis of Classroom Teacher-Made Test. Anderson, Lorin W. & David, R. Krathwohl, (eds.) (2001). A Taxonomy for Learning, Teaching, and A A Revision of Bloom's Taxonomy of Educational Objectives. New York: Addison Wesley Longman A Revision of Bloom's Taxonomy of Educational Objectives. New York: Addison Wesley Longman, Inc Armstrong, P. (2010) Bloom’s Taxonomy. Vanderbilt University Center for Teaching, Nashville. https://cft.vanderbilt.edu/guides-sub-pages/blooms-taxonomy f y f j y g , Armstrong, P. (2010) Bloom’s Taxonomy. Vanderbilt University Center for Teaching, Nashville. https://cft.vanderbilt.edu/guides-sub-pages/blooms-taxonomy p g p g y Banda, S., Mpolomoka, D.L., Mbono, D. & Sampa, R.L. (2017). Use of questions in qualitative research: How questions guided our study. International Journal of Development Research, 7, (12). q g y f p , , ( ) Bhandari, P. & Nikolopoulou, K. (2022, October 20). What Is a Likert Scale? | Guide & Examples. Scribbr. Retrieved November 3, 2022, from https://www.scribbr.com/methodology/likert-scale/ Bhandari, P. & Nikolopoulou, K. (2022, October 20). What Is a Likert Scale? | Guide & Examples. Scribbr. Retrieved November 3, 2022, from https://www.scribbr.com/methodology/likert-scale/ Chandio, T.M., Pandhaini, M.S. & Igbal, R. (2016). Bloom’s Taxonomy: Improving Assessment and Teaching- Bhandari, P. & Nikolopoulou, K. (2022, October 20). What Is a Likert Scale? | Guide & Examples. Scribbr. Retrieved November 3, 2022, from https://www.scribbr.com/methodology/likert-scale/ Chandio, T.M., Pandhaini, M.S. & Igbal, R. (2016). Bloom’s Taxonomy: Improving Assessment and Teaching- 12 Journal of Education and Practice www.iiste.org Learning Process. Journal of Education and Educational Development, 3(2), pg. 202-221. Garrison, D.R., Anderson, T. and Archer, W. (2001). Critical thinking and computer conferencing: A model an l i i A i J l f Di Ed i 15(1) 7 23 Learning Process. Journal of Education and Educational Development, 3(2), pg. 202-221. Learning Process. Journal of Education and Educational Development, 3(2), pg. 202-221. G i D R A d T d A h W (2001) C iti l thi ki d t f i A d l d Garrison, D.R., Anderson, T. and Archer, W. (2001). Critical thinking and computer conferencing: A m tool to assess cognitive presence. American Journal of Distance Education, 15(1), 7-23. Gluga, R., Kay, J., Lister, R. Simon & Kleitman. S. (2013). Mastering cognitive development theory in computer science education. Computer Science Education, 23(1):24–57. REFERENCES Iffat, N. & Tithi, U.M. (2018). Reflection of Bloom’s Taxonomy in the Learning, outcomes of Secondary Social Science Curriculum of Bangladesh. International Journal of Science and Research (IJSR), India Online ISSN: 2319-7064 Larson, B.M. & Lockee, B.B., (2019). Streamlined ID: A practical guide to instructional design. Routledge. Masapanta-Carrión, S. & Velázquez-Iturbide, J.A. (2018). A Systematic Review of the Use of Bloom’s Taxonomy in Computer Science Education. 441-446. Mpolomoka, D.L., Banda, S. & Dube, M. (2017). Invigorating the Teaching Profession for the 21stCentury in Zambia. Pyrex Journal of Educational Research and Reviews, 3(4), 40-43. Mpolomoka, D.L., Muyangana, A., Banda, S., Dube, M., Mabenga, M., Kangwa, K.N. & Muyoba, L. (2016). Teaching Experience of Student-Teachers in ODL Tertiary Institutions in Zambia. International Open & Distance Learning Journal 2016 Special Issue, pp. 48-55 g p pp Accessible on:http://www.iodlj.zou.ac.zw/ejournal/index.php/journal/issue/view/15/showToc Nkhoma, Z.M., Lam, K.T., Sriratanaviriyakul, N., Richardson, J., Kam, B. & Lau, H.K. (2017). Unpacking the revised Bloom’s Taxonomy: developing case-based learning activities. Education + Training, 59(3), pp.250- 264, https://doi.org/10.1108/ ET-03-2016-0061 , p g Robyn, E. (2014). The structure of the cognitive process dimension in the revised Taxonomy. Bloom's Taxonomy. Denver, Co: Expert Beacon. Retrieved from https://expertbeacon. com/blooms Taxonomy/#. XAap FtszaM9 Rentmeester, C. (2017). Adding academic rigor to introductory ethics courses using Bloom’s Taxonomy. International Journal of Ethics Education. [Online]. Available at: https://philpapers org/archive/RENAAR pdf [Accessed 17/03/2018] Semsar, K. & Casagrand, J. (2017). Bloom’s dichotomous key: a new tool for evaluating the cognitive difficulty of assessments. Advanced Physical Education, 41,170-177. Simui, F., Mpolomoka, D., Sakakombe, L. & Mhango, L. (2020). Exploring Presence of Interactivity in Distance Education Instructional Materials Using the Community of Inquiry Model at Four Universities in Zambia. Special Issue of Southern African Universities Learning and Teaching Forum (SAULT) in Zambian Journal of Educational Management, Administration and Leadership (ZJEMAL), 1(1), 202-222 Stanny, C.J. (2016). Reevaluating Bloom’s Taxonomy: What measurable verbs can and cannot say about student learning. Education Sciences, 6(37), 1–12. Worthen, J.R. & Sanders, R.J. (1987). Educational evaluation: Alternative approaches and practical guidelines. Longman: Publisher. 13
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Vertical Profiles of Pollution Particle Concentrations in the Boundary Layer above Paris (France) from the Optical Aerosol Counter LOAC Onboard a Touristic Balloon
Sensors
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10,492
Article Jean-Baptiste Renard 1,*, Vincent Michoud 2 and Jérôme Giacomoni 3 1 LPC2E, CNRS / Université d’Orléans, 45071 Orléans CEDEX 2, France 2 LISA, CNRS / Université Paris-Est-Créteil, Université de Paris, Institut Pierre Simon Laplace (IPSL), 94010 Créteil CEDEX, France; Vincent.Michoud@lisa.u-pec.fr 3 Aerophile SAS, 75015 Paris, France; giacomoni@aerophile.com * Correspondence: jean-baptiste.renard@cnrs-orleans.fr; Tel.: + 33-6-3291-7742 Jean-Baptiste Renard 1,*, Vincent Michoud 2 and Jérôme Giacomoni 3 Jean-Baptiste Renard 1,*, Vincent Michoud 2 and Jérôme Giacomoni 3 1 LPC2E, CNRS / Université d’Orléans, 45071 Orléans CEDEX 2, France 2 LISA, CNRS / Université Paris-Est-Créteil, Université de Paris, Institut Pierre Simon Laplace (IPSL), 94010 Créteil CEDEX, France; Vincent.Michoud@lisa.u-pec.fr 3 Aerophile SAS, 75015 Paris, France; giacomoni@aerophile.com * Correspondence: jean-baptiste.renard@cnrs-orleans.fr; Tel.: + 33-6-3291-7742 Received: 21 December 2019; Accepted: 15 February 2020; Published: 18 February 2020 Abstract: Atmospheric pollution by particulate matter represents a significant health risk and needs continuous monitoring by air quality networks that provide mass concentrations for PM10 and PM2.5 (particles with diameter smaller than 10 µm and 2.5 µm, respectively). We present here a new approach to monitor the urban particles content, using six years of aerosols number concentration measurements for particles in the 0.2−50 µm size range. These measurements are performed by the Light Optical Aerosols Counter (LOAC) instrument onboard the tethered touristic balloon “Ballon de Paris Generali”, in Paris, France. Such measurements have allowed us first to detect at ground a seasonal variability in the particulate matter content, due to the origin of the particles (anthropogenic pollution, pollens), and secondly, to retrieve the mean evolution of particles concentrations with height above ground up to 150 m. Measurements were also conducted up to 300 m above ground during major pollution events. The vertical evolution of concentrations varies from one event to another, depending on the origin of the pollution and on the meteorological conditions. These measurements have shown the interest of performing particle number concentrations measurements for the air pollution monitoring in complement with regulatory mass concentrations measurement, to better evaluate the intensity of the pollution event and to better consider the effect of smallest particles, which are more dangerous for human health. Keywords: particulate matter; urban pollution; number concentrations; tethered balloon sensors sensors sensors Sensors 2020, 20, 1111; doi:10.3390/s20041111 www.mdpi.com/journal/sensors 1. Introduction Atmospheric pollution by particulate matter (PM) is a growing concern, particularly in urban environments that concentrate a large portion of the population and the particle’s emission sources. These particles can be primary, directly coming from natural sources (dusts, salts, pollen) and from anthropogenic sources (transport, heating, industries, agriculture) or secondary, coming from chemical reactions involving sun light or atmospheric oxidants. Such particles represent a significant health risk [1–3]. The PM 10 fraction (particles with aerodynamic diameter smaller than 10 µm) can penetrate beyond the nasopharyngeal tract in the bronchi and up to the pulmonary alveoli. The smaller particles, below 1 µm, can diffuse in the body and be found in various human organs [4–6]. The smaller the particles are, the deeper they can penetrate and diffuse in the body. Thus, detecting and counting the submicronic pollution particles is a major area of interest for public health. Exceeding the WHO (World Health Organization) guideline values Sensors 2020, 20, 1111; doi:10.3390/s20041111 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors 2 of 15 Sensors 2020, 20, 1111 for PM2.5 (particles with aerodynamic diameter smaller than 2.5 µm) of 10 µg·m−3 is the cause for about 20,000 premature deaths or more each year in major European cities [7]. In particular, it has been estimated that if these standards would have been reached it would have resulted in a gain of six months of life expectancy for more than 11 million inhabitants of the Paris region [8]. The Paris region is characterized by relatively few industries. At a regional scale, the urban background conditions are driven by long-distance transport of pollutants, accounting for 70% in average of PM2.5 mass-concentration [9–13]. Local sources of primary particles are dominated in mass by traffic emission and by residential heating in winter, as in many other urban environments [14,15]. The spatial distribution of particles concentrations at the surface can strongly differ at the urban scale, also depending on air-mass dispersion constrained by the urban topography. At ground, the PM content is continuously monitored by air quality networks, which provide the mass concentrations of PM10 using microbalances or similar instruments. Some stations can also provide an estimate of PM2.5 mass concentrations. In Paris and the “Ile de France” region (which includes the city of Paris) these regulatory measurements are conducted by Airparif [16]. 1. Introduction However, the techniques deployed across the air quality monitoring networks are by law oriented toward the measurement of the aerosol mass concentration, while it is known that the smallest particles, which penetrate deeper into the human organism, mostly dominate the number concentration and contribute only weakly to the mass. Then, complementary measurements providing particle number concentrations in addition to mass concentrations can be proposed to monitor the urban particle content and better understand its distribution and dynamics. Vertical profiles measurements of the aerosols content above major cities, performed during specific campaigns, can be used to better understand the vertical transport and the dispersion of the particles [17–19]. The aim of this paper is to present six years of aerosols number concentration measurements performed by the Light Optical Aerosols Counter (LOAC) instrument onboard a tethered touristic balloon in Paris. Such conditions of measurements allow us to better determine the mean seasonal variations of particles content for different size classes, and also to evaluate the mean vertical evolution of particles and to focus on some specific pollution events. e u e o u ie o u a ae o o 3 M C di i h T i i “B ll d P i G li” i P i (F ) 3. Measurements Conditions at the Touristic “Ballon de Paris Generali” in Paris (France) 3. Measurements Conditions at the Touristic Ballon de Paris Generali in Paris (France) The LOAC has been mounted on the gondola of the touristic tethered balloon “Ballon de Paris Generali” (Figure 2) in the park “André Citroën” in the south-west of Paris, France (48.8414°N, 2.2740°E), since mid-2013 and works continuously [24]. Depending on the meteorological conditions, 150 to 200 days per year are favorable for flying. The height is measured by a GPS, with a vertical accuracy of about ±15 m. The balloon nominal maximum height above ground is 150 m and up to 50 flights can be performed per day. Some flights can also be conducted up to a height of 300 m when the wind speed is very low (< few m·s−1). The urban pollution events occur mainly during anticyclonic The LOAC has been mounted on the gondola of the touristic tethered balloon “Ballon de Paris Generali” (Figure 2) in the park “André Citroën” in the south-west of Paris, France (48.8414◦N, 2.2740◦E), since mid-2013 and works continuously [24]. Depending on the meteorological conditions, 150 to 200 days per year are favorable for flying. The height is measured by a GPS, with a vertical accuracy of about ±15 m. The balloon nominal maximum height above ground is 150 m and up to 50 flights can be performed per day. Some flights can also be conducted up to a height of 300 m when the wind speed is very low (< few m·s−1). The urban pollution events occur mainly during anticyclonic conditions when the wind speed is low; thus, the balloon can often fly during such events. conditions when the wind speed is low; thus, the balloon can often fly during such events. Due to the balloon location in one of the largest parks in Paris with no main highways in the direct vicinity, the ground-based measurements can be referred as background urban conditions (Figure 3). The highest buildings close to the park have a height below 50 m. Therefore, we can expect to measure mean persistent urban pollution when the balloon height is above 50 m. Nevertheless, these measurements are conducted just at one location in Paris, thus the results presented below Due to the balloon location in one of the largest parks in Paris with no main highways in the direct vicinity, the ground-based measurements can be referred as background urban conditions (Figure 3). 2. The Light Optical Aerosols Counter (LOAC) The LOAC is a prototype instrument developed for ground and balloon-based measurements [20]. Particles are drawn up to the optical chamber through an isostatic tube by a small pump and cross a laser beam working at 650 nm. The scattered light is recorded by two photodiodes at scattering angles of ~15◦and ~65◦, and photons travel directly to the photodiodes though pipes without a lens (Figure 1, updated from [21]). A total of 19 size classes are defined in the particle diameter range between 0.2 and ~50 µm. The size classes are chosen as a good compromise between the instrument sensitivity and the expected size distribution of ambient air particles. For a 10 min integration time, the uncertainty of total concentrations is about ±20% for concentrations higher than 10 particle·cm−3 up to ±60% for concentrations lower than 10−1 particle·cm−3. The uncertainties in the size determination is of ±0.025 µm for particles smaller than 0.6 µm, 5% for particles in the 0.7−2 µm range, and of 10% for particles greater than 2 µm. g The measurements at 15◦are almost insensitive to the refractive index of the irregular shaped aerosol particles and can be used to determine the concentrations [22]. On the other hand, measurements at side-scattering around 65◦are very sensitive to the refractive index of the irregular particles [23]. An indication of the typology of such particles can be obtained using a “speciation index”, retrieved by combining the 15◦and 65◦channels measurements. This index is sensitive to the imaginary part of the refractive index of the particles, and thus to their optical absorbing properties. Laboratory references for the speciation indexes have been determined with LOAC for 4 natures of particles: carbonaceous, mineral dust, salts, and liquid droplet [20]. The speciation indexes obtained from LOAC observations in the ambient air are compared to these laboratory data to derive the estimated dominant typology of particles in different size classes. The identification of the nature of the particles works well in case of a 3 of 15 3 f 15 Sensors 2020, 20, 1111 homogeneous medium but is more questionable in case of a heterogeneous medium that cause the speciation index to be more scattered. homogeneous medium but is more questionable in case of a heterogeneous medium that cause the speciation index to be more scattered. Figure 1. 2. The Light Optical Aerosols Counter (LOAC) Light Optical Aerosols Counter (LOAC) principle of measurements at two scattering angles (updated from Renard et al 2018) Figure 1. Light Optical Aerosols Counter (LOAC) principle of measurements at two scattering angles (updated from Renard et al., 2018). Figure 1. Light Optical Aerosols Counter (LOAC) principle of measurements at two scattering angles ( d t d f R d t l 2018) Figure 1. Light Optical Aerosols Counter (LOAC) principle of measurements at two scattering angles (updated from Renard et al., 2018). Finally, the concentrations can be converted to mass concentrations (in μg·m−3) assuming spherical particles and mass density between 1.2 and 2.2 g·cm−3 depending on the detected typology of the particles. For carbonaceous particles smaller than 1 μm, the mass density is assumed to be 2.0 g·cm−3, while the density is assumed to be of 1.2 g·cm−3 for particles greater than 1 m. For mineral particles the density is assumed to be of 2.2 g·cm−3; in case of no identification, the density is assumed to be of 2.0 g·cm−3 (these values are updated from [20]). The error bars are calculated considering the uncertainties in size determination. The LOAC average mass-concentrations accuracy is of about 5 3 h d i b l i l b [20] Finally, the concentrations can be converted to mass concentrations (in µg·m−3) assuming spherical particles and mass density between 1.2 and 2.2 g·cm−3 depending on the detected typology of the particles. For carbonaceous particles smaller than 1 µm, the mass density is assumed to be 2.0 g·cm−3, while the density is assumed to be of 1.2 g·cm−3 for particles greater than 1 m. For mineral particles the density is assumed to be of 2.2 g·cm−3; in case of no identification, the density is assumed to be of 2.0 g·cm−3 (these values are updated from [20]). The error bars are calculated considering the uncertainties in size determination. The LOAC average mass-concentrations accuracy is of about ±5 µg·m−3 when compared to microbalance measurements in laboratory [20]. ±5 μg·m−3 when compared to microbalance measurements in laboratory [20]. The performances of LOAC have been established during numerous sessions of inter-comparison with other instruments dedicated to the counting, size distribution, extinctions, and mass concentrations of solid aerosols in the atmosphere [20]. 2. The Light Optical Aerosols Counter (LOAC) These sessions have shown that LOAC can The performances of LOAC have been established during numerous sessions of inter-comparison with other instruments dedicated to the counting, size distribution, extinctions, and mass concentrations of solid aerosols in the atmosphere [20]. These sessions have shown that LOAC can be used for studies on urban aerosols. e u e o u ie o u a ae o o 3 M C di i h T i i “B ll d P i G li” i P i (F ) 3. Measurements Conditions at the Touristic “Ballon de Paris Generali” in Paris (France) The highest buildings close to the park have a height below 50 m. Therefore, we can expect to measure mean persistent urban pollution when the balloon height is above 50 m. Nevertheless, these measurements are conducted just at one location in Paris, thus the results presented below could be not representative of the general picture. could be not representative of the general picture. LOAC provides measurements every 10 s. During the balloon flight, data must be integrated over at least 30 s to reduce the measurement noise. Since the balloon ascent speed is of 1 m·s−1, the vertical resolution for an individual LOAC concentrations profile is of about 30 m. Individual profiles can be also daily averaged to reduce the natural dispersion of the concentration’s measurements. For measurements at ground, the data can be averaged over 10 min to reduce the dispersion of the measurements. 4 of 15 4 f 15 Sensors 2020, 20, 1111 O Figure 2. (a) LOAC mounted on the gondola of the “Ballon de Paris Generali”; (b) The balloon in the park André Citroën (Paris, France) Figure 2. (a) LOAC mounted on the gondola of the “Ballon de Paris Generali”; (b) The balloon in the park André Citroën (Paris, France). Figure 2. (a) LOAC mounted on the gondola of the “Ballon de Paris Generali”; (b) The balloon in the park André Citroën (Paris France) Figure 2. (a) LOAC mounted on the gondola of the “Ballon de Paris Generali”; (b) The balloon in the park André Citroën (Paris France) Figure 2. (a) LOAC mounted on the gondola of the “Ballon de Paris Generali”; (b) The balloon in the park André Citroën (Paris, France). Figure 2. (a) LOAC mounted on the gondola of the “Ballon de Paris Generali”; (b) The balloon in the k A d é Cit ë (P i F ) Figure 3. Map of the vicinity of the “Parc André Citroën” (the green dot represents the balloon location, map from mappy.com). Figure 3. Map of the vicinity of the “Parc André Citroën” (the green dot represents the balloon location, map from mappy.com). Figure 3. Map of the vicinity of the “Parc André Citroën” (the green dot represents the balloon location, map from mappy.com). Figure 3. Map of the vicinity of the “Parc André Citroën” (the green dot represents the balloon location, map from mappy.com). Figure 3. 4. Measurements at Ground 4. Measurements at Ground Temporal evolution of number concentrations for the 19 LOAC size classes when the balloon is at ground (the data have been smoothed by a sliding smoothing procedure with a width of 2 days). Figure 4. Temporal evolution of number concentrations for the 19 LOAC size classes when the balloon is at ground (the data have been smoothed by a sliding smoothing procedure with a width of 2 days). The PM10 mass concentrations obtained by the Airparif air quality network for a station in the suburb of Paris (Vitry, background urban conditions, 48.7778°N, 2.3779°E) and for a station in a rural area in the south of Paris region (Rural South, 48.3667°N, 2.2333°E) also show the concentrations enhancements during pollution events (Figure 5). The mass concentrations derived from the LOAC measurements are also plotted in Figure 5 and are globally in good agreement with the Airparif measurements (all the data have been also smoothed with the same procedure as for LOAC number concentrations). Excluding the 2013 period with the building activities close to the park, the mean LOAC mass concentrations measurements are 6.5 μg·m−3 and 0.5 μg·m−3 lower than the Vitry and Rural South stations with a standard deviation of 10 μg·m−3 and 9 5 μg·m−3 respectively The PM10 mass concentrations obtained by the Airparif air quality network for a station in the suburb of Paris (Vitry, background urban conditions, 48.7778◦N, 2.3779◦E) and for a station in a rural area in the south of Paris region (Rural South, 48.3667◦N, 2.2333◦E) also show the concentrations enhancements during pollution events (Figure 5). The mass concentrations derived from the LOAC measurements are also plotted in Figure 5 and are globally in good agreement with the Airparif measurements (all the data have been also smoothed with the same procedure as for LOAC number concentrations). Excluding the 2013 period with the building activities close to the park, the mean LOAC mass concentrations measurements are 6.5 µg·m−3 and 0.5 µg·m−3 lower than the Vitry and Rural South stations, with a standard deviation of 10 µg·m−3 and 9.5 µg·m−3 respectively. Rural South stations, with a standard deviation of 10 μg·m 3 and 9.5 μg·m 3 respectively. When considering the six years of measurements, a seasonal cycle seems to be present for the particles smaller than about 1 μm, with maximum concentrations in winter and minimum concentrations in summer (Figure 6). 4. Measurements at Ground 4. Measurements at Ground Figure 4 presents the 2013−2019 evolution of the particle number concentrations with time for the 19 size classes of LOAC when the balloon is at ground and outside major LOAC and/or balloon maintenance period. Data during fog and heavy rain events have been removed because of the presence of droplets that can skew the retrieval of the solid particles. For clarity reason, the data have been smoothed by a sliding smoothing procedure with a width of 2 days. The highest concentrations corresponding to strong pollutions events were recorded at the beginning of winter 2013, and at the beginning of 2014 and 2015. It can be noticed that the mean level of concentrations for particles smaller than 10 µm measured by LOAC is higher in the 2013 period than after (Figure 4), due to strong building activities close to the Parc André Citroën (unfortunately no Airparif station is close to this location to validate these observations). Figure 4 presents the 2013−2019 evolution of the particle number concentrations with time for the 19 size classes of LOAC when the balloon is at ground and outside major LOAC and/or balloon maintenance period. Data during fog and heavy rain events have been removed because of the presence of droplets that can skew the retrieval of the solid particles. For clarity reason, the data have been smoothed by a sliding smoothing procedure with a width of 2 days. The highest concentrations corresponding to strong pollutions events were recorded at the beginning of winter 2013, and at the beginning of 2014 and 2015. It can be noticed that the mean level of concentrations for particles smaller than 10μm measured by LOAC is higher in the 2013 period than after (Figure 4), due to strong building activities close to the Parc André Citroën (unfortunately no Airparif station is close to this location to validate these observations). Figure 4. Temporal evolution of number concentrations for the 19 LOAC size classes when the balloon is at ground (the data have been smoothed by a sliding smoothing procedure with a width of 2 days). Figure 4. Temporal evolution of number concentrations for the 19 LOAC size classes when the balloon is at ground (the data have been smoothed by a sliding smoothing procedure with a width of 2 days). Figure 4. e u e o u ie o u a ae o o 3 M C di i h T i i “B ll d P i G li” i P i (F ) 3. Measurements Conditions at the Touristic “Ballon de Paris Generali” in Paris (France) Map of the vicinity of the “Parc André Citroën” (the green dot represents the balloon location, map from mappy.com). Figure 3. Map of the vicinity of the “Parc André Citroën” (the green dot represents the balloon location, map from mappy.com). Sensors 2020, 20, 1111 measurements at measurements. 5 of 15 of the 4. Measurements at Ground 4. Measurements at Ground Temporal evolution of the PM10 mass concentrations from LOAC at the “Ballon de Paris Generali” and from the Airparif air quality network for stations in the suburb of Paris (Vitry) and in the rural area in the south of Paris region (the data have been smoothed by a sliding smoothing procedure with a width of 2 days) are indeed in the 15−50 μm size range and their seasonality is similar to the one detected by LOAC Figure 5. Temporal evolution of the PM10 mass concentrations from LOAC at the “Ballon de Paris Generali” and from the Airparif air quality network for stations in the suburb of Paris (Vitry) and in the rural area in the south of Paris region (the data have been smoothed by a sliding smoothing procedure with a width of 2 days). Figure 5. Temporal evolution of the PM10 mass concentrations from LOAC at the “Ballon de Paris Generali” and from the Airparif air quality network for stations in the suburb of Paris (Vitry) and in the rural area in the south of Paris region (the data have been smoothed by a sliding smoothing procedure with a width of 2 days). Figure 5. Temporal evolution of the PM10 mass concentrations from LOAC at the “Ballon de Paris Generali” and from the Airparif air quality network for stations in the suburb of Paris (Vitry) and in the rural area in the south of Paris region (the data have been smoothed by a sliding smoothing Figure 6. Annual evolution of the concentrations for the smallest particles (a) and the biggest particles (b) detected by LOAC for the six-year period; the vertical bars correspond to the mean absolute deviation of the concentrations. 5. Vertical Profiles 5.1. Daily Profiles Figure 6. Annual evolution of the concentrations for the smallest particles (a) and the biggest particles (b) detected by LOAC for the six-year period; the vertical bars correspond to the mean absolute deviation of the concentrations. Figure 6. Annual evolution of the concentrations for the smallest particles (a) and the biggest particles (b) detected by LOAC for the six-year period; the vertical bars correspond to the mean absolute deviation of the concentrations. Figure 6. Annual evolution of the concentration (b) detected by LOAC for the six-year period for the smallest particles (a) and the biggest particles the vertical bars correspond to the mean absolute deviation of the concentrations. 5. 4. Measurements at Ground 4. Measurements at Ground Vertical Profiles 5 1 Daily P ofile Figure 6. Annual evolution of the concentrations for the smallest particles (a) and the biggest particles (b) detected by LOAC for the six-year period; the vertical bars correspond to the mean absolute deviation of the concentrations. Figure 6. Annual evolution of the concentrations for the smallest particles (a) and the biggest particles (b) detected by LOAC for the six-year period; the vertical bars correspond to the mean absolute deviation of the concentrations. 4. Measurements at Ground 4. Measurements at Ground This could be related to the heating and traffic during winter but also to the seasonal variation of the boundary layer height [25] and to the meteorological conditions. The concentrations for particles larger than about 15 μm exhibit also a seasonal cycle, anticorrelated with the seasonal cycle of the smaller particles; the highest concentrations are obtained When considering the six years of measurements, a seasonal cycle seems to be present for the particles smaller than about 1 µm, with maximum concentrations in winter and minimum concentrations in summer (Figure 6). This could be related to the heating and traffic during winter but also to the seasonal variation of the boundary layer height [25] and to the meteorological conditions. The concentrations for particles larger than about 15 µm exhibit also a seasonal cycle, anticorrelated with the seasonal cycle of the smaller particles; the highest concentrations are obtained during summer (Figure 6). A possible explanation is the detection of pollens, since most of the pollens are indeed in the 15−50 µm size range and their seasonality is similar to the one detected by LOAC. 6 of 15 Sensors 2020, 20, 1111 during sum Sensors 2020, 20, x Figure 5. Temporal evolution of the PM10 mass concentrations from LOAC at the “Ballon de Paris Generali” and from the Airparif air quality network for stations in the suburb of Paris (Vitry) and in the rural area in the south of Paris region (the data have been smoothed by a sliding smoothing procedure with a width of 2 days). Figure 5. Temporal evolution of the PM10 mass concentrations from LOAC at the “Ballon de Paris Generali” and from the Airparif air quality network for stations in the suburb of Paris (Vitry) and in the rural area in the south of Paris region (the data have been smoothed by a sliding smoothing procedure with a width of 2 days). are indeed in the 15−50 μm size range and their seasonality is similar to the one detected by LOAC. Figure 5. 5.2. Background Conditions 5.2. Background Conditions The daily profiles exhibit a temporal variability of about two orders of magnitude depending on the weather and on the pollution conditions. As examples, Figure 7 presents two number concentrations profiles obtained in summertime, 8 July 2016 and 20 September 2017, during low pollution conditions (the LOAC PM10 mass concentrations were of about 10 µg·m−3 at ground for both cases and in the 5−15 µg·m−3 range in flight). The two profiles exhibit different vertical evolutions and size distributions; in particular, the profile of the 8 July 2016 presents an excess of large particles in the 10−30 µm size range, probably due to a pollen episode. The daily profiles exhibit a temporal variability of about two orders of magnitude depending on the weather and on the pollution conditions. As examples, Figure 7 presents two number concentrations profiles obtained in summertime, 8 July 2016 and 20 September 2017, during low pollution conditions (the LOAC PM10 mass concentrations were of about 10 μg·m−3 at ground for both cases and in the 5−15 μg·m−3 range in flight). The two profiles exhibit different vertical evolutions and size distributions; in particular, the profile of the 8 July 2016 presents an excess of large particles in the 10−30 μm size range, probably due to a pollen episode. Figure 7. Evolution with height of number concentrations for the 19 size classes of LOAC when the urban pollution is low. (a) 8 July 2016; (b) 20 September 2017. Figure 7. Evolution with height of number concentrations for the 19 size classes of LOAC when the urban pollution is low. (a) 8 July 2016; (b) 20 September 2017. Figure 7. Evolution with height of number concentrations for the 19 size classes of LOAC when the urban pollution is low (a) 8 July 2016; (b) 20 September 2017 Figure 7. Evolution with height of number concentrations for the 19 size classes of LOAC when the urban pollution is low. (a) 8 July 2016; (b) 20 September 2017. At each height range, the histogram of the number concentrations obtained during the six years of measurements do not follow a gaussian distribution. On the other hand, a gaussian distribution can be obtained when considering the logarithm of the concentrations. Four broad-range classes are defined: 0.2−1.0 μm, 1−3 µm, 3−10 μm, and 15−50 μm. Among the were cond 5.1. Daily Profi D h 5.1. Daily Profiles evolution of the concentrations with height. On the other hand, the few flights per month up to the height of 300 m can be used to study some specific events. During the 2013−2019 period, 976 days with flights performed during daytime are available. Among them 148 days with flights up to the height of 300 m were performed. Since most of the flights were conducted up to the height of 150 m, we will consider these flights to estimate the mean evolution of the concentrations with height. On the other hand, the few flights per month up to the height of 300 m can be used to study some specific events. During the 2013−2019 period, 976 days with flights performed during daytime are available. Among them 148 days with flights up to the height of 300 m were performed. Since most of the flights were conducted up to the height of 150 m, we will consider these flights to estimate the mean evolution of the concentrations with height. On the other hand, the few flights per month up to the height of 300 m can be used to study some specific events. To study the mean vertical trend, the concentrations are integrated over 7 height ranges: 0−20 m, 20−40 m, 40−60 m, 60−80 m, 80−100 m, 100−120 m, and 120−150 m. The vertical profiles are daily averaged to reduce the variability of the individual measurements. This variability originates from the measurement accuracy (Poisson distribution) but also from variations in the wind conditions. Globally, the daily standard deviation of the concentration profiles is in the 20%−60% range. Thus, the daily 7 of 15 s from ditions. Sensors 2020, 20, 1111 averaged to reduc the measurement profiles presented in the following subsections contains error bars that represent the standard deviation for each size class and height range. G oba y, e dai y s a da d de ia io o e co ce a io p o i es is i e 0% 60% a ge us, the daily profiles presented in the following subsections contains error bars that represent the standard deviation for each size class and height range. 5.2. Background Conditions 5.2. Background Conditions For the subm ntrations slightly decrease of about 20% in result of the particles sensitivity to upwar ies, but this analysis requires further inves of the more frequent concentrations for ese vertical profiles might not be indiv files but are a sequence of individual ronic particles, no evolution is detected, wh e first tens of meters for the largest particle diffusion, depending on their sizes, shap gation. Figure 8. Examples of the histogram of the logarithm of the concentrations at two height ranges for the six years of measurements, for the 1−3 μm size range. (a) measurements at ground; (b) measurements at the height of 120−150 m. Figure 8. Examples of the histogram of the logarithm of the concentrations at two height ranges for the six years of measurements, for the 1−3 µm size range. (a) measurements at ground; (b) measurements at the height of 120−150 m. Figure 9 presents the evolution with height of the more frequent concentrations for the 4 broad-range classes. It must be noticed that these vertical profiles might not be individually consistent and are not the modal vertical profiles but are a sequence of individual modal concentration for each height range. For the submicronic particles, no evolution is detected, while the concentrations slightly decrease of about 20% in the first tens of meters for the largest particles. This could result of the particles sensitivity to upward diffusion, depending on their sizes, shapes, and densities, but this analysis requires further investigation. Figure 8. Examples of the histogram of the logarithm of the concentrations at two height ranges for the six years of measurements, for the 1−3 μm size range. (a) measurements at ground; (b) measurements at the height of 120−150 m. Figure 9. Evolution with height of the mode of number concentrations for the six years of measurements. (a) broad-range class 0.2−1.0 µm; (b) broad-range class 1−3 µm; (c) broad-range class 3−10 µm; (d) broad-range class 15−50 µm. Figure 9. Evolution with height of the mode of number concentrations for the six years of measurements. (a) broad-range class 0.2−1.0 µm; (b) broad-range class 1−3 µm; (c) broad-range class 3−10 µm; (d) broad-range class 15−50 µm. Figure 9. Evolution with height of the mode of number concentrations for the six years of measurements. (a) broad-range class 0.2−1.0 µm; (b) broad-range class 1−3 µm; (c) broad-range class 3−10 µm; (d) broad-range class 15−50 µm. Figure 9. 5.2. Background Conditions 5.2. Background Conditions Evolution with height of the mode of measurements. (a) broad-range class 0.2−1.0 µm; (b) b 3−10 µm; (d) broad-range class 15−50 µm. number concentrations for the six years o broad-range class 1−3 µm; (c) broad-range class Figure 9. Evolution with height of the mode of number concentrations for the six years of measurements. (a) broad-range class 0.2−1.0 µm; (b) broad-range class 1−3 µm; (c) broad-range class 3−10 µm; (d) broad-range class 15−50 µm. Figure 9. Evolution with height of the mode of number concentrations for the six years of measurements. (a) broad-range class 0.2−1.0 µm; (b) broad-range class 1−3 µm; (c) broad-range class 3−10 µm; (d) broad-range class 15−50 µm. 5.2. Background Conditions 5.2. Background Conditions Then, for each height range and for the 4 broad-range classes, the histogram of the logarithm of the concentrations is fitted by a gaussian function, to estimate the mode of number concentrations (corresponding to the most frequent concentrations over the full time period of measurements examples are given in Figure 8) At each height range, the histogram of the number concentrations obtained during the six years of measurements do not follow a gaussian distribution. On the other hand, a gaussian distribution can be obtained when considering the logarithm of the concentrations. Four broad-range classes are defined: 0.2−1.0 µm, 1−3 µm, 3−10 µm, and 15−50 µm. Then, for each height range and for the 4 broad-range classes, the histogram of the logarithm of the concentrations is fitted by a gaussian function, to estimate the mode of number concentrations (corresponding to the most frequent concentrations over the full time period of measurements, examples are given in Figure 8). 8 of 15 s d Sensors 2020, 20, 1111 concentrations could result o Figure 8. Examples of the histogram of the logarithm of the concentrations at two height ranges for the six years of measurements, for the 1−3 μm size range. (a) measurements at ground; (b) measurements at the height of 120−150 m. Figure 8. Examples of the histogram of the logarithm of the concentrations at two height ranges for the six years of measurements, for the 1−3 µm size range. (a) measurements at ground; (b) measurements at the height of 120−150 m. Sensors 2020, 20, x FOR PEER REVIEW 8 of 15 Figure 9 presents the evolution with height of the more frequent concentrations for the 4 broad-range classes. It must be noticed that these vertical profiles might not be individually consistent and are not the modal vertical profiles but are a sequence of individual modal concentration for each height range. For the submicronic particles, no evolution is detected, while the concentrations slightly decrease of about 20% in the first tens of meters for the largest particles. This could result of the particles sensitivity to upward diffusion, depending on their sizes, shapes, and densities, but this analysis requires further investigation. 2020, 20, x FOR PEER REVIEW igure 9 presents the evolution with heig -range classes. It must be noticed that tent and are not the modal vertical p ntration for each height range. 5.3.1. Winter Event 5.3.1. Winter Event Pollution is often encountered in Paris during anticyclonic conditions in winter, typically in December, with particulates originating from traffic and heating [26]. During such events, the concentration of the particles can be from 10 to 100 times higher than during the summer background conditions. The stronger winter event recorded in the 2013−2019 period by LOAC occurs in December 2013. Figure 10 presents the profiles for the strong pollution event in 11 December 2013. The profile exhibits a strong concentration increase at a height of 200 m for the smallest particles <0.4 µm while the concentrations are about ten times lower above, due to a temperature inversion layer resulting from the anticyclonic conditions. The LOAC PM10 mass concentrations at ground are of about 50 µg·m−3 at ground, of about 60 µg·m−3 at a height of 130 m due to an increase of concentrations of particles in the 5−10 µm size range, only of 45 µg·m−3 at a height of 200 m although the increase of submicronic particles concentrations, and of about 35 µg·m−3 above. Thus, the height variability in mass concentrations do not fully reflect the more complex height variability of the number concentrations for the various size classes. Pollution is often encountered in Paris during anticyclonic conditions in winter, typically in December, with particulates originating from traffic and heating [26]. During such events, the concentration of the particles can be from 10 to 100 times higher than during the summer background conditions. The stronger winter event recorded in the 2013−2019 period by LOAC occurs in December 2013. Figure 10 presents the profiles for the strong pollution event in 11 December 2013. The profile exhibits a strong concentration increase at a height of 200 m for the smallest particles <0.4 μm while the concentrations are about ten times lower above, due to a temperature inversion layer resulting from the anticyclonic conditions. The LOAC PM10 mass concentrations at ground are of about 50 μg·m−3 at ground, of about 60 μg·m−3 at a height of 130 m due to an increase of concentrations of particles in the 5−10 µm size range, only of 45 μg·m−3 at a height of 200 m although the increase of submicronic particles concentrations, and of about 35 μg·m−3 above. Thus, the height variability in mass concentrations do not fully reflect the more complex height variability of the number concentrations for the various size classes. Figure 10. 5.3. Main Pollution Events During most of the pollution events, the winds were low, thus flights up to a height of 300 m were available. We present below the main pollution events observed by LOAC in the 2013−2019 period. Sensors 2020, 20, 1111 During most o il bl W 9 of 15 m were i d 5.3.1. Winter Event 5.3.1. Winter Event Evolution with height of LOAC concentrations for the 11 December 2013 pollution event. (a) number concentrations; (b) PM10 mass concentration. Figure 10. Evolution with height of LOAC concentrations for the 11 December 2013 pollution event. (a) number concentrations; (b) PM10 mass concentration. Figure 10. Evolution with height of LOAC concentrations for the 11 December 2013 pollution event. (a) number concentrations; (b) PM10 mass concentration Figure 10. Evolution with height of LOAC concentrations for the 11 December 2013 pollution event. (a) number concentrations; (b) PM10 mass concentration. 5 3 2 S i E t 5.3.2. Spring Event 5.3.2. Spring Event Another type of pollution is often encountered in beginning of spring, typically in March, mainly originating from the combination of local pollution and the transport of pollutants from agricultural activities around Paris [27,28]. The vertical profiles in Figure 11 for the 14 March 2014 and 17 March 2015 differ from those measured in winter (Figure 10). The concentrations of the particles are more constant with height, because boundary layer is thicker due to more vertically mixed air masses, while concentrations of the biggest particles slightly decrease with height. The LOAC PM10 mass concentrations decrease from 110 μg·m−3 at ground to about 65 μg·m−3 at 300 m for the 17 March 2015 and decrease from about 40 μg m−3 at ground to about 20 μg m−3 at 300 m (Figure 12) Another type of pollution is often encountered in beginning of spring, typically in March, mainly originating from the combination of local pollution and the transport of pollutants from agricultural activities around Paris [27,28]. The vertical profiles in Figure 11 for the 14 March 2014 and 17 March 2015 differ from those measured in winter (Figure 10). The concentrations of the particles are more constant with height, because boundary layer is thicker due to more vertically mixed air masses, while concentrations of the biggest particles slightly decrease with height. The LOAC PM10 mass concentrations decrease from 110 µg·m−3 at ground to about 65 µg·m−3 at 300 m for the 17 March 2015 and decrease from about 40 µg·m−3 at ground to about 20 µg·m−3 at 300 m (Figure 12). 10 of 15 10 f 15 10 of 15 Sensors 2020, 20, 1111 S 2020 20 FOR Sensors 2020, 20, x FOR Figure 11. Evolution with height of LOAC number concentrations during the spring pollution events. (a) 14 March 2014; (b) 17 March 2015. Figure 11. Evolution with height of LOAC number concentrations during the spring pollution events. (a) 14 March 2014; (b) 17 March 2015. Figure 11. Evolution with height of LOAC number concentrations during the spring pollution events. (a) 14 March 2014; (b) 17 March 2015. Figure 11. Evolution with height of LOAC number concentrations during the spring pollution events. (a) 14 March 2014; (b) 17 March 2015 Figure 11. Evolution with height of LOAC number concentrations during the spring pollution events. (a) 14 March 2014; (b) 17 March 2015. Figure 11. 5.3.3. Summer Event the 100−200 m height r This event was not d Pollution events can be also present during summer, depending on the weather conditions. Although no major pollution event was detected at ground during the 2014−2019 period, an increase in aerosol concentrations was detected from 24 to 30 June above a height of about 100 m. For the 25 June 2019 profile, the number concentrations were up to 10 times higher for particles smaller than 5 µm in the 100−200 m height range than at ground, the mass concentration were five times higher (Figure 13). This event was not detected from ground-based station, showing the interest of balloon-borne measurements. This event was not detected from ground based station, showing the interest of balloon borne measurements. No explainable extra-source (smoke, industrial activities) was identify during this period at the vicinity of the measurement location. The LOAC typology indication differs strongly from those of the other pollution events and do not correspond to the usual LOAC typology for conventional urban pollution particles (carbonaceous, mineral, sulfate, ammonium nitrate). At present no explanation can be proposed for the origin of this event and for the nature of the particles, and further investigations are needed. Figure 13. Evolution with height of LOAC concentrations for the 25 June 2019 pollution event. (a) number concentrations; (b) PM10 mass concentration. Figure 13. Evolution with height of LOAC concentrations for the 25 June 2019 pollution event. (a) number concentrations; (b) PM10 mass concentration. Figure 13. Evolution with height of LOAC concentrations for the 25 June 2019 pollution event. (a) number concentrations; (b) PM10 mass concentration Figure 13. Evolution with height of LOAC concentrations for the 25 June 2019 pollution event. (a) number concentrations; (b) PM10 mass concentration. 6. Discussion The concentrations of the particles and their vertical evolution varies from one pollution episode to another. To better evaluate the variability, we can consider the previous 4 broad-range classes, now applied to the 6 vertical profiles presented in Figures 7, 10, 11 and 13. A factor of more than 50 occurs between low pollution and high pollution events for the 3 first broad-range classes (particles with No explainable extra-source (smoke, industrial activities) was identify during this period at the vicinity of the measurement location. The LOAC typology indication differs strongly from those of the other pollution events and do not correspond to the usual LOAC typology for conventional urban pollution particles (carbonaceous, mineral, sulfate, ammonium nitrate). 5 3 2 S i E t 5.3.2. Spring Event Evolution with height of LOAC number concentrations during the spring pollution events. (a) 14 March 2014; (b) 17 March 2015. ( ) ; ( ) Figure 12. Evolution with height of LOAC PM10 mass concentrations during the spring pollution t ( ) 14 M h 2014 (b) 17 M h 2015 Figure 12. Evolution with height of LOAC PM10 mass concentrations during the spring pollution events. (a) 14 March 2014; (b) 17 March 2015. Figure 12. Evolution with height of LOAC PM10 mass concentrations during the spring pollution events. (a) 14 March 2014; (b) 17 March 2015. Figure 12. Evolution with height of LOAC PM10 mass concentrations during the spring pollution Figure 12. Evolution with height of LOAC PM10 mass concentrations during the spring pollution events. (a) 14 March 2014; (b) 17 March 2015. Figure 12. Evolution with height of LOAC PM10 mass concentrations during the spring pollution events. (a) 14 March 2014; (b) 17 March 2015. 11 of 15 rease in 25 June 11 of 15 rease in 25 June Sensors 2020, 20, 1111 Although no major aerosol concentrati 5.3.3. Summer Event the 100−200 m height r This event was not d At present no explanation can be proposed for the origin of this event and for the nature of the particles, and further investigations are needed. diameter small submicronic pa 6. Discussion Number concentration measurements as those presented above, mainly for submicronic that can be the most dangerous for health [4–6], may be more efficient than mass concentrations to evaluate the health impact of pollutions events and to establish the temporal trend of number concentration of (anthropogenic) pollution particles. Obviously, such trends cannot be retrieved from measurements only at one location; thus, a network of (optical) counter instruments must be implanted in parallel with regulatory mass concentration instruments. Figure 14. Evolution with height of aerosols concentrations. (a) broad-range class 0.2−1.0 µm; (b) broad-range class 1−3 µm; (c) broad-range class 3−10 µm; (d) broad-range class 15−50 µm Figure 14. Evolution with height of aerosols concentrations. (a) broad-range class 0.2−1.0 µm; (b) broad-range class 1−3 µm; (c) broad-range class 3−10 µm; (d) broad-range class 15−50 µm. Figure 14. Evolution with height of aerosols concentrations. (a) broad-range class 0.2−1.0 µm; (b) Figure 14. Evolution with height of aerosols concentrations. (a) broad-range class 0.2−1.0 µm; (b) broad-range class 1−3 µm; (c) broad-range class 3−10 µm; (d) broad-range class 15−50 µm. broad range class 1 3 µm; (c) broad range class 3 10 µm; (d) broad range class 15 50 µm. 7. Conclusions The six years of number concentration measurements performed in Paris, France, by the LOAC instrument for particles in the 0.2−50 μm size range allowed us to detect a seasonal variability in the When considering the mass concentrations values from LOAC, the highest pollution events were on March 2014 and March 2015 (with maximum values up to 110 µg·m−3 and up to 90 µg·m−3, respectively). When considering the number of all particles larger than 0.2 µm, the highest concentrations were detected on December 2013, although the mass concentrations were only of up to 80 µg·m−3. These differences could be due to the origin of the pollution, typically primary (carbonaceous) particles versus secondary aerosols. Mass concentration variability from low pollution to high pollution levels in Paris is about one order of magnitude, while the variability is of two orders of magnitude for number concentration. Most of the studies on the pollution trend in cities are based on mass concentrations [29]. It is often established that the mass-concentration of PM10 is decreasing in European cities, mainly due to changes in diesel engines and particles filters [30]. diameter small submicronic pa 6. Discussion the particles in the 1−3 μm size range, the highest concentrations were detected during the March 2014 event, during the March 2015 event, and during the June 2019 event in flight. In particular, the March 2015 event was dominated by secondary aerosols (more particularly of ammonium nitrate, as reported in [28]). The concentration of the 1−3μm particles during the December 2013 event is significantly lower than for the March 2014 event. The tendency for the 3−10 μm particles is relatively similar to the one for the 1−3 μm particles, except for the March 2015 concentrations that are closer to The concentrations of the particles and their vertical evolution varies from one pollution episode to another. To better evaluate the variability, we can consider the previous 4 broad-range classes, now applied to the 6 vertical profiles presented in Figures 7, 10, 11 and 13. A factor of more than 50 occurs between low pollution and high pollution events for the 3 first broad-range classes (particles with diameter smaller than 10 µm), as shown in Figure 14. As expected, the highest concentrations of the submicronic particles occur for the December 2013 event and for the March 2014 and 2015 events. For the particles in the 1−3 µm size range, the highest concentrations were detected during the March 2014 event, during the March 2015 event, and during the June 2019 event in flight. In particular, the March 2015 event was dominated by secondary aerosols (more particularly of ammonium nitrate, as reported in [28]). The concentration of the 1−3 µm particles during the December 2013 event is 12 of 15 ith such pending Sensors 2020, 20, 1111 as possible increa Al significantly lower than for the March 2014 event. The tendency for the 3−10 µm particles is relatively similar to the one for the 1−3 µm particles, except for the March 2015 concentrations that are closer to the background conditions. Finally, no obvious correlation between pollution and concentrations of the largest particles (15−50 µm) can be pointed out, since, as previously mentioned, these particles are likely mainly originating from pollen events. on the origin of the pollution events. 7. Conclusions The six years of number concentration measurements performed in Paris, France, by the LOAC instrument for particles in the 0.2−50 µm size range allowed us to detect a seasonal variability in the PM content, expectedly due to the origin of the particles (anthropogenic pollution, pollens). Since the instrument is mounted onboard the touristic tethered balloon “Ballon de Paris Generali”, the mean evolution of particles concentrations with height was obtained for background conditions up to a height of 150 m. The concentrations of submicronic particles remain constant with height, while the concentrations slightly decrease in the first tens of meters for the largest particles. Measurements were also conducted during major pollution events, with particles concentrations several tens of times higher than during background conditions. The vertical evolution of concentrations varies from one event to another one, depending on the origin of the pollution and on the meteorological conditions. In particular, an accumulation layer was sometimes detected at a height of about 200 m in winter due to temperature inversion layer. These measurements have shown the interest of performing particle number concentrations measurements for the air pollution monitoring in complement with regulatory mass concentrations measurements, to better evaluate the intensity of the pollution event and to better consider the effect of smallest particles. We have presented here the first results obtained with LOAC at the Paris touristic balloon. Future studies could be conducted using this LOAC database, as modeling works on the vertical transport of particles, studies on the correlation of the variability of the particle’s concentrations and their size distribution with the meteorological conditions, and studies on the evolution of the particle concentrations after rain. Finally, a LOAC is also mounted in another touristic balloon, the “Ballon Terra Botanica” in a smaller city, Angers (West of France); its measurements will be compared soon to those of Paris to better establish the similarities and the differences for the vertical evolution of the pollution particles depending on the measurement locations. New measurements could be proposed by mounting the LOAC instrument onboard the various touristic tethered balloons available in the world, to better evaluate the vertical transport of the urban pollution particles. Author Contributions: The instrument conception and the data analysis were conducted by J.-B.R. The measurements interpretation was done by J.-B.R. and V.M. The funding acquisitions were conducted by V.M., J.-B.R. and J.G. The “Ballon de Paris” facilities were provided by J.G. diameter small submicronic pa 6. Discussion Nevertheless, possible change in the size distribution, as possible increase in number concentration of submicronic particles, cannot be estimated with such measurements. Also, the vertical evolution of particles number concentrations can change depending on the origin of the pollution events. Number concentration measurements as those presented above, mainly for submicronic that can be the most dangerous for health [4–6], may be more efficient than mass concentrations to evaluate the health impact of pollutions events and to establish the temporal trend of number concentration of (anthropogenic) pollution particles. Obviously, such trends cannot be retrieved from measurements only at one location; thus, a network of (optical) counter instruments must be implanted in parallel with regulatory mass concentration instruments. Sensors 2020, 20, 1111 13 of 15 13 of 15 7. Conclusions All authors have read and agreed to the published version of the manuscript. Funding: This project was partly funded by ADEME (project MESUrPOP) CORTEA / contract number 1762C0004 and by the Labex “Étude des géofluides et des VOLatils–Terre, Atmosphère et Interfaces – Ressources et Environnement” (VOLTAIRE) (ANR-10-LABX-100-01) managed by the University of Orleans. Funding: This project was partly funded by ADEME (project MESUrPOP) CORTEA / contract number 1762C0004 and by the Labex “Étude des géofluides et des VOLatils–Terre, Atmosphère et Interfaces – Ressources et Environnement” (VOLTAIRE) (ANR-10-LABX-100-01) managed by the University of Orleans. 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Characterising an intense PM pollution episode in March 2015 in France from multi-site approach and near real time data: Climatology, variabilities, geographical origins and model evaluation. Atmos. Environ. 2017, 155, 68–84. [CrossRef] 29. Font, A.; Guiseppin, L.; Blangiardo, M.; Ghersi, V.; Fuller, G.W. A tale of two cities: is air pollution improving in Paris and London. Environ. Poll. 2019, 249, 1–12. [CrossRef] 30. Fiebig, M.; Wiartalla, A.; Holderbaum, B.; Kiesow, S. Particulate emissions from diesel engines: correlation between engine technology and emissions. J. Occup. Med. Toxicol. 2014, 9, 6. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Jurnal Ilmiah DIDAKTIKA Agustus 2011 VOL. XII NO. 1, 149-162 Jurnal Ilmiah DIDAKTIKA Agustus 2011 VOL. XII NO. 1, 149-162 Abstrak Media pembelajaran merupakan hal yang mutlak digunakan dalam proses belajar mengajar. Seorang guru harus mampu memilih media yang sesuai dengan konsep yang akan disampaikan dan dapat menunjang keberhasilan proses belajar mengajar. Dalam pembelajaran biologi khususnya konsep Keanekaragaman Hayati guru harus dapat memberikan pemahaman dan pengenalan kepada siswa terhadap berbagai jenis tumbuhan termasuk tumbuhan herba. Pemanfaatan keanekaragaman tumbuhan yang ada di alam merupakan salah satu solusi yang paling tepat bagi guru untuk mencapai tujuan pembelajaran yang lebih optimal. Keberhasilan pembelajaran khususnya bidang studi biologi yang dapat dilihat dari ketiga aspek yakni, kognitif, afektif dan psikomotorik diharapkan bisa tercapai apabila peran guru sebagai pendidik dapat membimbing siswa pada saat pengenalan tumbuhan di lapangan. Kata Kunci: pemanfaatan, media, pembelajaran Biologi PEMANFAATAN MEDIA DALAM PEMBELAJARAN BIOLOGI DI SEKOLAH Amna Emda Dosen tetap pada Fakultas Tarbiyah IAIN Ar-Raniry Abstract In teaching learning process, teachers have to use some media of instruction. They have to be able to select appropriate media with the concept that they want to teach. In biology, especially the concept of biodiversity, the teacher should explain and introduce the students to various plants available around them including herb plants. It will be one of solution for a teacher to achieve an optimum goal if they can use the diverse plant around them. The successful of learning especially in the field of biology can be seen from cognitive, affective and psychomotoric aspects which is hoped to be achieved if the teachers as educators can guide their students to know about the plants in the field. 2Wibawa, B. dan F. Mukti, Media Pengajaran, Jakarta: Departemen Pendidikan dan Kebudayaan, 1992. PENDAHULUAN Pembelajaran merupakan suatu proses yang dilakukan baik melalui pendidikan secara formal maupun non-formal. Dalam penyelenggaran pendidikan secara formal maka pendidik dan peserta didik dituntut untuk lebih kreatif agar tercapai tujuan pendidikan yang diharapkan. Seorang pendidik harus mampu menguasai metode dan pemilihan media yang tepat dalam mengajar. 1Dalyono, M., Psikologi Pendidikan, Jakarta: Renika Cipta, 1997, hal. 55. PEMANFAATAN MEDIA DALAM PEMBELAJARAN BIOLOGI DI SEKOLAH Peningkatan prestasi siswa pada setiap semester terkait dengan beberapa faktor yang secara garis besar dikelompokkan menjadi dua, yaitu: faktor internal dan faktor eksternal. Yang termasuk faktor internal antara lain kesehatan, intelegensi, bakat, minat, motivasi dan cara belajar. Sedangkan faktor eksternal antara lain keluarga, kualitas guru, metode mengajar, kesesuaian kurikulum dengan kemampuan siswa, fasilitas/perlengkapan sekolah, keadaan ruangan, jumlah murid perkelas, masyarakat, lingkungan sekitar dan lain-lain.1 Kedua faktor tersebut secara simultan mempengaruhi prestasi belajar siswa. Sebagai contoh seorang siswa yang memiliki intelegensi yang baik belum tentu menguasai materi pembelajaran, jika minat dan motivasinya terhadap pembelajaran rendah, dan sebaliknya seorang seorang siswa yang inteligensinya tidak terlalu baik dapat meningkat penguasaan terhadap materi pembelajaran karena dorongan minat dan motivasi yang tinggi. Dengan demikian, minat dan motivasi merupakan faktor penting yang secara langsung dapat mempengaruhi prestasi belajar siswa. Minat dan motivasi yang sudah dimiliki oleh siswa masih saja dapat dipengaruhi oleh faktor eksternal. Seperti antara lain cara guru mengajar, media yang digunakan dan suasana lingkungan kelas. Wibawa dan Mukti, mengemukakan bahwa verbalisme, kekacauan makna, dan persepsi yang kurang tepat dapat menurunkan minat dan motivasi peserta didik sehingga akan berdampak pada prestasi belajar.2 Penggunaan media yang tepat dalam pembelajaran biologi merupakan salah satu solusi dari berbagai masalah yang terkait dengan minat dan motivasi belajar siswa. Penggunaa media yang tepat akan meningkatkan perhatian siswa pada topik yang akan dipelajari, dengan bantuan media minat dan motivasi siswa dapat ditingkatkan, siswa akan lebih konsentrasi dan diharapkan proses pembelajaran menjadi lebih baik sehingga pada akhirnya prestasi belajar siswa dapat ditingkatkan. Oleh karena itu penggunaan media sebagai alat bantu dalam pembelajaran harus dipilih yang sesuai dan benar-benar dapat membantu siswa dalam memahami materi yang disampaikan. Dengan demikian diharapkan Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 150 Amna Emda mampu memacu kepada pengambangan aspek kognitif, afektif dan psikomotor siswa. Dalam pembelajaran biologi pada Sekolah Menengah Tingkat Pertama dan juga Sekolah Menengah Tingkat Atas terdapat konsep Keanekaragaman Hayati yang merupakan salah satu materi yang harus diajarkan kepada siswa. Sesuai dengan kurikulum tahun 2004, kompetensi dasar yang ingin dicapai adalah siswa mampu mengaplikasikan pemahaman pentingnya keanekaragaman dan klasifikasi tumbuhan, serta mampu mengaplikasikan prinsip-prinsip pengelompokan makhluk hidup untuk mempelajari keanekaragaman hayati bagi kehidupan. Keanekaragaman Hayati merupakan keanekaragaman organisme yang menunjukkan keseluruhan variasi gen, jenis dan ekosistem pada suatu daerah. PEMANFAATAN MEDIA DALAM PEMBELAJARAN BIOLOGI DI SEKOLAH Keanekaragaman tumbuhan secara umum dan tumbuhan herba secara khusus yang terdapat di alam merupakan salah satu media alami yang dapat digunakan sebagai media pembelajaran dalam penyampaian materi pada konsep tersebut. Tumbuhan herba merupakan salah satu jenis tumbuhan yang dapat dibedakan dengan jenis tumbuhan lainnya berdasarkan ciri-ciri yang dimilikinya. Siswa dapat diajak ke alam menyaksikan keanekaragaman jenis tumbuhan dan ekosistemnya serta dapat mengenal langsung tumbuhan yang ada sehingga tujuan pembelajaran dapat lebih optimal. Kriteria Tumbuhan Herba Tumbuhan herba tergolong ke dalam tumbuhan biji (Divisio Spermatophyta) dan kebanyakan termasuk kedalam tumbuhan biji tertutup (Subdivisio Angiospermae), baik dari kelas Monocotyledoneae (tumbuhan berkeping satu) maupun kelas Dycotyledoneae (tumbuhan berkeping dua). Tumbuhan Angiospermae meliputi berbagai jenis tanaman pertanian dan pekarangan, baik annual (tumbuhan semusim) maupun perennual (tumbuhan tahunan). Tumbuhan herba bersifat kosmopolit di alam karena sangat mudah menyebar dan tumbuh hampir di semua tempat atau habitat, sehingga mempunyai jumlah yang sangat besar di alam. Tumbuhan angiospermae merupakan tumbuhan kormus sejati, yakni tumbuhan yang telah dapat dengan jelas dibedakan dalam tiga bagian pokok yaitu Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 151 4Steenis, C.G.J. Van, Flora Untuk Seolah di Indonesia, Jakarta: Pradnya Paramita, 1978, ha 1268. 3Tjitrosoepomo, Morfologi Tumbuhan (Spermatologi), Yogyakarta: Gajah Mada Universi Press, 1989, hal. 2 PEMANFAATAN MEDIA DALAM PEMBELAJARAN BIOLOGI DI SEKOLAH Pepaya (Carica papaya, L), ciri-cirinya : herba berbentuk pohon,berakar tunggang, berbatang lurus bulat silendris, tangkai daun berongga, helaian daun berlekuk, berbiji banyak, bunga beraturan, kelopak bertajuk lima, daun mahkota lima, benang sari sepuluh. 3. Kunyit (Curcuma domestica, Val.) , cirri-cirinya : berakar rimpang, batang tegak, pelepah memeluk batang, daun sering jelas dua baris, bunga berkelamin dua, kelopak berbentuk tabung, daun mahkota tiga yang melekat pada pangkalnya. 3. Kunyit (Curcuma domestica, Val.) , cirri-cirinya : berakar rimpang, batang tegak, pelepah memeluk batang, daun sering jelas dua baris, bunga berkelamin dua, kelopak berbentuk tabung, daun mahkota tiga yang melekat pada pangkalnya. 5Deshmuk, Ian, Penerjemah Kartawinata Kuswata dan Danimiharja Sarkat, Ekologi dan Biologi Tropika, Edisi Pertama, Jakarta: Yayasan Obor Indonesia, 1992, hal. 5. 6Reosoedarmo, S. Kartawinata Kuswata dan Apriliani. S, Pengantar Ekologi, Jakarta: Remaja Rosdakarya, 1985, hal.40. PEMANFAATAN MEDIA DALAM PEMBELAJARAN BIOLOGI DI SEKOLAH akar (radix), batang (caulis) dan daun (folium). Daunnya tergolong type makrofil dengan bentuk dan susunan tulang-tulang daun beraneka ragam. Embrionya bersifat bipolar atau dwipolar. Tidak hanya kutub batang yang tumbuh dan berkembang membentuk batang, cabang-cabang dan daun, tetapi kutub akar pun tumbuh dan berkembang membentuk system perakaran.3 Adapun kriteria tumbuhan herba secara umum adalah sebagai berikut : - Batangnya lunak, berair dan tidak berkayu (batang basah), berbentuk bulat atau kebanyakan bersegi. - Sistem perakaran tunggang dan serabut atau akar rimpang di bawah tanah. - Daun berjejal-jejal pada pangkal batang atau berjarak - Pelepah daun ada, lidah tidak ada. - Helaian daun duduk berbentuk garis dengan urat daun membujur kadang- kadang tidak tumbuh sempurna atau tidak ada sama sekali. - Bunga berkelamin satu atau dua, tersusun dalam anak bulir yang kemudian berkumpul lagi menjadi berbeda-beda karangan bunga. - Bunga berdiri sendiri dalam ketiak sekam. Bunga berdiri sendiri dalam ketiak sekam. - Tenda bunga tidak ada - Benang sari satu sampai tiga - Tangkai sari bebas, kepala sari beruang dua - Bakal buah beruang satu, bakal biji satu - Tangkai putik satu kebanyakan bercabang dua sampai tiga - Buah tidak membuka, biji lepas dari dinding buah - Umumnya berkembang biak dengan biji.4 Berdasarkan kategori di atas, maka berikut ini beberapa contoh tumbuhan yang tergolong kedalam tumbuhan herba. 1. Pisang (Musa paradisiaca, L) ciri-cirinya : hidupnya menahun, dan berumpun dengan akar rimpang, helaian daun berbentuk lanset memanjang, mudah koyak, pada bagian bawah daun berlilin, bungan berkelamin satu dalam tandan yang bertandan. Daun pelindung merah tua 3Tjitrosoepomo, Morfologi Tumbuhan (Spermatologi), Yogyakarta: Gajah Mada University Press, 1989, hal. 2 3Tjitrosoepomo, Morfologi Tumbuhan (Spermatologi), Yogyakarta: Gajah Mada University Press, 1989, hal. 2 2 | Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 Amna Emda berlilin, masing-masing dalam ketiaknya dengan banyak bunga yang tersusun dalam dua baris melintang. 2. Pepaya (Carica papaya, L), ciri-cirinya : herba berbentuk pohon,berakar tunggang, berbatang lurus bulat silendris, tangkai daun berongga, helaian daun berlekuk, berbiji banyak, bunga beraturan, kelopak bertajuk lima, daun mahkota lima, benang sari sepuluh. 2. Pepaya (Carica papaya, L), ciri-cirinya : herba berbentuk pohon,berakar tunggang, berbatang lurus bulat silendris, tangkai daun berongga, helaian daun berlekuk, berbiji banyak, bunga beraturan, kelopak bertajuk lima, daun mahkota lima, benang sari sepuluh. 2. PEMANFAATAN MEDIA DALAM PEMBELAJARAN BIOLOGI DI SEKOLAH Keanekaragaman tumbuhan herba serta keberadaannya dalam suatu komunitas sangat dipengaruhi oleh faktor ekologi seperti iklim yang meliputi cahaya, suhu, curah hujan, daya penguapan dan angin, dan faktor tanah atau edafik yang meliputi fragmen-fragmen mineral, air tanah, udara dalam tanah, bahan organik dan organisme hidup, seperti faktor biotik, yang meliputi makhluk hidup, hewan, tumbuhan dan manusia. Tumbuhan herba yang tergolong kedalam Angiospermae (tumbuhan biji tertutup) mempunyai sifat yang beranekaragam, yakni herba menahun, herba kokoh, herba berumpun, herba bergetah, herba menjalar, herba batangnya merayap, dan herba bentuk pohon. Penyebaran /pemencaran tumbuhan herba dapat terjadi dengan berbagai cara, antara lain disebabkan oleh angina, biasanya tumbuhan yang memiliki biji bersayap, penyebaran oleh air, khususnya bagi tumbuhan yang hidup di air atau dekat air, penyebaran oleh hewan dan juga manusia. Keanekaragaman Tumbuhan Herba Keanekaragaman merupakan suatu istilah untuk menyatakan jumlah jenis atau jumlah individu, baik itu hewan maupun tumbuhan yang hidup dan menempati suatu tempat tertentu yang sudah menjadi habitat. Menurut Deshmukh, Keanekaragaman merupakan gabungan antara jumlah jenis dan jumlah individu masing-masing jenis dalam satu komunitas5 Selanjutnya Resosoedarmo menyatakan keragaman (diversity) adalah jumlah jenis tumbuhan atau tumbuhan yang hidup pada suatu tempat tertentu.6 Komunitas merupakan bagian dari ekosistem yang terdiri dari kumpulan tumbuhan dan hewan yang secara bersama-sama membentuk suatu masyarakat hewan dan tumbuhan. Keanekaragaman jenis tumbuhan herba menunjukkan banyaknya jenis tumbuhan yang memiliki ciri-ciri seperti tersebut di atas yang hidup, tumbuh dan berkembang biak pada habitatnya dalam suatu komunitas. Keanekaragaman jenis tumbuhan pada suatu komunitas akan berbeda dengan komunitas lainnya. Hal ini dapat terjadi karena kemampuan tumbuhan untuk menyesuaikan diri terhadap lingkungannya akan berbeda pula. Banyak faktor yang dapat mempengaruhi keberadaan suatu jenis tumbuhan dalam suatu komunitas. Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 153 Peran Media Dalam Kegiatan Pembelajaran Secara umum media pembelajaran adalah semua saluran pesan yang dapat digunakan sebagai sarana komunikasi dalam proses belajar mengajar. Penggunaan media dalam proses belajar mengajar dapat menghindari dari verbalisme. Peran media dalam proses belajar mengajar antara lain : a. Sebagai alat bantu belajar 8Sadiman, Arief K., Media Pendidikan, Pengerian Pengembangan dan Pemanfaatannya, Jakarta: Raja Grafindo Persada, 2002, hal.16. 7Hamalik, Oemar, Media Pendidikan, Bandung: Alumni, 1986, hal. 27. a. Sebagai alat bantu belajar Media pembelajaran dapat memberikan pengalaman pendidikan yang bermakna bagi siswa. Media dapat memberikan pengalaman yang nyata dalam belajar karena mengikutsertakan seluruh indra dan akal pikirannya. Sebagaimana yang dikemukakan oleh Hamalik, ada beberapa faktor keuntungan atau kegunaan media, yaitu : 1. Meletakkan dasar-dasar yang konkret untuk berfikir sehingga mengurangi verbalisme. 2. Memperbesar perhatian siswa. 2. Memperbesar perhatian siswa. 3. Meletakkan dasar yang penting untuk perkembangan belajar, sehingga membuat pelajaran lebih menetap. 154 | Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 Amna Emda 4. Memberikan pengalaman yang nyata dapat menumbuhkan kegiatan berusaha sendiri di kalangan siswa. 4. Memberikan pengalaman yang nyata dapat menumbuhkan kegiatan berusaha sendiri di kalangan siswa. 5. Menumbuhkan pikiran yang teratur dan kontinyu terutama terdapat dalam gambaran hidup. 6. Membantu tumbuhnya pengertian dan perkembangan kemampuan berbahasa. 7. Memberikan pengalaman yang tidak mudah diperoleh dengan cara lain serta membantu berkembangnya efesiensi yang lebih mendalam serta keragaman yang lebih banyak dalam belajar.7 b. Sebagai alat komunikasi Dengan adanya media , penyampaian pesan dari sumber pesan (pendidik) kepada penerima pesan (anaka didik) akan lebih mudah dipahami. b. Sebagai alat komunikasi Dengan adanya media , penyampaian pesan dari sumber pesan (pendidik) kepada penerima pesan (anaka didik) akan lebih mudah dipahami. c. Sebagai alat untuk menumbuhkan ciptaan baru. 7Hamalik, Oemar, Media Pendidikan, Bandung: Alumni, 1986, hal. 27. 9 Moore, M.G dan Kearsley, G., Distance Education . A System View, Washington DC: Woodgeworth Publishing Company. c. Sebagai alat untuk menumbuhkan ciptaan baru. c. Sebagai alat untuk menumbuhkan ciptaan baru. Agar siswa dapat terangsang untuk mengikuti pelajaran, maka guru perlu menciptakan suasana belajar yang menyenangkan. Penyajian materi pelajaran tidak secara monoton, tetapi menggunakan media yang bervariasi dan sesuai, maka perhatian anak didik akan terpusat pada pelajaran yang disajikan. Arif K. Sadiman menjelaskan, dengan menggunakan media pendidikan secara tepat dan bervariasi dapat mengatasi sikap pasif anak didik, karena media pendidikan berguna : Agar siswa dapat terangsang untuk mengikuti pelajaran, maka guru perlu menciptakan suasana belajar yang menyenangkan. Penyajian materi pelajaran tidak secara monoton, tetapi menggunakan media yang bervariasi dan sesuai, maka perhatian anak didik akan terpusat pada pelajaran yang disajikan. Arif K. Sadiman menjelaskan, dengan menggunakan media pendidikan secara tepat dan bervariasi dapat mengatasi sikap pasif anak didik, karena media pendidikan berguna : 1. Menumbuhkan gairah belajar. 2. Memungkinkan interaksi yang lebih langsung antara anak didik dengan lingkungan dan kenyataan. 3. Memungkinkan anak didik belajar sendiri-sendiri menurut kemampuan dan minatnya.8 Dari uraian di atas tampak bahwa media merupakan alat bantu pembelajaran yang diharapkan dapat membantu peserta didik untuk belajar lebih baik. Dengan demikian suatu media dikategorikan baik jika media media tersebut dapat membantu siswa untuk mempelajari sesuatu dengan lebih baik. Untuk dapat memenuhi tujuan tersebut maka pemilihan suatu media hendaknya memperhatikan tujuh hal berikut : 8Sadiman, Arief K., Media Pendidikan, Pengerian Pengembangan dan Pemanfaatannya, Jakarta: Raja Grafindo Persada, 2002, hal.16. Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 155 156 | Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 PEMANFAATAN MEDIA DALAM PEMBELAJARAN BIOLOGI DI SEKOLAH 1. Kesesuaian media dengan tujuan pembelajaran yang ingin dicapai. 1. Kesesuaian media dengan tujuan pembelajaran yang ingin dicapai. 2. Kesesuaian karakteristik media dengan karakteristik pelajaran. 3. Kecanggihan media dibandingkan dengan tingkat perkembangan peserta didik. 4. Kesesuaian media pembelajaran dengan minat, kemampuan dan wawasan siswa. 5. Kesesuaian karakteristik media dengan latar belakang sosial budaya. 6. Kemudahan memperoleh dan menggunakan media pembelajaran di sekolah. 7. Kualitas teknis media pembelajaran yang membuat pembelajaran yang disajikan menjadi lebih mudah dicerna siswa.9 Lebih spesifik lagi dapat dijelaskan bahwa pengetahuan yang diperoleh oleh seseorang secara persentase yakni mencapai 83 % melalui penglihatan dan hanya sekitar 11 % diperoleh dari pendengaran serta sekitar 6 % dari yang lainnya. Kemampuan daya ingat terhadap pengetahuan yang telah diperoleh tersebut sekitar 20 % dari apa yang didengar dan sekitar 50 % dari apa yang dilihat. Dengan demikian peranan penglihatan lebih dominan dalam menentukan kemampuan kognitif siswa. Oleh sebab itu penggunaan media yang lebih banyak menggunakan penglihatan daripada pendengaran akan menunjang pencapaian tujuan pembelajaran. Lebih spesifik lagi dapat dijelaskan bahwa pengetahuan yang diperoleh oleh seseorang secara persentase yakni mencapai 83 % melalui penglihatan dan hanya sekitar 11 % diperoleh dari pendengaran serta sekitar 6 % dari yang lainnya. Kemampuan daya ingat terhadap pengetahuan yang telah diperoleh tersebut sekitar 20 % dari apa yang didengar dan sekitar 50 % dari apa yang dilihat. Dengan demikian peranan penglihatan lebih dominan dalam menentukan kemampuan kognitif siswa. Oleh sebab itu penggunaan media yang lebih banyak menggunakan penglihatan daripada pendengaran akan menunjang pencapaian tujuan pembelajaran. Dari beberapa pendapat yang telah diuraikan di atas , maka dapat dijelaskan bahwa media berfungsi : 1. Meletakkan dasar yang konkret untuk berfikir dan dapat mengurangi verbalisme serta dapat melampaui batas pengalaman pribadi siswa. 2. Media dapat melampaui batas ruangan kelas. 2. Media dapat melampaui batas ruangan kelas. 3. Media memungkinkan terjadinya interaksi langsung antara siswa dan lingkungannya. 4. Media memberikan informasi dan kesamaan dalam pengamatan. 5. Media akan memberikan pengertian atau konsep yang sebenarnya secara realistis dan teliti. 156 | Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 Amna Emda 6. Media membangkitkan keinginan dan minat-minat baru. 7. media membangkitkan rangsangan belajar. 8. Media akan memberikan pengalaman yang menyeluruh. 8. Media akan memberikan pengalaman yang menyeluruh. PEMANFAATAN MEDIA DALAM PEMBELAJARAN BIOLOGI DI SEKOLAH Pemakaian media pembelajaran dalam proses belajar mengajar dapat membangkitkan keinginan, minat baru, motivasi, rangsangan kegiatan belajar, bahkan dapat membawa pengaruh psikologis terhadap siswa.10 Media pembelajaran menumbuhkan semangat dan gairah belajar, terjadinya interaksi langsung antara peserta didik dengan lingkungannya. Selanjutnya dapat memberikan kesempatan kepada peserta didik untuk menggali pengalaman langsung, sehingga peserta didik tidak cepat bosan dan sangat mendukung terhadap peencapaian tujuan pendidikan yang diharapkan. Suhardi mengemukakan bahwa pengamatan langsung terhadap objek studi alami ternyata sangat menarik, objek dan gejala kehidupan sangat bervariasi, lagi pula jarang ditemui di ruang laboratorium.11 10Hamalik, Oemar, Media Pendidikan…, hal. 27. 11Suhardi, Media Pendidikan Biologi Avertebrata, Jakarta: Depdikbud Direktorat Jenderal Pendidikan Tinggi, Proyek Pengembangan Lembaga Pendidikan Tenaga Pendidik, 1992, hal.6. 13 Oemar Hamalik, Media Pendidikan, Bandung : Citra Aditya Bakti, 1994, hal 63. Manfaat Keanekaragaman Tumbuhan Herba Sebagai Media Dalam kurikulum pembelajaran Biologi terdapat konsep-konsep yang sangat baik dan menujang proses pembelajaran dengan memanfaatkan tumbuhan yang ada di alam sekitar sebagai media, khususnya Konsep Keanekaragaman Hayati. Sebagaimana kompetensi dasar yang diharapkan berdasarkan kurikulum 2004 bahwa siswa mampu mengenal dan memahami keanekaragaman hayati yang ada di alam sehingga mereka mampu mengelompokkan tumbuhan tersebut sesuai dengan jenis dan ciri-ciri yang dimilikinya. . Pengenalan langsung berbagai tumbuhan , khususnya tumbuhan herba akan merangsang panca indra anak didik secara maksimal untuk merekam bentuk dan gejala-gejala yang terjadi pada objek yang diamati. Suhardi mengatakan bahwa sebagai pendidik dalam bidang biologi guru harus mampu pula menggunakan lingkungan sekitar yang ada di alam Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 | 157 PEMANFAATAN MEDIA DALAM PEMBELAJARAN BIOLOGI DI SEKOLAH sebagai media belajar siswa, karena dapat memberi stimulus multi sensoria dan multi kesan.12 Tumbuhan herba merupakan jenis tumbuhan yang menjadi bagian dari materi pembelajaran Keanekaragaman Hayati. Tumbuhan herba yang tergolong kosmopolit paling mudah ditemukan karena dapat hidup hampir disemua tempat. Guru tidak perlu bersusah payah dan menghabiskan waktu dan biaya karena tumbuhan herba juga dapat dijumpai di lingkungan sekolah. Tumbuh-tumbuhan yang ada di lingkungan kita dan tergolong tumbuhan herba biasanya tumbuh baik di pekarangan sekolah, pekarangan rumah, di hutan, sawah, bantaran sungai, atau tempat-tempat lain yang ada di alam. Tumbuhan herba yang terdapat di semua tempat tersebut dapat dimanfaatkan sebagai media dalam pembelajaran biologi. Sebagaimana telah disebutkan sebelumnya bahwa dalam proses pembelajaran guru harus berupaya dan kreatif demi tercapainya tujuan pembelajaran yang diharapkan. Pemanfaatan tumbuhan yang ada sebagai media merupakan salah satu cara yang paling mudah dan juga sangat tepat dan cepat bagi peseta didik untuk memahami dan mengenal tumbuhan yang dipelajari dalam pembelajaran biologi. 12 Suhardi, Media Pendidikan Biologi Avertebrata,…hal 6. | Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 Macam-Macam Media Dalam Pembelajaran Biologi Secara umum media pembelajaran dapat diklasifikasikan kedalam empat jenis, yaitu : 1. Media yang dapat dilihat, seperti : papan tulis, bulletin board, gambar- gambar, grafik, peta, dan lain-lain. 2. Media yang hanya di dengar, seperti : radio, tape recorder, dan lain-lain. 3. Media yang dapat dilihat dan didengar, seperti :film dan televisi. 4. Media yang berupa benda tiga dimensi yang biasanya dipertunjukkan, misalnya model bak pasir, peta elektris, dan pertunjukan melalui pameran museum.13 Penggunaan berbagai jenis media pendidikan sangat diperlukan dalam proses belajar mengajar, khususnya yang sesuai dengan konsep apa yang diajarkan. 158 | Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 Amna Emda Terdapat beberapa jenis media pendidikan yang sesuai dengan pembelajaran di sekolah. Adapun media pendidikan yang dapat digunakan dalam pembelajaran biologi antara lain : a. Media asli dan media tiruan (model). Media asli merupakan benda yang masih dalam keadaan utuh dan asli, bukan tiruan. Macam-macam media asli antara lain: 1. Spesimen makhluk hidup yang masih hidup, seperti: aquarium dengan ikan dan tumbuhan, terrarium dengan hewan darat dan tumbuhan, kebun binatang dengan segala yang ada, kebun percobaan dengan berbagai tumbuhan, insektarium berupa kotak kaca yang berisi serangga. 2. Benda asli yang bukan makhluk hidup, seperti : kereta api, radio, pesawat terbang, teropong, mobil, dan lain-lain. Media tiruan (model) adalah benda-benda yang dibuat menyerupai benda asli, karena benda asli tidak dapat dipakai atau karena terlalu besar atau terlalu kecil, terlalu rumit, tempat yang terlalu jauh, sehingga tidak memungkinkan benda asli diperlihatkan kepada peserta didik. Macam-macam media tiruan antara lain : Macam-macam media tiruan antara lain : Macam-macam media tiruan antara lain : Macam-macam media tiruan antara lain : 1. Spesimen makhluk yang telah mati, seperti herbarium (bagian tumbuban yang sudah dikeringkan atau diawetkan), diaroma;pameran hewan dan tumbuhan yang telah dikeringkan dengan kedudukan seperti asli dialamnya, taksidermi; kulit hewan yang dibentuk kembali sesuai dengan aslinya setelah dikeringkan dan isi tubuhnya diisi dengan benda lain, awetan dalam botol; makhluk yang telah mati diawetkan dalam botol yang berisi larutan formalin,alkohol, awetan dalam cairan plastik; makhluk yang sudah mati disimpan dalam cairan plastik yang semula cair kemudian membeku. 2. Spesimen dan benda yang tak hidup, seperti berbagai jenis batuan, mineral dan lain-lain. 14 Winarno Surachmad, Dasar-Dasar dan Teknik InteraksiBelajar Mengajar, Bandung: Jammars, 1973, hal. 150. b. Media Grafis Media grafis yaitu bahan pelajaran yang menyajikan ringkasan informasi dan pesan dalam bentuk lukisan, sketsa, kata-kata, symbol gambar tiruan Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 159 c. Media bentuk papan c. Media bentuk papan Media bentuk papan yaitu media yang menggunakan benda berupa papan sebagai sarana komunikasi, seperti : papan tulis, papan tempel, papan planel, papan pameran (visual), papan magnet, papan demonstrasi, papan paku. d. Media yang disorot atau alat pandang Media ini baru dapat dimanfaatkan oleh siswa setelah diproyeksikan, seperti : media sorot yang bergerak dan media sorot mikro. e. Media dengar (audio) Berkaitan dengan indra pendengaran, yakni dapat didengar, seperti : radio, piringan hitam. f. Media pandang dengar (audiovisual) Media ini dapat didengar dan dilihat, seperti : televisi, computer. f. Media pandang dengar (audiovisual) Media ini dapat didengar dan dilihat, seperti : televisi, computer. Media ini dapat didengar dan dilihat, seperti : televisi, computer. g. Media cetak (printer material) Media cetak merupakan hasil cetak dari bahan instruksional, seperti : buku, komik, majalah, koran. g. Media cetak (printer material) PEMANFAATAN MEDIA DALAM PEMBELAJARAN BIOLOGI DI SEKOLAH yang mendekati aslinya, diagram,grafik, dan lan-lain. Media grafis meliputi: media bagan (chart), media grafik (media gambar), media poster, media karikatur, media komik, media gambar bersambung atau berseri. c. Media bentuk papan Media bentuk papan yaitu media yang menggunakan benda berupa papan sebagai sarana komunikasi, seperti : papan tulis, papan tempel, papan planel, papan pameran (visual), papan magnet, papan demonstrasi, papan paku. g. Media cetak (printer material) g. Media cetak (printer material) Media cetak merupakan hasil cetak dari bahan instruksional, seperti : buku, komik, majalah, koran. Media cetak merupakan hasil cetak dari bahan instruksional, seperti : buku, komik, majalah, koran. Pembelajaran akan berhasil bila dibarengi dengan media yang sesuai. Surachmad mengatkan dalam mengajar perlu menggunakan berbagai alat bantu untuk memudahkan guru dalam mengajar dan juga memahaminya.14 Penggunaan media yang tepat dalam proses belajar mengajar selain membantu guru dalam menjelaskan permasalahan yang sedang dikaji, juga sangat berperan dalam menimbulkan stimulasi bagi siswa serta menjadikan media sebagai sumber belajar. Dengan demikian menjadikan media tersebut sebagai bahan pelajaran yang konkret, baik secara individual maupun berkelompok sehingga akan sangat banyak membantu tugas guru dalam kegiatan belajar mengajar. Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 160 Amna Emda Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 | 161 SIMPULAN Pencapaian hasil belajar siswa setiap semesternya sangat dipengaruhi oleh faktor-faktor internal (meliputi kesehatan, intelejensi dan bakat, minat dan motivasi dan cara belajar) sera faktor-faktor eksternal (meliputi keluarga, sekolah, kualitas guru, metode mengajar, kesesuaian kurikulum dengan kemampuan anak, fasilitas dan perlengkapan sekolah, keadaan ruangan, jumlah murid perkelas, masyarakat, lingkungan dan lain-lain). Penggunaan media pembelajaran yang baik dan tepat akan memberikan keuntungan bagi guru dan siswa karena dapat membantu kelancaran proses belajar mengajar serta sangat bermanfaat bagi siswa karena sangat membantu meningkatkan pengenalan dan pemahaman terhadap materi yang diajarkan. Keanekaragaman tumbuhan secara umum dan tumbuhan herba secara khusus yang terdapat di alam, merupakan media yang dapat dimanfaatkan dan dikembangkan untuk membantu siswa dalam memahami dan juga mengenal jenis-jenis tumbuhan yang tergolong herba, khususnya yang terkait dengan materi Keanekaragaman Hayati. Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 | 161 Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 | 161 162 | Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011 DAFTAR PUSTAKA Dalyono, M., Psikologi Pendidikan, Jakarta: Renika Cipta, 1997. Deshmuk, Ian, Penerjemah Kartawinata Kuswata dan Danimiharja Sarkat, Ekologi dan Biologi Tropika, Edisi Pertama, Jakarta: Yayasan Obor Indonesia, 1992. Hamalik, Oemar, Media Pendidikan, Bandung: Alumni, 1986. ______, Media Pendidikan, Bandung: Citra Aditya Bakti, 1994. Moore, M.G dan Kearsley, G., Distance Education . A System View, Washington DC: Woodgeworth Publishing Company. Reosoedarmo, S., Kuswata, Kartawinata dan Apriliani. S, Pengantar Ekologi, Jakarta: Remaja Rosdakarya, 1985. Sadiman, Arie K., Media Pendidikan, Pengerian Pengembangan dan Pemanfaatannya, Jakarta: Raja Grafindo Persada, 2002. Steenis, C.G.J. Van, Flora Untuk Seolah di Indonesia, (Jakarta: Pradnya Paramita, 1978), hal. 1268. Suhardi, Media Pendidikan Biologi Avertebrata, Jakarta: Depdikbud Direktorat Jenderal Pendidikan Tinggi, Proyek Pengembangan Lembaga Pendidikan Tenaga Pendidik, 1992. Surachmad, Winarno, Dasar-Dasar dan Teknik InteraksiBelajar Mengajar, Bandung: Jammars, 1973. Tjitrosoepomo, G., Morfologi Tumbuhan (Spermatologi), Yogyakarta: Gajah Mada University Press, 1989. Wibawa, B. dan Mukti, F., Media Pengajaran, Jakarta: Departemen Pendidikan dan Kebudayaan, 1992. 162 | Jurnal Ilmiah Didaktika Vol. XII, No. 1, Agustus 2011
https://openalex.org/W3110934868
https://eprints.qut.edu.au/206969/1/Creative_Hotspots_Vic_Geelong_Surf_Coast_FINAL_20201125.pdf
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Australian Cultural and Creative Activity: A Population and Hotspot Analysis: Geelong and Surf Coast
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Australian Cultural and Creative Activity: A Population and Hotspot Analysis Geelong and Surf Coast Victoria Geelong and Surf Coast Victoria Geelong and Surf Coast Victoria Australian Cultural and Creative Activity: A Population and Hotspot Analysis Geelong and Surf Coast This report is an output of an Australian Research Council Linkage project (LP160101724) led by Queensland University of Technology in partnership with the University of Newcastle, Arts Queensland, Create NSW, Creative Victoria, Arts South Australia and the WA Department of Culture and the Arts. Suggested citation: Kerrigan, S., McIntyre, P. and McCutcheon, M. 2020. Australian Cultural and Creative Activity: A Population and Hotspot Analysis: Geelong and Surf Coast, Brisbane. Digital Media Research Centre. Available https://research.qut.edu.au/creativehotspots/. Australian Cultural and Creative Activity: A Population and Hotspot Analysis Geelong and Surf Coast This report is an output of an Australian Research Council Linkage project (LP160101724) led by Queensland University of Technology in partnership with the University of Newcastle, Arts Queensland, Create NSW, Creative Victoria, Arts South Australia and the WA Department of Culture and the Arts. Suggested citation: Kerrigan, S., McIntyre, P. and McCutcheon, M. 2020. Australian Cultural and Creative Activity: A Population and Hotspot Analysis: Geelong and Surf Coast, Brisbane. Digital Media Research Centre. Available https://research.qut.edu.au/creativehotspots/. s an output of an Australian Research Council Linkage project (LP160101724) led by Queensland University of Technology in partnership wi y of Newcastle, Arts Queensland, Create NSW, Creative Victoria, Arts South Australia and the WA Department of Culture and the Arts. Suggested citation: Kerrigan, S., McIntyre, P. and McCutcheon, M. 2020. Australian Cultural and Creative Activity: A Population and Hotsp Geelong and Surf Coast, Brisbane. Digital Media Research Centre. Available https://research.qut.edu.au/creativehotspots/. Strategic summary Geelong has a diverse creative scene with a relative strength in Creative Services, especially Software and Digital Content. In 2016, 4,386 people earned their primary income in the Creative Industries, an average increase of 6.25% each year since 2011. ● Geelong’s Creative Services sub-sector is a key growth area for both specialists and embe ● Geelong’s Creative Services sub-sector is a key growth area for both specialists and embedde Cultural Production is mature with a lively night time music economy and a strong performi ● Geelong’s Creative Services sub-sector is a key growth area for both specialists and embedded creativ Cultural Production is mature, with a lively night-time music economy and a strong performing arts sc ● Geelong’s Creative Services sub-sector is a key growth area for both specialists and embedded creatives. Cultural Production is mature, with a lively night-time music economy and a strong performing arts scene. ● Geelong is being referred to as ‘Australia's Silicon Valley’, with a developing boutique app industry. ● Geelong has repurposed many decommissioned industrial sites as co-working hubs, connecting the city’s ‘maker spirit’ with entrepreneurship, innovation and fabrication. ● Geelong is being referred to as ‘Australia's Silicon Valley’, with a developing boutique app industry. ● Geelong has repurposed many decommissioned industrial sites as co-working hubs, connecting the city’ ‘maker spirit’ with entrepreneurship, innovation and fabrication. The Surf Coast Shire has high levels of specialist employment in Design. In 2016, 490 people earned their primary income in the Creative Industries, an average increase of 3.47% each year since 2011. Creative migrants are flowing from Melbourne into Geelong and the Surf Coast because G21 is big enough to accommodate global and aspirational businesses yet small enough for an interconnected ecosystems ● Geelong’s size, proximity to Melbourne, and ‘can-do attitude’ are its strengths. ● The Surf Coast is the original sea-change location, and with the successful delivery o ● The Surf Coast is the original sea-change location, and with the successful delivery of the National Broadband Network (NBN), entrepreneurship and lifestyle choices can thrive. Broadband Network (NBN), entrepreneurship and lifestyle choices can thrive. ● Geelong’s ‘big shiny’ cultural hotspots are identified as Queenscliff and the Surf Coast, especially Torquay Rutland Street, Newtown, Pakington Street. he Victorian Government has a range of programs to support business, and programs and policies specifically o support Creative Industries, such as Creative State, a policy of Creative Victoria. The Victorian Government has a range of programs to support business, and progr The Victorian Government has a range of programs to support business, and programs and policies specifically o support Creative Industries, such as Creative State, a policy of Creative Victoria. o support Creative Industries, such as Creative State, a policy of Creative Victoria. ● The State Government has heavily invested in Geelong’s arts and cultural facilities, and this has allow opportunities for creatives to generate intellectual property for future success. ● Creative Victoria has an astute understanding of the value of the Creative Industries for regional economies, and that may also be the case at the local government level. ● Geelong is a UNESCO creative city, an international city of design, focused on Creative Industries an encouraging networking across CI sectors. ● The Council has fostered sustainable design and architecture and has built major public infrastructur such as Green Spine. ● Geelong and the Surf Coast community have realised that creative growth is less about having an audience, and more about the cost of living and sharing artistic experiences within the community. Recommendations: o-working spaces, collective practices and entrepreneurial activities are evident throughout the region. ● Connections are enhanced through multiple co-working spaces – two fostered by Australia Post and National Australia Bank; creative hubs in locations such as Federal Mills Park, which has attracted digita innovation businesses; Deakin University, for advanced manufacturing; Little Malop and Makers Hub for creative arts, and Pakington Street, Newtown, for creative services. ● Renew Geelong is activating empty retail spaces in central Geelong. ● An activated cultural hub on Little Malop St and in Moorabool is the result of civic policy and an active property developer and it has been able to attract a diversity of thriving retail business. ● An activated cultural hub on Little Malop St and in Moorabool is the result of civic policy and an active property developer and it has been able to attract a diversity of thriving retail business. ● Surf Coast collaboration is occurring through co-worker spaces in Torquay, the Workers Hut ● Surf Coast collaboration is occurring through co-worker spaces in Torquay, the Workers Hut and the old Quicksilver building, as well as Ashmore Arts, a commercial hub for artists. ● Surf Coast collaboration is occurring through co-worker spaces in Torquay, the Workers Hut and the old Quicksilver building, as well as Ashmore Arts, a commercial hub for artists. Quicksilver building, as well as Ashmore Arts, a commercial hub for artists. Creative migrants are flowing from Melbourne into Geelong and the Surf Coast because G21 is big enough to accommodate global and aspirational businesses yet small enough for an interconnected ecosystems The Surf Coast Shire has high levels of specialist employment in Design. In 2016, 490 people earned their primary income in the Creative Industries, an average increase of 3.47% each year since 2011. ● ‘Zombie urbanism’, a globally homogenised way to develop creative cities, as pitched in a tourism video Geelong Reinvented, has been used to reframe Geelong’s post-industrial identity. Co-working spaces, collective practices and entrepreneurial activities are evident throughout the region. The Surf Coast Shire has high levels of specialist employment in Design. In 2016, 490 people earned their primary income in the Creative Industries, an average increase of 3.47% each year since 2011. ● The Surf Coast’s Creative Services sub-sector has the highest rates of specialists and embedded employment in comparison to those in Cultural Production in the region. The Cultural Production sector has the highest number of Visual Artists in this region, closely followed by Music and Performing Arts, while many embedded creatives are Writers/Publishers. ● The Surf Coast Shire is striving for art to be seen in economic development terms; art should not be see as simply an add-on but crucial as a placemaking activity. ● Creatives in Torquay are embedded in the high-profile and economically dominant surfin ● Creatives in Torquay are embedded in the high-profile and economically dominant surfing industry. The Geelong region has a vision to become clever and creative by encouraging an economic transformation that embraces Creative Industries to help it move away from heavy manufacturing to a more mature mixed economy. ● Geelong is positioning itself as one of the leading non-capital Australian cities, and the 2020 City Deal agreement commits $3 billion over 10 years to improve regional infrastructure and community projects, supported by federal, state and local governments. ● Geelong is positioning itself as one of the leading non-capital Australian cities, and the 2020 City Deal agreement commits $3 billion over 10 years to improve regional infrastructure and community projects, supported by federal, state and local governments. ● Geelong is pivoting away from the car industry, by uniting community groups such as Creative Geelong, businesses organisations such as G21, and the Council to deliver the city’s creative vision. ● Cultural tourism is a strength for the Surf Coast, with a seasonal population that almost doubles during the summer holidays and a string of year-round events and festivals that feed into the economy. ● Cultural tourism is a strength for the Surf Coast, with a seasonal population that almost doubles during the summer holidays and a string of year-round events and festivals that feed into the economy. Final 25 November 2020 1 ● ‘Zombie urbanism’, a globally homogenised way to develop creative cities, as pitched in a tourism video Geelong Reinvented, has been used to reframe Geelong’s post-industrial identity. ● ‘Zombie urbanism’, a globally homogenised way to develop creative cities, as pitched in a tourism video Geelong Reinvented, has been used to reframe Geelong’s post-industrial identity. Recommendations: • Ensure that wider Victoria understands that the digital infrastructure offered in Geelong is known to accelerate the ongoing diversification of the broader economy through the Creative Industries. • Celebrate the cultural diversity in Geelong and the Surf Coast and amplify this through the Creative • Ensure that wider Victoria understands that the digital infrastructure offered in Geelong is known to accelerate the ongoing diversification of the broader economy through the Creative Industries. • Celebrate the cultural diversity in Geelong and the Surf Coast and amplify this through the Creative Industries. • The City of Greater Geelong and the Surf Coast Shire need to create focused Creative Industries strategic plans to harness its economic development opportunities. ● Geelong’s CBD needs further redevelopment, as central Geelong has struggled for decades to have a vibrant retail street offering. ● Creative migration is occurring in both the Surf Coast Shire and Greater Geelong, and both Councils need to maintain their awareness of the importance of career development pathways for their communities. Final 25 November 2020 2 2 Table of Contents STRATEGIC SUMMARY 1 ACKNOWLEDGEMENTS 5 BACKGROUND AND CONTEXT 6 POPULATION................................................................................................................................................................................... 6 ECONOMY ...................................................................................................................................................................................... 7 GEELONG’S POST-INDUSTRIAL IDENTITY ............................................................................................................................................... 9 CREATIVE ECONOMY......................................................................................................................................................................... 9 LOCAL GOVERNMENT POLICY CONTEXT .............................................................................................................................................. 12 CREATIVE VICTORIA, LAUNCH VIC AND REGIONAL DEVELOPMENT VICTORIA ............................................................................................. 13 CREATIVE GEELONG ....................................................................................................................................................................... 14 GRANT FUNDING – LOCAL, STATE AND FEDERAL ................................................................................................................................. 15 STRATEGIC THEME 1 WHAT ARE THE INTERRELATIONSHIPS ACROSS THE SUB-SECTORS OF THE CREATIVE INDUSTRIES? 17 CREATIVE SERVICES 17 ADVERTISING AND MARKETING = BRANDING AND DESIGN .................................................................................................................... 17 DIGITAL CONTENT VISUALISATIONS AS ANIMATION, AUGMENTED REALITY (AR) AND VIRTUAL REALITY (VR) ................................................. 18 CODEACIOUS ................................................................................................................................................................................ 18 XGAMEDEV – TORQUAY ................................................................................................................................................................. 19 ARCHITECTURE .............................................................................................................................................................................. 19 CULTURAL PRODUCTION 20 LIVE MUSIC AND RETAIL ................................................................................................................................................................. 20 BAY CITY EVENTS ........................................................................................................................................................................... 22 BACK TO BACK THEATRE ................................................................................................................................................................. 22 COURT HOUSE YOUTH ARTS ............................................................................................................................................................ 23 THE CREATIVE ENGINE – GEELONG ARTS CENTRE ................................................................................................................................ 23 COMMERCIAL AND CORPORATE SCREEN BUSINESSES ............................................................................................................................. 23 A LOCATION FOR SCREEN PRODUCTION .............................................................................................................................................. 24 GEELONG RADIO ............................................................................................................................................................................ 24 GEELONG ADVERTISER AND SURF COAST TIMES .................................................................................................................................. 24 LOCAL WRITERS AND PUBLISHERS ..................................................................................................................................................... 25 THE DOME - GEELONG LIBRARY AND HERITAGE CENTRE ....................................................................................................................... 25 NON-FICTION LITERARY FESTIVAL ...................................................................................................................................................... 25 INDEPENDENT ARTISTS – CERAMICISTS, POTTERS, PAINTERS AND ILLUSTRATORS ........................................................................................ 26 GEELONG GALLERY ........................................................................................................................................................................ 26 HUBS AND CO-WORKING SPACES 27 ASHMORE ARTS - THE SUM IS GREATER THAN THE PARTS ....................................................................................................................... 27 ANGLESEA ART HOUSE ................................................................................................................................................................... Recommendations: 27 SURF COAST CO-WORKING SPACES .................................................................................................................................................... 28 WATHAURONG ABORIGINAL COOPERATIVE ........................................................................................................................................ 28 BOOM GALLERY AND CO-WORKING SPACES ........................................................................................................................................ 28 RENEW GEELONG .......................................................................................................................................................................... 28 CREATIVE GEELONG’S MAKERS HUB ................................................................................................................................................. 29 FEDERATION MILLS – PIVOT CITY INNOVATION DISTRICT ...................................................................................................................... 29 STRATEGIC THEME 2: THE RELATIONSHIP OF CULTURAL AND CREATIVE ACTIVITY TO THE WIDER ECONOMY 30 SUPPORTING REGIONAL DEVELOPMENT IN GEELONG ........................................................................................................................... 30 Table of Contents STRATEGIC SUMMARY 1 ACKNOWLEDGEMENTS 5 BACKGROUND AND CONTEXT 3 RUNWAY GEELONG ........................................................................................................................................................................ 30 TRIBAL – GLOBAL EDUCATION SOFTWARE ........................................................................................................................................... 31 FE TECHNOLOGIES – RADIO FREQUENCY IDENTIFICATION LABELS ........................................................................................................... 31 CARBON FIBRE INDUSTRIAL DESIGN ................................................................................................................................................... 31 FORTEK GEELONG .......................................................................................................................................................................... 32 THE COMMITTEE FOR GEELONG ....................................................................................................................................................... 32 TAFE AND TERTIARY EDUCATION ..................................................................................................................................................... 32 OXYGEN – CREATIVE ARTS COLLEGE.................................................................................................................................................. 32 CULTURAL TOURISM 33 TOURISM AND CULTURAL POLICY ...................................................................................................................................................... 33 FALLS FESTIVAL ON THE SURF COAST ................................................................................................................................................. 33 MOUNTAIN TO MOUTH WALK ......................................................................................................................................................... 34 GEELONG AFTER DARK FESTIVAL ...................................................................................................................................................... 34 AUSTRALIAN NATIONAL SURFING MUSEUM ....................................................................................................................................... 35 SURF COAST ARTS TRAIL ................................................................................................................................................................. 35 LORNE SCULPTURE BIENNALE........................................................................................................................................................... 36 NATIONAL WOOL MUSEUM ............................................................................................................................................................ 36 Final 25 November 2020 Final 25 November 2020 Acknowledgements The research team gratefully acknowledge the assistance of the following people and organisations for providing the information and insights that made this report possible: The research team gratefully acknowledge the assistance of the following people and organisations for providing the information and insights that made this report possible: Geelong Participants Mark Sanders - Third Ecology Architects Jason Smith - Geelong Gallery Rochelle Smith - Word for Word writers festival Jarred Steenvoorden - FE Technologies Harrison Tribe - TAFE Graduate Catherine Ward - Ceramicists Hilton Winiecki - Oxygen Music Amy Wright - Multimedia Artist Geelong Participants Final 25 November 2020 5 Background and context Djillong, meaning ‘tongue of land’, describes the region where the Wathaurong community lived before European settlement. That community is still there, but their country is now known as Geelong and the Surf Coast (Wadawurrung Aboriginal Corporation, 2020). The geography of the region is a unique blend of coast, mountain, farmland, rainforest and urban landscapes. Geelong and the Surf Coast are treated here as one entity, although there are marked differences between them. Geelong serviced a wool industry on its western plains and, with a manufacturing and seaport past, it is now a post-industrial city. The Surf Coast has benefitted from the ‘sea change’ phenomenon. Both communities have fast-growing populations and have benefitted from their proximity to Melbourne. They are deeply integrated with this major urban centre. The region is a crucial part of the G21 Geelong Regional Alliance, which comprises the City of Greater Geelong, Surf Coast Shire, Colac Otway Shire, the borough of Queenscliff and the south-west portion of the Golden Plains Shire (Walker & Cromarty, 2018). Established in the 1830s, Geelong developed into one of the largest manufacturing regions in Victoria, with mills for wool, paper and rope. Geelong became a pivot point where cargo moved from rail to sea, and sea to rail, earning the nickname ‘The Pivot’. Population Outside of metropolitan Melbourne, the region's population, at 324,067, is the fastest growing in Victoria, with a growth rate higher than the national average. The region is increasingly sought-after by people seeking a 'sea- change' or 'tree-change', and it is a popular tourist and holiday destination. Population increases over the past decade were due to growth in service and tourism industries, as the manufacturing sector has declined. On average, the region has an older population than the whole of Victoria; with 18.1% of residents over 65 years, compared to 15.6% for Victoria (G21, 2020, p. 36). Figure 1 Demographic profile by place of residence, Greater Geelong and Surf Coast local government areas compared with greater Melbourne and regional Victoria, 2016 Sources: Australian Bureau of Statistics (2016) Geelong’s population is 263,280 and, in 2011, 65% of Greater Geelong residents lived in urban areas. The inner city has been redeveloped and inner suburbs gentrified. Many commute from Geelong to greater Melbourne for work, with ‘17,000’ workers commuting by train each day, while ‘6,500 come the other way’ (G21, 2020, p. 36). Figure 1 Demographic profile by place of residence, Greater Geelong and Surf Coast local government areas compared with greater Melbourne and regional Victoria, 2016 Figure 1 Demographic profile by place of residence, Greater Geelong and Surf Coast local government areas compared with greater Melbourne and regional Victoria, 2016 Sources: Australian Bureau of Statistics (2016) Geelong’s population is 263,280 and, in 2011, 65% of Greater Geelong residents lived in urban areas. The inner city has been redeveloped and inner suburbs gentrified. Many commute from Geelong to greater Melbourne for Sources: Australian Bureau of Statistics (2016) Sources: Australian Bureau of Statistics (2016) Geelong’s population is 263,280 and, in 2011, 65% of Greater Geelong residents lived in urban areas. The inner city has been redeveloped and inner suburbs gentrified. Many commute from Geelong to greater Melbourne for work, with ‘17,000’ workers commuting by train each day, while ‘6,500 come the other way’ (G21, 2020, p. 36). Sources: Australian Bureau of Statistics (2016) Geelong’s population is 263,280 and, in 2011, 65% of Greater Geelong residents lived in urban areas. The inner city has been redeveloped and inner suburbs gentrified. Many commute from Geelong to greater Melbourne for work, with ‘17,000’ workers commuting by train each day, while ‘6,500 come the other way’ (G21, 2020, p. 36). Population Final 25 November 2020 6 6 The Surf Coast has a population of 33,381 with a 2.6% growth rate that is ‘focused in Torquay and neighbouring Jan Juc’ (Surf Coast Shire, 2017, p. 13). The coastal lifestyle is attractive to young families and retiring ‘baby boomers’, and during summer the population doubles as ‘holiday-home residents, seasonal visitors, event crowds and daytrippers’ visit (Surf Coast Shire, 2017 p. 13). Economy The G21 Region, of which the City of Greater Geelong and the Surf Coast Shire are a part, has a diverse economy, with manufacturing, agriculture, horticulture, aquaculture, tourism, education, research, health, and services. Manufacturing still dominates, with more than 50% of regional exports, ‘about $7.9 billion’, coming from the manufacturing sector (G21, 2020, p. 36). Traditionally heavy manufacturing had been the main employment sector, based around companies such as Ford, Viva Energy and Alcoa. The 2016 closure of Ford Australia’s Geelong engine plant and Broadmeadows assembly plant put 1,200 people out of work (Beer, 2018). In Geelong, the Ford closure, combined with Alcoa closure, meant 1,000 jobs were lost between 2014 and 2016 (Mills, 2020). As the international economic climate impacted on large-scale manufacturers, leading to downsizing or closure, the region had been planning and diversifying its employment base. New investment opportunities were grasped and fostered, creating growth in new industries. Future growth areas are scientific research, advanced manufacturing (such as composite fibre), innovative agribusiness, specialist insurance and personal injury services, ICT, tourism and Creative Industries services and production (G21 2020). G21 is the Strategic Planning Committee which provides advice to Regional Development Australia (RDA) and the State and Federal Governments on the region’s needs and priorities. G21 identified regional challenges as: very high levels of socioeconomic disadvantage in specific areas; low high school completion rates and lower levels of tertiary qualifications; higher levels of unemployment and more women working part-time; and lower household and individual mean incomes (Vaughan, 2014, p. 17). The City Deal agreement is aimed at addressing many of these challenges, through a combined government commitment of $3 billion over 10 years for ‘infrastructure and community projects across the region’ (G21, 2020, p. 5). The City Deal has secured a financial commitment of $370 million: $183.8 million from the Commonwealth, $172 million from the Victorian Government, and $14.2 million from the City of Greater Geelong (G21, 2020, p. 9). Figure 2 Economic activity by ANZSIC subdivision, Greater Geelong local government area Greater Geelong The Greater Geelong region is doing well, with growth in all key economic indicators. From 2011 to 2016 the regional population grew by 2.1%, and Gross Regional Product grew 4.6%, higher than the state average. Economy Economic activity by ANZSIC subdivision, Greater Geelong local government area Greater Geelong onomic activity by ANZSIC subdivision, Greater Geelong local government area Greater Geelong The Greater Geelong region is doing well, with growth in all key economic indicators. From 2011 to 2016 the regional population grew by 2.1%, and Gross Regional Product grew 4.6%, higher than the state average. 7 7 Final 25 November 2020 Table 1 Economic indicator summary, Greater Geelong and Surf Coast local government area, 2016 Population Gross regional product ($m, 2017-18) Total employed Total businesses Greater Geelong Measure 233,426 12,340 95,058 56,982 Avg annual growth, 2011-2016 2.1% 4.6% 3.7% 2.3% % of state 3.9% 3.2% 3.9% 3.0% Surf Coast Measure 29,402 1,512 8,694 10,320 Avg annual growth, 2011-2016 2.6% 8.2% 4.0% 2.9% % of state 0.5% 0.4% 0.3% 0.6% Sources: Australian Bureau of Statistics (2016), (Australian Bureau of Statistics, 2018a), Australian Bureau of Statistics (2018b), .id (2019) or summary, Greater Geelong and Surf Coast local government area, 2016 Table 1 Economic indicator summary, Greater Geelong and Surf Coast local government area, 2016 The economy of the Surf Coast Shire is unique, and distinctly different to that of Victoria, Geelong and surrounding regions, with the surfing industry representing almost 27% of all jobs and 26.5% ($217 million) of all ‘Value Add’ activity (Surf Coast Shire, 2017 p. 31). Regional Product grew by 8.2% between 2011 and 2016. The Surf Coast Shire Council Plans for 2017-2021 report there are ‘3,000 businesses, which provide more than 7,000 local jobs’, worth more than ‘$1 billion annually’, with a 4% growth rate (Surf Coast Shire, 2017 p. 32). Small business drives the economy and the top three employment sectors are accommodation and food, construction, and retail (Surf Coast Shire, 2017 p. 32). The unemployment rate is very low; approximately half of that of the broader G21 region and Victoria itself. Shire residents are employed outside of the shire with ‘employment options including telecommuting, working part-time or working in the broader G21 area’ (Surf Coast Shire, 2017 p. 32). Locally based employment opportunities will need to increase to retain current levels of employment between 2017 and 2021 (Surf Coast Shire, 2017 p. 32). Creative economy The role and impact of the Creative Industries at a global, national, state and regional level has been examined for the G21 region, finding that Creative Industries provides growing economic benefits. The G21 Arts, Heritage and Culture Pillar aims to foster artistic creativity, cultural leadership and involvement, and the recognition and use of heritage assets by: promoting culture for all by engaging the community; developing artists, ideas and knowledge; building creative industries; acknowledging, celebrating and creating places and spaces; and promoting cultural leadership (G21, 2019). Geelong’s Creative Industries data from 2017, in the Creative Industries Environmental Scan 2018 (Walker & Cromarty, 2018), shows increased growth since 2015 for regional output (up by 0.9%), regional exports (up by 1%), total employment (up by 16.4%) and wages and salaries (Walker & Cromarty, 2018, p. 10). It draws on Cultural and Creative Industries (CCI) UNESCO definitions using 11 sectors: ‘advertising, architecture, books, gaming, movies, music, newspapers/magazines, performing arts, radio, television and visual arts’ (Walker & Cromarty, 2018, p. 18). In those CCI sectors the G21 region has 3,730 working individuals. It is male dominated, with 2,447 men to 1,283 women, and shows growth of 16.7% from 2011. The report found workers from Engineering Design and Engineering Consulting Services, Computer System Design and Related Services, and Architectural Services, had the highest incomes, the highest levels of post-graduate qualifications and worked the longest hours, probably because ‘these people may quite likely be owner/operators’ (Walker & Cromarty, 2018, p. 49). Those working in Market Research and Statistical Services and Arts Education had the lowest incomes, probably due to the ‘part-time nature of some of this work’ (Walker & Cromarty, 2018, p. 49). Employment in Arts Education; Creative Artists, Musicians, Writers and Performers; and Advertising Services sectors, represented those working less than ‘15 hours per week … these people may be freelancing or only interested in part-time work’ (Walker & Cromarty, 2018, p. 47). Regional cultural festivals are ‘strongly connected to local communities through employment, volunteerism and participation’ (Gibson, Waitt, Walmsley, & Connell, 2010, p. 280). Geelong hosts 52 festivals, with two – the Pako Festa and Geelong Show – claiming audiences of 100,000 (Gibson et al., 2010, p. 285). These small-scale festivals produce big direct and indirect economic benefits for communities (Gibson et al., 2010). Geelong’s post-industrial identity Geelong is a city searching for its ‘post-industrial identity’ (Gray, Garduno-Freeman, & Novacevski, 2017, p. 68). Academics from Deakin’s School of Architecture and Built Environment have explored the cultural re-invention of Geelong in a series of articles (Gray et al., 2017; Gray & Novacevski, 2017) and examined aggressive antisocial behavior in the CBD. The research examines the form, function and nature of Geelong’s Market Square Mall over the past 30 years (Gray & Novacevski, 2015), examines the consequences of the termination of heavy manufacturing, and seeks to explain how Geelong has repurposed industrial sites and embraced the city’s ‘maker spirit’ by connecting with innovation and fabrication (Gray et al., 2017, p. 61). The concept of ‘zombie urbanism’ (Gray & Novacevski, 2017, p. 328), where a certain globalised and homogenized effect is evident in urban planning particularly in regard to creative cities, as pitched in a tourism video Geelong Reinvented, has been used as a way to reframe Geelong’s post-industrial identity (Robot Army Productions, 2014). Geelong continues to pivot away from the car industry, even though its built environments and ‘spatial fabric remains dependent on the automobile’ (Gray & Novacevski, 2017, p. 328). Economy Tourism plays a vital role, with more than two million visitors in 2016, enjoying major surfing and musical events, generating ‘$105 million annually in expenditure’ and drawing ‘an annual viewer audience of over 32 million globally’ (Surf Coast Shire 2017, p. 35). gure 3 Economic activity by ANZSIC subdivision, Surf Coast local Government area. Figure 3 Economic activity by ANZSIC subdivision, Surf Coast local Government area. Figure 3 Economic activity by ANZSIC subdivision, Surf Coast local Government area. Final 25 November 2020 8 Creative economy Research into Australian car manufacturing and its employment of embedded creative workers found that 52% of embedded creative workers are performing the same tasks as specialist creatives (Rodgers, 2015). This research should be relevant for Geelong, because of its automobile manufacturing past, however, the de-identified case studies means it is impossible to locate these experiences as being specific to the G21 region. So, it is not clear whether any of these embedded creatives benefited from Federal Government programs to support redundant workers and supply chain businesses affected by the wider automotive manufacturing closures, such as the Geelong Region Innovation and Investment Fund, or the Ford company’s own program, Ford Workers In Transition Project (Australian Government, 2020). Regardless, Rodgers’ overall finding confirms that ‘embedded Final 25 November 2020 9 creatives are critical to innovation in manufacturing, and thus they play a key role in the future of Australian manufacturing’ (Rodgers, 2015, p. 20). High levels of employment in the Creative Services sub-sector, as described through the Creative Industries Environmental Scan 2018, (Walker & Cromarty, 2018) are visible when looking at employment by industry and occupation (see Figure 4). Greater Geelong’s Creative Services sub-sector has grown from 2011 to 2016, and there is an increasing dominance in employment in Creative Services for other industries. Cultural production sectors employ fewer people overall. The Surf Coast follows similar trends, but surprisingly the number of jobs in Creative Services in other industries fell between 2011 and 2016. Figure 4 Creative service and cultural production employment by industry and occupation, 2011 and 2016, Greater Geelong and Surf Coast local government areas Sources: Australian Bureau of Statistics (2016) Figure 4 Creative service and cultural production employment by industry and occupation, 2011 and 2016, Greater Geelong and Surf Coast local government areas Sources: Australian Bureau of Statistics (2016) Australian 2016 Census data for Geelong identifies 4,386 people who earned their primary income in the Creative Industries sector, with a creative employment intensity of 4.61% and an overall growth of 6.25% (See Appendix A.1). In particular, the Creative Services (CS) sub-sector are key growth areas for both creative specialists and embedded creatives: for Architecture and Design (8% growth), Advertising and Marketing (7.5%) and Software and Digital content, which includes software design, app development and games (6.3%). Creative economy For the Cultural Production sub-sector (CP) there is a strong performing arts and music scene (9% growth), followed by Film, TV and Radio (7.2%), a supportive network of writers, and support for the sector known as the GLAM sector (Galleries, Libraries, Archives, Museums) through the performing arts, theatres, and Geelong Library. Figure 4 Creative service and cultural production employment by industry and occupation, 2011 and 2016, Greater Geelong and Surf Coast local government areas Sources: Australian Bureau of Statistics (2016) Australian 2016 Census data for Geelong identifies 4,386 people who earned their primary income in the Creative Industries sector, with a creative employment intensity of 4.61% and an overall growth of 6.25% (See Appendix A.1). In particular, the Creative Services (CS) sub-sector are key growth areas for both creative specialists and embedded creatives: for Architecture and Design (8% growth), Advertising and Marketing (7.5%) and Software and Digital content, which includes software design, app development and games (6.3%). For the Cultural Production sub-sector (CP) there is a strong performing arts and music scene (9% growth), followed by Film, TV and Radio (7.2%), a supportive network of writers, and support for the sector known as the GLAM sector (Galleries, Libraries, Archives, Museums) through the performing arts, theatres, and Geelong Library. The Surf Coast Australian Census data for 2016 identifies 490 people earning a primary income in the Creative Industries, with a creative employment intensity of 5.58% and overall growth of 3.47% (See Appendix A.1). Creative Services has the highest level of employment of creative specialists in Architecture and Design, and strong employment levels for embedded creatives, that is those employed in other industries. For Cultural Production on the Surf Coast, the highest levels of specialist employment are in Visual Arts, closely followed by Music and Performing Arts, with other industries employing writers and those with publishing skills. The heatmaps (Figure 5 and 6) show that concentration of CS and CP professionals, measured as a proportion of total employment, differs slightly across the region. Creative services professionals are concentrated in Geelong CBD, while there is also a mild concentration of creative services professionals in Ocean Grove and Barwon Heads, which could be due to lifestyle choices. For Cultural Production, there is a concentration again in Ocean Grove and Barwon Heads, and along the Surf Coast in Lorne and Anglesea. 10 Final 25 November 2020 10 Figure 5 Creative services employment by SA2 region Employment counts Figure 5 Creative services employment by SA2 region Employment counts As a portion of total employment Figure 6 Cultural production employment by SA2 region Employment counts as a proportion of total employment Employment counts Similar but much smaller increases can also be seen in Visual and Performing Arts. Publishing shows a significant change in mean income, while the number of employed persons shows no change. Growth in the numbers of larger GST-paying businesses is subdued, except for Software and Digital Content, and Architecture and Design. Given the strong level of Victorian investment in facilities for the Cultural Production sub-sectors, it is disappointing to see no growth in earning and employment. However, it is likely that the growth is there, but not visible in this data as it draws on primary income Census data, and any secondary income, casual earnings or volunteerism is not visible. The Cultural Planner at City of Greater Geelong Council, Sahn Cramer,explains ‘a lot of people may not feel confident that they’d be able to support themselves in a creative career, and so they have another career that supports them … Most artists have another job until such time that they can be successful’ (Cramer iv, 18 June 2019). In this regard we note the economic contribution made through part-time work is invisible when drawing on Census data. with no GST registration and turnover of less than $75,000), compared with larger businesses (those registered for GST). Examination by sector indicates significant growth in Architecture and Design, and Software and Digital Content, for both micro businesses and larger businesses, for employed persons and total earnings; self- employment is stronger than employment in larger GST-paying business. with no GST registration and turnover of less than $75,000), compared with larger businesses (those registered for GST). Examination by sector indicates significant growth in Architecture and Design, and Software and Digital Content, for both micro businesses and larger businesses, for employed persons and total earnings; self- employment is stronger than employment in larger GST-paying business. Employment counts Figure 7 Creative industry employment, total earnings and mean income by place of work compared with business registrations, 2011 and 2016, Greater Geelong and Surf Coast local government areas Figure 7 Creative industry employment, total earnings and mean income by place of work compared with business registrations, 2011 and 2016, Greater Geelong and Surf Coast local government areas Sources: Australian Bureau of Statistics (2016), Australian Business Register (2019) Sources: Australian Bureau of Statistics (2016), Australian Business Register (2019) There are several creative industries sectors in the Greater Geelong and Surf Coast local government areas where there is no growth, or negative growth, such as employment and earnings for Advertising and Marketing, Film, TV and Radio, and Publishing. Further, mean incomes earned by people working in Advertising and Marketing occupations fell between 2011 and 2016. Looking at the ABN and employment data together shows increases in employment in Architecture and Design and Software and Digital Content coinciding with increases in business registrations, particularly micro businesses. Similar but much smaller increases can also be seen in Visual and Performing Arts. Publishing shows a significant change in mean income, while the number of employed persons shows no change. Growth in the numbers of larger GST-paying businesses is subdued, except for Software and Digital Content, and Architecture and Design. Given the strong level of Victorian investment in facilities for the Cultural Production sub-sectors, it is disappointing to see no growth in earning and employment. However, it is likely that the growth is there, but not visible in this data as it draws on primary income Census data, and any secondary income, casual earnings or volunteerism is not visible. The Cultural Planner at City of Greater Geelong Council, Sahn Cramer,explains ‘a lot of people may not feel confident that they’d be able to support themselves in a creative career, and so they have another career that supports them … Most artists have another job until such time that they can be successful’ (Cramer iv, 18 June 2019). In this regard we note the economic contribution made through part-time work is invisible when drawing on Census data. Employment counts Figure 6 Cultural production employment by SA2 region Employment counts as a proportion of total l production employment by SA2 region Employment counts as a proportion of total employment oyment counts as a proportion of total employment Figure 6 Cultural production employment by SA2 region Employment counts as a proportion of total employment Changes in Creative Industries employment, total earnings, mean income and business registrations can be tracked. The comet chart provides a comparison between 2011 and 2016 (See Figure 7), revealing for Geelong and the Surf Coast there has been some significant sector increases in the number of micro businesses (those Changes in Creative Industries employment, total earnings, mean income and business registrations can be tracked. The comet chart provides a comparison between 2011 and 2016 (See Figure 7), revealing for Geelong and the Surf Coast there has been some significant sector increases in the number of micro businesses (those Changes in Creative Industries employment, total earnings, mean income and business registrations can be tracked. The comet chart provides a comparison between 2011 and 2016 (See Figure 7), revealing for Geelong and the Surf Coast there has been some significant sector increases in the number of micro businesses (those Final 25 November 2020 Final 25 November 2020 11 with no GST registration and turnover of less than $75,000), compared with larger businesses (those registered for GST). Examination by sector indicates significant growth in Architecture and Design, and Software and Digital Content, for both micro businesses and larger businesses, for employed persons and total earnings; self- employment is stronger than employment in larger GST-paying business. Figure 7 Creative industry employment, total earnings and mean income by place of work compared with business registrations, 2011 and 2016, Greater Geelong and Surf Coast local government areas Sources: Australian Bureau of Statistics (2016), Australian Business Register (2019) There are several creative industries sectors in the Greater Geelong and Surf Coast local government areas where there is no growth, or negative growth, such as employment and earnings for Advertising and Marketing, Film, TV and Radio, and Publishing. Further, mean incomes earned by people working in Advertising and Marketing occupations fell between 2011 and 2016. Looking at the ABN and employment data together shows increases in employment in Architecture and Design and Software and Digital Content coinciding with increases in business registrations, particularly micro businesses. Local Government policy context The Council also has a Live Music Action Plan for central Geelong, helping to grow ‘networks and align community, commercial and government efforts towards a more active, creative and sustainable live music culture in Geelong’ (Greater City of Geelong, 2017). Renew Geelong is also operating in Geelong’s central shopping precinct through a matched funding partnership between Creative Victoria and Geelong Council (Renew Geelong, 2018). The Surf Coast Shire’s Council Plan for 2017-2021 (Surf Coast Shire, 2017) identifies population growth as a major issue, with ‘the permanent population forecast to rise from 30,048 in 2017 to almost 44,000 by 2036’ (Surf Coast Shire, 2017, p. 13). Anticipated growth in its permanent and seasonal population means the sustainable management of infrastructure and environment is critical. The Council accepts that more could be done regarding affordable housing, housing density, traffic, parking, tourism, alternative inland routes for the Great Ocean Road, the attraction of major employers, and digital technology (Surf Coast Shire 2017). The Surf Coast Shire Council recognised the challenges in a wide variety of employment opportunities, but indicated focus could be placed on promoting professional, non-seasonal, home-based and youth employment opportunities (Surf Coast Shire 2018, p. 18). Transport for those employed outside the region could be improved while the Shire’s major events were recognised as providing ‘immediate economic contributions’ to the community. The Council is also working to increase off-peak season events (Surf Coast Shire 2017, p. 32). While Surf Coast Shire Council has limited ability to directly impact the unemployment rate, it is able to support business development and attraction, create business networks and facilitate connections between education and business (Surf Coast Shire 2018, p. 30). ‘Creative Places 2019-2021 A New Vision’ presents the Shire’s development strategies which recognises ‘the vital role arts, culture and heritage plays in helping our communities and environment to thrive’ (Surf Coast Shire, 2019 p. 2). The Creative Places vision has four key themes that align with Council objectives: celebrating Aboriginal cultural heritage; placemaking; enhancing creative industries; and showcasing the best and brightest (Surf Coast Shire, 2019, p. 3). The aim is to align work in these areas with ‘the next Arts, Culture and Heritage strategy’ to be adopted in 2021 (Surf Coast Shire, 2019). Local Government policy context Arts practitioners and arts workers are supported through the Surf Coast Arts Space, the Surf Coast Arts Trail, and Surf Coast Arts Calendar, alongside the conservation of local heritage (Surf Coast Shire, 2019). Arts Development Officer at Surf Coast Shire, Harriet Gaffney, wants to work with Council’s economic development team to ‘ensure that art is not seen as simply an add-on’ but to ‘recognise the crucial need for placemaking activity within the shire’ and that developer contributions are put toward placemaking (Gaffney iv, 19 June 2019). Local Government policy context In 2018, Geelong Council delivered its ‘Council Plan 2018-22 – Putting Our Community First’, focused on improving ‘the City of Greater Geelong’s resources to deliver infrastructure, services and programs to the community in a sustainable way’ (City of Greater Geelong, 2018, p. 3). With 11 strategic priorities – four related to the Creative Industries – it is focused on incorporating arts and cultural initiatives into infrastructure development to enhance a sense of place, with a specific focus on a recognition of First Nations culture, sharing Final 25 November 2020 12 heritage, improving public art, and the GLAM sector. The delivery of creative outcomes were framed through the UNESCO Creative City of Design, bestowed on Geelong in 2017 (Creative Cities Network, 2017) and include strengthening connections with the ‘National Wool Museum; the Geelong Gallery redevelopment; Mountain to Mouth; Geelong After Dark; [and] cultural trails’ (City of Greater Geelong, 2018, p. 11). Success measures are laid out for each of these priorities, leading to increased access to cultural collections and libraries, maintaining visitations to cultural facilities, improved online engagement and ‘increased employment in creative occupations’ (City of Greater Geelong, 2018, p. 11). The Council’s specific aims for 2018-2019 included the development of a cultural strategy, attracting more national art exhibitions, supporting music and performing arts programs, commencing Osborne House remediation and conservation plans for significant infrastructure (City of Greater Geelong, 2018, p. 11) In terms of the development of Innovative Finances and Technology, the Council aims to achieve digital growth by attracting more technology-focused businesses, through enhancing smart technology and digital tools to improve service delivery such as increasing free Wi-Fi in public spaces (City of Greater Geelong, 2018, p. 15). The Council also has a Live Music Action Plan for central Geelong, helping to grow ‘networks and align community, commercial and government efforts towards a more active creative and sustainable live music culture in In terms of the development of Innovative Finances and Technology, the Council aims to achieve digital growth by attracting more technology-focused businesses, through enhancing smart technology and digital tools to improve service delivery such as increasing free Wi-Fi in public spaces (City of Greater Geelong, 2018, p. 15). Creative Victoria, Launch Vic and Regional Development Victoria Victoria has stolen the march on other Australian states in attracting Creative Industries. The Victorian Government has an advanced understanding of 21st century Creative Industries; how they are constituted and how they contribute to highly interlinked state and regional economies. Capitalising on this knowledge, the Final 25 November 2020 13 Government gives agency to Creative Victoria, Launch Vic and Regional Development Victoria, who work across a dispersed network that creates an ecosystem to support the totality of regional Creative Industries. Creative Victoria’s schemes have enabled a rich creative ecosystem to exist by offering programs that ‘stimulate high quality, diverse creative activity across the state’ (Creative Victoria, 2020). Their Creative Activation Fund, Innovation in Marketing Fund, Quick Response Grants, Unlocking Capacity Grants, and First People’s Creative Industries Traineeship program offer real creative opportunities (Creative Victoria, 2020). Launch Vic is generating a globally connected start-up ecosystem. Since 2016, it has dispersed $6.5 million in funding to support 26 Victorian start-ups (Launch Vic, 2020) and has conducted mapping of the Victorian Start-up Ecosystem, Angel Networks, Sports Tech, Health Tech, Victorian Tech start-ups and digital marketplaces (Launch Vic, 2020). Regional Development Victoria (RDV) supports industries and new investments that lead to jobs in the regions. Its industry specialists work directly with businesses, and its economic development teams work with non-business organisations such as councils, chambers of commerce and universities, ‘groups that work across multiple areas’ (Ryan iv, 18 June 2019). RDV leads engagement through the government’s Regional Partnerships initiative which includes a committee of community representatives, the ‘community’s voice into government’ (Ryan iv, 18 June 2019). RDV works closely with G21 in the pillars of economic development and education and training. The GROW (G21 Regional Opportunities for Work) Program was established in Geelong with stakeholders including G21, the Chamber of Commerce and the Committee for Geelong, as well as industry focused groups such as Geelong Manufacturing Council and Creative Geelong. Grant Funding – Local, State and Federal Grant Funding – Local, State and Federal Each level of government has strategies designed to fund the Creative Industries and, when examined holistically, it is possible to see the ecosystem at work. Regional Development Victoria and Creative Victoria contributed to Stage two of the Geelong Arts Centre (GAC) redevelopment, The Creative Engine, which opened late 2019. The $38.5 million redevelopment (Brereton, 2019) saw the redesign of the former Mechanics Institute and sandstone church on Ryrie Street become a ‘massive glass and steel cantilevered building’ providing ‘state-of-the-art dance studios’ and improved accessibility (Ryan iv, 18 June 2019). Stephen Ryan, RDV Manager for Economic Development, says the GAC has ‘nurtured and developed a number of performing arts companies and other enterprises and businesses’ over the years in ‘an ad hoc way’ in ‘higgledy-piggledy’ old buildings, and this more recent redevelopment aims to provide ‘dedicated co-working spaces’ for creatives. RDV emphasises the benefits that will come from the co-location of artists with the GAC (Ryan iv, 18 June 2019). Stage three of the Geelong Arts Centre redevelopment will be a $128 million theatre redevelopment, funded by Creative Victoria. Through ongoing Creative Victoria funding, The Creative Engine offers opportunities of cash and in-kind projects, between $5000 and $20,000, to support ideas where artists meet audiences (GAC, 2020). Figure 8 Cultural grants by investment type, Geelong & Surf Coast Figure 9 Federal, state and local cultural grant and infrastructure funding Geelong & Surf Coast Geelong Council provides ongoing administrative support for the gallery, the library and the Courthouse youth arts centre. It provides $220,000 in small grants annually – including the Creative Community Grants (Geelong City Council, 2020) program in which $120,000 goes towards arts grants and $100,000 towards festivals to ‘support arts development and to impact community development through art works and art projects’ (Esler iv, Figure 8 Cultural grants by investment type, Geelong & Surf Coast Figure 8 Cultural grants by investment type, Geelong & Surf Coast Figure 8 Cultural grants by investment type, Geelong & Surf Coast Figure 9 Federal, state and local cultural grant and infrastructure funding Geelong & Surf Coast Figure 9 Federal, state and local cultural grant and infrastructure funding Geelong & Surf Coast Geelong Council provides ongoing administrative support for the gallery, the library and the Courthouse youth arts centre. Creative Geelong Creative Geelong Inc., an industry focus group supported by RDV, was founded in 2015 by Jen Cromarty, also the CEO of the Committee for Geelong, ‘to give Geelong a sense of identity as a creative centre’ (Menaul iv, 18 June 2019). Creative Geelong is involved in a range of projects and seeks support from Councils, the Victorian Government, non-government agencies, business and community to collaborate and partner with them. The board for Creative Geelong included members of the Civic Collective, the Committee for Geelong, Netgain, Geelong After Dark/Pako Festa, Auld Planning and Projects, Codeacious, Rogue Academy and BATForce. Creative Geelong strives to provide a forum for local creative collaboration, understand the work of the future, to tell stories of Geelong’s creativity and to solve problems in the community (Creative Geelong, 2019). Creative Geelong began as a social network on Facebook, and the not-for-profit continues to demonstrate the importance of the Creative Industries to the region’s transformation, through research, advocacy, filmmaking and crowdfunding. It has created connections that change lives: ‘People who weren’t in work, got work. People who had a business, have grown their business’ (Cromarty iv 17 June 2019). Educational pathways for creative youth are also important, but not all creative development is linear. Padraic Lee Fisher, the National Wool Museum Director at Geelong Gallery, suggests that the ‘linear progression of career opportunity’ is ‘not how it’s happening’ and ‘not the only way it happens, particularly not in the 21st century’ (Fisher iv, 18 June 2019). Instead, ‘mid-career artists’ or ‘creatives’ are ‘finding themselves transitioning from one thing to another. They may be working as a seamstress someplace and that might be considered creative, but they are actually a performing artist who’s doing other things, doing installation art’ (Fisher iv, 18 June 2019). People may learn ‘employable trades’, such as automotive painting, and then transition those skills into creative industries (Fisher iv, 18 June 2019). Some transition at the end of their career or ‘as a secondary career’ (Fisher iv, 18 June 2019). ‘Very few are lucky enough to make their passion their career’ (Fisher iv, 18 June 2019). 14 Final 25 November 2020 14 18 June 2019). Biennially and triennially, the Council offers opportunities for larger projects and festivals. In 2017- 2018 the allocated budget was $184,300 with another round in 2019-2020 (Geelong City Council, 2019). The grant program is also ‘an interface with our community, and an opportunity for hearing more about their projects’ which can help local organisations in ‘touring and helping them get connected’ (Esler iv, 18 June 2019). Surf Coast Shire offers an annual Arts Development Seed funding round. Advertising and Marketing = Branding and Design The global trend where there is a merging of Advertising and Marketing with the Digital Design sub-sectors is reflected in Geelong and the Surf Coast. These sub-sectors offer specialist creative services for other CI businesses as well as businesses in the wider economy where Geelong’s largest source of creative employment is in advertising and marketing. It accounts for the largest numbers of jobs by both industry and occupation and has the highest earnings by occupation, which has doubled from 2006 to 2016. However, marketers are more likely to be embedded in non-creative industries than employed in specialist marketing firms, whereas advertising and design creatives can work in very small or medium businesses which offer creative services in digital collateral creation through website design and social media management. Geelong and the Surf Coast have some leading agencies including Fluid, a branding agency located in Torquay and South Melbourne, and Pace, in Geelong, the oldest operating advertising agency in Australia. Pace, established in 1964 (Pace, 2019), emerged from the advertising department of the Ford car dealership group (Heath iv 17 June 2019). Today, it employs 15 people and is a full-service agency, offering traditional advertising services, digital strategies and marketing services including websites, apps and digital production. Pace owner Nicholas Heath said the company’s employment profile has changed in line with trends. In the 1960s there was an Art Department, with artists who would create newspaper advertisements, and they also employed writers and musicians. These employees would have been included as occupations within Cultural Production whereas today they employ ‘marketers’, ‘designers’ and ‘code developers’, who are part of the Creative Services. Digital marketing includes leveraging Search Engine Marketing (SEM) and Search Engine Optimisation (SEO), and social media, while digital production includes ‘writing blog content and EDM development to YouTube video production’ (Pace, 2019). The employee talent pool has changed dramatically. Today, Pace employs university graduates as designers and coders, and freelancers to make television commercials, and that relationship has been stable for 20 years (Heath iv 17 June 2019). Choc Chip, a digital marketing agency specialising in app development, was recently purchased by Pace, with the intention of maintaining it as a separate entity. Pace runs an internship with Deakin University which helps identify potential employees (Heath iv 17 June 2019). Fluid, a branding agency recognised locally for producing very high-quality work, presented at the G21 Pivot Summit in 2019. What are the interrelationships across the sub-sectors of the creative industries? The makeup of the populations of Geelong and the Surf Coast means all occupations across the sub-sectors have an existence in the region and this breadth and depth, in terms of business longevity and/or highly skilled mature practitioners, is where the interconnections between the CI sub-sectors occur. With people working in large firms, micro businesses or as freelance creatives, there are high quality international services and products available in the G21 region. The diverse nature of work within and between these sub-sectors includes occupations that would not normally be associated with creative work. Conversely, many local firms, such as real estate agencies, have marketing, website and social media teams. The most obvious interrelationships occur between the Creative Services sub- sectors, and examples demonstrate a highly mature and dynamic sector existing in this region. Grant Funding – Local, State and Federal It provides $220,000 in small grants annually – including the Creative Community Grants (Geelong City Council, 2020) program in which $120,000 goes towards arts grants and $100,000 towards festivals to ‘support arts development and to impact community development through art works and art projects’ (Esler iv, Final 25 November 2020 15 18 June 2019). Biennially and triennially, the Council offers opportunities for larger projects and festivals. In 2017- 2018 the allocated budget was $184,300 with another round in 2019-2020 (Geelong City Council, 2019). The grant program is also ‘an interface with our community, and an opportunity for hearing more about their projects’ which can help local organisations in ‘touring and helping them get connected’ (Esler iv, 18 June 2019). Surf Coast Shire offers an annual Arts Development Seed funding round. 18 June 2019). Biennially and triennially, the Council offers opportunities for larger projects and festivals. In 2017- 2018 the allocated budget was $184,300 with another round in 2019-2020 (Geelong City Council, 2019). The grant program is also ‘an interface with our community, and an opportunity for hearing more about their projects’ which can help local organisations in ‘touring and helping them get connected’ (Esler iv, 18 June 2019). Surf Coast Shire offers an annual Arts Development Seed funding round. 18 June 2019). Biennially and triennially, the Council offers opportunities for larger projects and festivals. In 2017- 2018 the allocated budget was $184,300 with another round in 2019-2020 (Geelong City Council, 2019). The grant program is also ‘an interface with our community, and an opportunity for hearing more about their projects’ which can help local organisations in ‘touring and helping them get connected’ (Esler iv, 18 June 2019). Surf Coast Shire offers an annual Arts Development Seed funding round. 16 Final 25 November 2020 Final 25 November 2020 16 CREATIVE SERVICES Advertising and Marketing = Branding and Design Codeacious Geelong’s Arts and Culture Trails app was designed by Codeacious, a creative software studio. Codeacious builds business apps and does ‘fun, interesting integration-type projects with other artists’ (Priddle iv, 17 June 2019), such as the augmented reality project based on a mural of the late singer Chrissy Amphlett in Little Malop Street, commissioned by the City of Greater Geelong. Their goal long-term is to be a ‘product business’, not necessarily a ‘services business’ (Priddle iv, 17 June 2019) and head of design Ian Priddle said they are working on a ticketing product that will allow event organisers to ‘create experiences for attendees’ (Priddle iv, 17 June 2019). Priddle said connection between local creatives is vital, and ‘a level of government policy’, local and state, is ‘contributing to those interconnections’ through small grants and broader economic development (Priddle iv, 17 June 2019). Good internet has been vital. Priddle said Codeacious doesn’t use ‘the NBN, the product provided by the government’ (Priddle iv, 17 June 2019) because Geelong had HFC fibre installed by a private company, now operating as iiNet. This provides internet access speeds of ‘250 megabit down and 50 megabit up … We couldn’t run our business without internet like that’ (Priddle iv, 17 June 2019). Geelong’s Arts and Culture Trails app was designed by Codeacious, a creative software studio. Codeacious builds business apps and does ‘fun, interesting integration-type projects with other artists’ (Priddle iv, 17 June 2019), such as the augmented reality project based on a mural of the late singer Chrissy Amphlett in Little Malop Street, commissioned by the City of Greater Geelong. Their goal long-term is to be a ‘product business’, not necessarily a ‘services business’ (Priddle iv, 17 June 2019) and head of design Ian Priddle said they are working on a ticketing product that will allow event organisers to ‘create experiences for attendees’ (Priddle iv, 17 June 2019). Priddle said connection between local creatives is vital, and ‘a level of government policy’, local and state, is ‘contributing to those interconnections’ through small grants and broader economic development (Priddle iv, 17 June 2019). Good internet has been vital. Priddle said Codeacious doesn’t use ‘the NBN, the product provided by the government’ (Priddle iv, 17 June 2019) because Geelong had HFC fibre installed by a private company, now operating as iiNet. Digital Content Visualisations as Animation, Augmented Reality (AR) and Virtual Reality (VR Specialist software and digital content businesses exist in Geelong and the Surf Coast, operating as IT specialists, games developers, AR and VR, and animation studios. Local businesses include Enterprise Monkey, Hanalei Studios, Codeacious, XGameDev and Pillowfort Creative and each appears to be a key example of the interconnections between cultural production and creative services. Pillowfort Creative is a small animation studio whose services include ‘2D traditional, 2D digital, 3D, motion graphics, architectural visualization, AR and a bit of VR’ (Murray iv, 18 June 2019). Co-founded by duo Leigh Ryan and Stuart Murray, their animation services are unique to Geelong, and there is no local competition (Ryan iv, 18 June 2019). Drawing on their 15-year freelance careers as ‘animators and illustrators’ for kids TV shows for broadcast, they provide ‘a crazy mixed bag’ of creative services (Murray iv, 18 June 2019). Local jobs include animations for the National Wool Museum, motion graphics for Deakin University, and a visualisation of products for designer Penny Scallon. Globally, they have worked on a web series for Zuru, and produced ‘nine episodic, two-minute shorts for kids, based on a toy franchiser in China’ (Ryan iv, 18 June 2019). Located in the Creative Geelong Makers Hub, their aim is to ‘establish the animation industry in Geelong’ by pitching ideas for broadcasters to fund a series and employ locals (Murray iv, 18 June 2019). Enterprise Monkey is co-located in Melbourne and North Geelong. They aim to solve complex business problems through simple digital solutions such as web applications, web design, and development solutions that are achieved through mobile applications. Hanalei Studios is run by Luke Harris, a freelance game developer and XR developer. He works with Extend Reality Tech, including mixed reality, virtual reality and augmented reality. Clients include the University of Melbourne, Deakin University, German manufacturer Bosch Rexroth, and local start-ups such as Pillowfort. Advertising and Marketing = Branding and Design The Executive Creative Director, Clifton Daniell, asserts that ‘authenticity is the currency of today’s branding’ (iv, 19 June 2019). Fluid has a large corporate client base in Melbourne, including Carlton United Beverages and BHP. Established in 2001, Fluid employs 15 people across two offices. Elk Creative is a small design agency, with two full-time workers. Core business is designing and branding through websites for small to medium-sized local businesses; ‘bread and butter jobs’ are around $20,000 for trades Final 25 November 2020 17 businesses (Dodd iv, 17 June 2019). Elk Creative designs everything in-house and specialises in brand consistency across all business assets: logos, brochures, business cards, vehicles and digital collateral. Owner Corey Dodd’s sales pitch is that a polished business website helps to build ‘trust’ with clients because it delivers an accurate reflection of what each business actually is (Dodd iv, 17 June 2019). businesses (Dodd iv, 17 June 2019). Elk Creative designs everything in-house and specialises in brand consistency across all business assets: logos, brochures, business cards, vehicles and digital collateral. Owner Corey Dodd’s sales pitch is that a polished business website helps to build ‘trust’ with clients because it delivers an accurate reflection of what each business actually is (Dodd iv, 17 June 2019). Geelong and Surf Coast have a number of smaller advertising and marketing firms such as Video Vault, Flip Switch Media, and The Tribe, as well as freelancers such as marketer Jo Smith, who does work for big retailers such as Myer and Energy Australia. XGameDev – Torquay XGameDev has designed 18 games but their core work is engineering for others with a specialisation in ‘multi- player game development’ (Leemans, J. iv, 19 June 2019). Clients, including Pubbly, Aerobo and Creative Curriculum International, are worldwide including from the United States, Canada, Denmark, New Zealand, Egypt and South Africa. South African-born husband and wife Jacques and Melanie Leemans run the business from their Torquay home, a location they chose for their children’s schooling as much as for its gaming reputation and creative community. His experience as a software engineer and hers in Microsoft service and support combined to create their business, but it almost faltered at the start because of poor internet. Arriving in 2015, Torquay did not have the NBN: ‘That was the last thing we even thought of, coming from Third World, going to First World. The internet speed was five download, one upload’ (Leemans, J. iv, 19 June 2019). Added to the shock that their assumption about connectivity in a supposed First World country was mistaken, the slow speeds soon affected their business, a business premised on reliable digital infrastructure. Within two months they had no clients left and Jacques had to find a job and was fortunate to be employed at Deakin University: ‘I worked there for two years and then midway through that the NBN came in’ (Leemans, J. iv, 19 June 2019). Now with fibre to the node they achieve 40 Mbps download, 10 Mbps upload; ‘not ideal, but it’s fine’ (Leemans, J. iv, 19 June 2019). Fast internet is essential because they act as developers and project managers for clients who generally provide the assets required –narration, animation, text and music files. Their suppliers are also international. Their audio editor works from South Africa and they outsource some graphic design, but it’s ‘in the cloud’ and freelancers can be anywhere in the world. Their long-term goal is to move away from a service provider business to make more of their own games (Leemans, J. iv, 19 June 2019). The challenge is paying the bills. While they have researched venture capital, ‘they want you to build a product where they can get a big return on investment in a year or two’ (Leemans, J. iv 19 June 2019). For them, seeking government grants requires time to ‘build a prototype’ or spend ‘a week on documents unbilled’ which they ‘just financially cannot’. XGameDev – Torquay ‘It’s the balance of having clients right now [and] trying to build your own thing on the sideline’ (Leemans, M. iv, 19 June 2019). Codeacious This provides internet access speeds of ‘250 megabit down and 50 megabit up … We couldn’t run our business without internet like that’ (Priddle iv, 17 June 2019). Final 25 November 2020 18 Live Music and Retail Considering its proximity to Melbourne, the music scene in Geelong is well serviced by performers, booking agents, retailers, suppliers, recording studios and venues. The Surf Coast has the very successful Falls Festival, while Geelong hosts music festivals such as the Queenscliff Music Festival, Meredith Music Festival, Offshore Festival, Poppykettle Festival, and National Celtic Festival. Notable musicians from Geelong include Chrissy Amphlett, Barry Crocker, Gyan Evans, Magic Dirt, Jeff Lang and Denis Walter. The City of Greater Geelong has a Live Music Action Plan which is important, as music shop owner Deniel Bee indicates, audiences are hard to come by: ‘People just aren’t going out during the week’; it’s more expensive, and they have the ‘distractions’ of streaming services such as Netflix and Stan (Bee iv, 17 June 2019). The Live Music Action Plan focuses on ‘four elements’: venues, artists, audiences, and a ‘broader community that are in principle supportive of the arts, whether or not they attend the arts’ (Esler iv, 18 June 2019): City of Greater Geelong Coordinator of Arts and Cultural Development Duncan Esler asserts that ‘if you have a community that are complaining because there is too much loud music and too much noise, or they see the city festivals interfering with their day-to-day, it makes it more difficult to deliver’ (Esler iv, 18 June 2019). Geelong is in ‘a growth phase’ and will ‘hit a threshold’ where it can potentially ‘sustain programs and nightlife entertainment and live music’ (Cramer iv, 18 June 2019). Cultural Planner at City of Greater Geelong Council, Sahn Cramer, suggested ‘that’s what we will work towards, and it’s just a balancing game of trying to attract that population out of Melbourne at the same time build investment in arts and culture’ (Cramer iv, 18 June 2019). The Live Music Action Plan recognised that Geelong based musicians will need to leave Geelong to establish themselves with metropolitan audiences (Esler iv, 18 June 2019). The plan also seeks to encourage established artists to become mentors for other local musicians. In terms of venues the Piano Bar Geelong is an interesting one. It employs local musicians who draw an over 35 crowd that is 80% female – ‘the three Gs – the grannies, the girlies and the gays’ (Pobjoy iv, 17 June 2019). Architecture Geelong typically has sole practitioners or very small firms of architects employing between two and five staff. There are a few that have between five and 10 employees, but most of the large firms are in Melbourne. The architecture sector employs two types of CI specialists, Architects, and Software and Digital Content specialists. Architecture also employs more support staff than other Creative Industry sub-sectors. Clients are dispersed throughout the broader economy, with commercial, retail, education and healthcare customers, as well as developers. Melbourne firms win most of Geelong’s big architectural jobs, which have budgets of more than $2 million, and it’s common for Geelong architects to commute to Melbourne. Deakin University’s Geelong Campus has a thriving School of Architecture. Local firms, such as Select Architects, offer ‘end-to-end’ services for commercial, retail and town planning clients. Others, such as Cotter Reid, and James Deans & Associates, have been operating in Geelong for nearly 20 years. Practices such as Zohdy & Associate Architects, and 3iD Architecture, operate in Geelong and the Surf Coast. It’s also become common for Melbourne firms to set up with a Geelong address, while never actually operating in Geelong (Sanders iv, 18 June 2019). Third Ecology Architects is a solo architectural practice, but pre-GFC it employed 15 staff. Today, it specialises in design and project management of new homes, renovations and multi-residential projects for owner-occupiers, investors and developers across Geelong, the Surf Coast, Otways and the rural hinterland. Third Ecology has a good reputation and owner Mark Sanders said ‘word of mouth’ is the main way of attracting new clients (Sanders iv, 18 June 2019). Final 25 November 2020 19 19 On the Surf Coast there are high quality residential architects, such as Peter Winkler Architects, established in 2005 (Winkler, 2019). The practice specialises in medium- to high-end residential dwellings, multi-residential and office fit-outs. In 2017, Winkler won the Timber Design Awards, Recycled Timber House of the Year (Winkler iv, 19 June 2019). Winkler, a Deakin University graduate, runs a three-person practice, implementing architectural solutions that realise both function and form, requiring a unique creative design response to each site. Operating from a home office on the Surf Coast, Winkler has calculated that with ‘one staff I can just survive. With two staff, I start to move ahead really well’ which means they can be profitable and efficient (Winkler iv, 19 June 2019). Architecture Their smallest projects are around $650,000, for a renovation, with the average project sitting at $1 million to $1.2million. In 2019 Winkler was working on an Apollo Bay, ‘four-bedroom architectural-designed home priced around $2.1 million’ (Winkler iv, 19 June 2019). CULTURAL PRODUCTION Live Music and Retail Live Music and Retail Owner and jazz musician Andy Pobjoy employs two staff, 25 hours a week to handle social media and bookings which includes touring performers such as Rick Price and Dolly Diamond, tribute bands, local bands and drag shows, but ‘core business’ three nights a week is a piano man who takes requests. The Piano Bar guarantees local musicians ‘three-and-a-half, gigs a week’ (Pobjoy iv, 17 June 2019). Business growth hinges on ‘keeping overheads at a minimum’ which is possible in the regions (Pobjoy iv, 17 June 2019). The bar is decorated with paintings by local artist Jae Allen. The recording studio, don’t Poke the BEAR, is located in Geelong West close to the heart of the city. Competing with Melbourne’s entrenched and historically successful recording studios, it offers production services with a Final 25 November 2020 20 roster of freelance professional engineers and producers who cater to a diverse range of musical projects. These recording projects come from up-and-coming artists through to fully established music professionals as well as from other parts of the Cultural Production sector. For example, a number of radio plays have been recorded there which were completed in conjunction with the Greater Geelong Creative Inc, the City of Greater Geelong and Skin of Our Teeth Productions. One of these, ‘The Machine Stops’, was originally written by EM Forster and adapted by E. Watson, directed by R. O’Connor and produced by Someone New Theatre. It was engineered in the studio by the award-winning Travis Dom. The studio also has rehearsal suites and live sound equipment for hire. It is owned by three brothers who also own a private creative arts college. Oxygen – Creative Arts College is a private provider in Geelong offering vocational tertiary programs in music, photography, digital media, visual arts, film and design. It is supported through the Victorian Government’s Skills First program. Founded in 2006 by musician brothers Tony, Joe and Dominic Monea, it grew out of a music school they started in 1986 as a way ‘to supplement their performance income via regular teaching’ (Monea iv, 17 June 2019). The school continues, with private instrument tuition for 100 students. While most of their students stay local, there are people ‘doing international work’, such as musicians Xavier Rudd and Evangeline, and Aboriginal artist Yirrmal. Geelong businesses have been supportive in terms of offering internships and work experience to students. Live Music and Retail As Dominic Monea claims, ‘there’s no substitute for experience, and getting in and getting your hands dirty’, and Geelong Workers Club is a strong promoter of local original music (Monea iv, 17 June 2019). They’ve found dealing with local government ‘a lot of red tape’. They employ a digital content specialist and a blogger to help tell their story. The internet has been vital to their business, especially teaching photography and graphic design where applications such as Adobe are ‘in the cloud’, and they were fortunate that ‘the street was picked up by network cable’ nine years ago, prior to the NBN. These brothers also started the music retailer Oxygen Music. This retail outlet sources instruments through wholesalers in Sydney, Brisbane and Melbourne, from manufacturers in China and Japan, with some from the United States and Europe (Winiecki iv, 18 June 2019). They have an in-house string instrument repairer and outsource bigger jobs (Winiecki iv, 18 June 2019). They have an online store, but most business is in-store, with promotion through social media and email (Winiecki iv, 18 June 2019). They partner with local schools in ‘battles of the bands’ and provide ‘sponsorship dollars’ in prizes. For 10 years, they ran Drumania, a festival featuring student drummers, teachers and touring professionals ‘from all over the world’, workshops, expos and a family sausage sizzle (Winiecki iv, 18 June 2019). Apart from some local TV coverage, ‘the council never came on board with any support’ (Winiecki iv, 18 June 2019) and it was discontinued. Music Workshop Geelong is a retail, production and installation company servicing Geelong and the western district. Its ongoing existence indicates the strength and depth of the music scene in this regional centre. Predominantly guitar-based, it’s a 40-year-old family business that employs six full-time staff, four casuals, and seasonally up to 30 contractors; ‘once the sun’s out, it all happens’ (Bee iv, 17 June 2019). General manager Deniel Bee said the ‘wholesale model’ means it can be faster to get replacement ‘parts from New York than from Perth to Geelong’ because ‘retail in this country’s not big enough to demand the ability to purchase direct from the manufacturer’ (Bee iv, 17 June 2019). Customers are savvier than they used to be; ‘pre-internet, customers came in wanting to source a product based on your advice. It’s very much shifted now. Bay City Events Bay City Events is a Geelong events company located on Little Malop St that has been in business for 40 years focused primarily around musical events. Having been previously involved with Queenscliff Music Festival, Echuca Winter Blues, Motor City Music Festival and the Royal Geelong Show, their focus is now on promoting the Queenscliff Blues Train dinner and show, and music programming for the Festival of Sails and venues such as the Flying Brick Cider House. Director Hugo Armstrong is proud to use all Geelong suppliers for staging and screens, security, fencing, marquees and toilets, and local creative services professionals for web design, graphic design and advertising (Armstrong iv, 20 June 2019). Musical performers are a ‘mix of local and Melbourne, a little bit of interstate’, and punters come from Melbourne with a ‘reasonable amount from Geelong, depending on the event’ and ’strong numbers out of Ballarat, Bendigo, Shepparton and Waurn Ponds’ (Armstrong iv, 20 June 2019). Armstrong said promotion and coverage of events is an issue: ‘We can’t get any media coverage because we’re relying on Melbourne’ and big media players ‘don’t cover Geelong except for the AFL’ (Armstrong iv, 20 June 2019). Figure 10 – Back to Back Theatre Income and Expenses 2014-2019 36% 31% 15% 14% 4% 0% 10% 20% 30% 40% PERFORMANCE INCOME OPERATION GRANTS PROJECT GRANTS PRIVATE … SERVICE FEES Back to Back Theatre Average Income 2014-2019 29% 26% 19% 16% 9% 3% 0% 10% 20% 30% 40% ARTISTS WAGES CORE EMPLOYEE WAGES PRODUCTION COSTS PROJECT DELIVERY WAGES OVERHEADS MARKETING COSTS Back to Back Theater Average Expenses 2014-2019 Back to Back Theater Average Expenses 2014-2019 Back to Back Theatre Average Income 2014-2019 Established 30 years ago, they operate on the presenter model: ‘We don't hold the ticket sale responsibility because we sell out our shows to presenters’ (Branch iv 18 June 2019) Creating original work Back to Back Established 30 years ago, they operate on the presenter model: ‘We don't hold the ticket s years ago, they operate on the presenter model: ‘We don't hold the ticket sale responsibility because we sell out our shows to presenters (Branch iv, 18 June 2019). Creating original work, Back to Back Theatre’s ‘selling point is ourselves, to the presenters of a festival ... that could be locally, nationally or internationally’ (Harvey iv, 18 June 2019). Employing the presenter model effectively shows the artistic directors of the company have a mature understanding of how to develop their intellectual property. The raising of private income and investment began in a concerted way in 2014, and the outcome was approximately $160,000 (Back to Back Theatre, 2014, p. 30). The largest source of income is performances (36%) followed by operational grants (31%). In terms of expenses, artists’ wages are highest (29%) followed by core employee wages (26%) and production costs (19%). Interviews revealed how the company has achieved these financial outcomes as well as continuing to grow their international performance reputation. Funded through all levels of government, the quality of their work and international reputation is due to the dedication of Executive Producer Alice Nash and Artistic Director Bruce Galdwin. This production team builds ‘those relationships and negotiates those performance fees. And the marketing team supports us by providing us with collateral that we can use to sell the because we sell out our shows to presenters (Branch iv, 18 June 2019). Creating original work, Back to Back Theatre’s ‘selling point is ourselves, to the presenters of a festival ... that could be locally, nationally or internationally’ (Harvey iv, 18 June 2019). Back to Back Theatre Back to Back is a unique theatre company employing an ensemble of six artists with intellectual disabilities. A mainstay of the international circuit, Back to Back creates extraordinary contemporary work in Geelong which is commissioned and co-funded by international theatre presenters, festivals and venues. The company exists ‘because of those artists, not for those artists’ (Harvey iv, 18 June 2019). Annual reports since 2012 lay out an impressive body of internationally recognised performances (Back to Back Theatre, 2020, p. Publications; Holden, 2016). Figure 10 – Back to Back Theatre Income and Expenses 2014-2019 Established 30 years ago, they operate on the presenter model: ‘We don't hold the ticket sale responsibility because we sell out our shows to presenters’ (Branch iv, 18 June 2019). Creating original work, Back to Back Theatre’s ‘selling point is ourselves, to the presenters of a festival ... that could be locally, nationally or 36% 31% 15% 14% 4% 0% 10% 20% 30% 40% PERFORMANCE INCOME OPERATION GRANTS PROJECT GRANTS PRIVATE … SERVICE FEES Back to Back Theatre Average Income 2014-2019 29% 26% 19% 16% 9% 3% 0% 10% 20% 30% 40% ARTISTS WAGES CORE EMPLOYEE WAGES PRODUCTION COSTS PROJECT DELIVERY WAGES OVERHEADS MARKETING COSTS Back to Back Theater Average Expenses 2014-2019 Figure 10 – Back to Back Theatre Income and Expenses 2014-2019 Figure 10 – Back to Back Theatre Income and Expenses 2014-2019 Established 30 years ago, they operate on the presenter model: ‘We don't hold the ticket sale responsibility because we sell out our shows to presenters’ (Branch iv, 18 June 2019). Creating original work, Back to Back Theatre’s ‘selling point is ourselves, to the presenters of a festival ... that could be locally, nationally or internationally’ (Harvey iv, 18 June 2019). Employing the presenter model effectively shows the artistic directors of the company have a mature understanding of how to develop their intellectual property. The raising of private income and investment began in a concerted way in 2014, and the outcome was approximately $160,000 (Back to Back Theatre, 2014, p. 30). The largest source of income is performances (36%) followed by operational grants (31%). In terms of expenses, artists’ wages are highest (29%) followed by core employee wages (26%) and production costs (19%). Interviews revealed how the company has achieved these financial outcomes as well as continuing to grow their international performance reputation. Live Music and Retail There is such a massive amount of information available via blogs, and product research is so easily done now… [Customers] come in saying “this is the product that I want. This is the price I want to pay for it. If you can make that and supply it now, I’ll take it”’ (Bee iv, 17 June 2019). The hire business helps with sales, supplying venues, acts and festivals in the region such as Queenscliff Music Festival and Port Fairy Folk Festival (Bee iv, 17 June 2019). National and international touring acts might also use their gear. In terms of audiences, ‘people just aren’t going out Monday, Tuesday, Wednesday, Thursday’ (ibid); it’s more expensive, and they have the ‘distractions’ (ibid) of streaming services such as Netflix and Stan. 21 Final 25 November 2020 21 Bay City Events Back to Back Theatre Funded through all levels of government, the quality of their work and international reputation is due to the dedication of Executive Producer Alice Nash and Artistic Director Bruce Galdwin. This production team builds ‘those relationships and negotiates those performance fees. And the marketing team supports us by providing us with collateral that we can use to sell the 36% 31% 15% 14% 4% 0% 10% 20% 30% 40% PERFORMANCE INCOME OPERATION GRANTS PROJECT GRANTS PRIVATE … SERVICE FEES Back to Back Theatre Average Income 2014-2019 29% 26% 19% 16% 9% 3% 0% 10% 20% 30% 40% ARTISTS WAGES CORE EMPLOYEE WAGES PRODUCTION COSTS PROJECT DELIVERY WAGES OVERHEADS MARKETING COSTS Back to Back Theater Average Expenses 2014-2019 The Creative Engine – Geelong Arts Centre Geelong Arts Centre (GAC) was re-branded in 2019. (Formerly known as GPAC, the ‘performing’ adjective has now been dropped). The rebranding coincided with the redevelopment and opening of The Creative Engine, with ‘beautiful rehearsal, incubator, and flexible spaces’ that will help GAC fast-track the curation and development of new work (Rush iv, 17 June 2019). The Creative Engine aims to be a ‘home for all creative industries and a hub for young artists and creative businesses across a range of disciplines’ (Coldwell-Ross, 2019). GAC is moving away from being a ‘venue for hire’ offering performance spaces for touring, local and community performances, to focusing on developing new work that will generate intellectual property. The Creative Engine offers small grants as part of this push, which signifies a shift from Performing Arts into Creative Industries. Unfortunately, the opening and use of The Creative Engine has been affected by COVID-19 restrictions (GAC, 2020). Court House Youth Arts Courthouse Youth Arts (CHYA) defines youth in two ways: ‘It can be youth in terms of age, or it can be in terms of practice. So, youth can also be people emerging in their practice’ (Russell iv, 18 June 2019). They began as The Creative Collective in 2015, working with a group of six young people who brought unique individual skills to the overall team. Voicebox was a multidisciplinary performance held on the waterfront precinct to showcase their talents. In 2016 and 2017 the collective took on four more members, focusing on writing, dance, visual arts and theatre (CHYA, 2019). The Council is a major funder of CHYA providing ‘$250,000 of core operational funding’ on a multi-year scheme (Russell iv, 18 June 2019). Other funders include Creative Victoria, through their Organisational Innovation Implement (OIP) scheme, which has a four-year funding cycle of $67,000 annually. Geelong Central Marketing and the Department of Human Services also provided project grants (Russell iv, 18 June 2019). The Council acknowledges the challenges in providing career pathways in the performance sector, realising that ‘deeper engagement’ will lead to ‘development and through to emerging artists and career pathways’ (Esler iv, 18 June 2019). CHYA has just completed its strategic three-year plan. Previously, they hired their venue and space to ‘corporate, community and creative bookings’ (ibid), behaving more as a service provider, but in 2019 they changed that practice, now offering creatively led programs. Figure 10 – Back to Back Theatre Income and Expenses 2014-2019 Employing the presenter model effectively shows the artistic directors of the company have a mature understanding of how to develop their intellectual property. The raising of private income and investment began in a concerted way in 2014, and the outcome was approximately $160,000 (Back to Back Theatre, 2014, p. 30). The largest source of income is performances (36%) followed by operational grants (31%). In terms of expenses, artists’ wages are highest (29%) followed by core employee wages (26%) and production costs (19%). Interviews revealed how the company has achieved these financial outcomes as well as continuing to grow their international performance reputation. Funded through all levels of government, the quality of their work and international reputation is due to the dedication of Executive Producer Alice Nash and Artistic Director Bruce Galdwin. This production team builds ‘those relationships and negotiates those performance fees. And the marketing team supports us by providing us with collateral that we can use to sell the Final 25 November 2020 22 organisation and sell the shows. Our marketing point is between us and the presenters’ (Harvey iv, 18 June 2019). Due to the COVID-19 pandemic, Back to Back Theatre has ‘temporarily suspended all programming and activities, indefinitely’ (Back to Back Theatre, 2020). organisation and sell the shows. Our marketing point is between us and the presenters’ (Harvey iv, 18 June 2019). Due to the COVID-19 pandemic, Back to Back Theatre has ‘temporarily suspended all programming and activities, indefinitely’ (Back to Back Theatre, 2020). Geelong radio Radio is a mature Creative Industries sector, and regional radio contributes to a whole series of interrelationships both within Creative Industries itself and beyond. Geelong’s radio stations continue to compete with Melbourne radio, which can be received clearly in the region. Despite this, there are six local commercial, public and community stations broadcasting across a range of formats;; rock and pop music (K-Rock), a community station (The Pulse) a Christian community station (Rhema FM), country music (Hot Country Radio), adult contemporary (Bay FM) and a radio reading service (3GPH). As an example The Pulse is a community FM station (94.7 FM), which is underwritten by Geelong Ethnic Communities Council. The remit is broad, with programs in Dutch, Russian, South Sudanese, Afrikaans and Croatian. Announcer Mitchell Dye says, ‘we try to throw open the doors to as many people as possible because that’s our job as a community radio station’ (Dye iv, 18 June 2019). Analytics show their internet stream has listeners in ‘all parts of the world’ (Dye iv, 18 June 2019). Dye said, ‘community radio is a tough environment and we operate on a shoestring budget’, with only three paid staff and 100 volunteers (Dye iv, 18 June 2019). Although it has been ‘challenging’, presenters have adapted to making podcasts because ‘with the changing media landscape, people expect stuff to be available on demand’ (Dye iv, 18 June 2019). Dye himself presents a ‘Geelong-centric’ current affairs program. The station has programs dedicated to the arts, and outside broadcasts finished with a local artist ‘giving these people platforms that they otherwise wouldn’t get’ (Dye iv, 18 June 2019). A location for screen production Geelong and the Surf Coast have attractive locations for feature and television serial productions. For example, the highly popular 1990s series Sea Change was filmed on location at Barwon Heads between 1998 and 2002 (Millar, 2007). Film attraction is beneficial for regional locations, as international or interstate productions might film for weeks at a time, providing economic benefits for the broader community, particularly in accommodation, catering and employment of local people. Geelong and the Surf Coast continue to feature in Australian productions; for example, The Dressmaker (2015) was filmed in the region. Commercial and corporate screen businesses Most of the screen production businesses in Geelong are commercially focused, primarily because Geelong is part of Melbourne’s television licence area, so all commercial and community television is made and broadcast from the state’s capital. It is not surprising, then, that Melbourne employs 5,000 television workers and Geelong employs 50. In Geelong, there are commercial and corporate screen production businesses who make commissioned work for clients from the corporate, not-for-profit and government sectors. Acetate Base, Love Letter Films and Crafty Fox Media are three commercial screen production businesses owned and operated by business partners Andy Marriott and Jennifer Kidd. Crafty Fox Media focuses on social media advertising, Love Letter Films offers bridal and wedding video packages, and Acetate Base offers video production and post-production services for TV and high-end corporate productions (Kidd iv, 20 June 2019). The businesses ‘cater for different audiences’ and separate agendas help attract the right sort of clients (Kidd iv 20 June 2019). There are two types of local film production. Those who leverage Film Victoria’s Production Attraction and Regional Assistance Fund (Film Vic) typically shoot films in the region and do post-production in Melbourne or Final 25 November 2020 23 Sydney, which Kidd finds disappointing ‘because there's a massive pool of world-class talent’ in Geelong (Kidd iv 20, June 2019). The second type, emerging local productions, include independently funded TV series (Boshier & Moore, 2017; Pobjie, 2019) and low-budget feature films (Groves, 2019) Sydney, which Kidd finds disappointing ‘because there's a massive pool of world-class talent’ in Geelong (Kidd iv 20, June 2019). The second type, emerging local productions, include independently funded TV series (Boshier & Moore, 2017; Pobjie, 2019) and low-budget feature films (Groves, 2019) Geelong Advertiser and Surf Coast Times As far as the Publishing sector goes in Geelong, the daily paper, the Geelong Advertiser (circulation in 2018 was 46,276), is produced by News Corp Australia (Gee, 2020). Established in 1840, it is the oldest newspaper title in Victoria and the second oldest in Australia. The free Geelong Independent and Geelong News are the city's other major newspapers. The Geelong Advertiser is part of Rupert Murdoch’s international News Corp group and, as such, services such as IT are managed in-house through ‘vertical integrated’ systems. Advertisers use ‘two layers’ of engagement with the paper – nationally through national advertising agencies, and locally through local agencies, and the Geelong Advertiser has ‘ad reps on the ground’ (Cairns iv, 17 June 2019). The newspaper’s business editor David Cairns notes that businesses, particularly retail, have ‘all had to develop a digital footprint and some capacity to operate online’ (Cairns iv, 17 June 2019), and that this requires ‘a bigger investment for a local player than someone who is a national or international player’ (Cairns iv, 17 June 2019). Regional newspapers have been impacted by the economic downturn caused by the COVID-19 pandemic (Media Entertainment and Arts Alliance, 2020), but fortunately the printing of the Geelong Advertiser was unaffected by News Corp shake-ups in April and May (Doran, 2020). However, the Star News Group, which publishes the free weekly Geelong Independent, has reduced its print run by 70%. The Times News Group is still home-delivering Final 25 November 2020 24 and bulk-dropping its weekly newspapers, the Surf Coast Times and the Armstrong Creek Times. Newspaper jobs in the region could be affected when the Federal JobKeeper program ends. The Dome - Geelong Library and Heritage Centre The Geelong Library and Heritage Centre, known as The Dome, is the central library for a network of 17 libraries across four local government areas (Geelong Regional Libraries, 2019). The integration of the heritage centre and the library has made an historical catalogue of images, artefacts and records accessible internationally, increased visitor numbers 17-fold, and improved accessibility to the community. The building is also a tourist attraction for local, national and international visitors, and was shortlisted for the international Library of the Year award in 2016 (Smith Iv, 17 June 2019). Patti Manolis, the CEO of Geelong Regional Library Corporation, explains the library is building a ‘digital repository’ that will connect the collection with historical societies making their information ‘accessible to anyone around the world’ (iv, 17 June 2019). Creative Services professionals are helping staff create the repository and develop a library app. Local Writers and Publishers Creative specialists such as writer Ross Mueller exploit all opportunities to earn an income, and that means working across all media and performing arts areas. Mueller’s income is generated from being a playwright, radio and television series writer, a grants writer and a journalist. Based in Geelong, Mueller has had eight plays published which have been performed at Melbourne Theatre Company (MTC) and Sydney Theatre Company (STC). As a playwright, Mueller is employed by commission: ‘With MTC the commission is just the starting point. The payday is the royalties, which can be substantially bigger. So, I've had a show at STC, which did really well, and that was a big payday for me, which ended up half of it going [in tax] to John Howard’ (Mueller iv, 18 June 2019). In 2019, Mueller was working as a grants writer for Geelong Ethnic Communities Council, while writing a column for the Geelong Advertiser, freelancing for Crikey.com and ‘working on a series for ABC Radio. That pays too, which is good!’ (Mueller iv, 18 June 2019). To help other writers in the region Geelong Writers Inc was set up as ‘a non-profit community group formed to promote support for writers’ (Geelong Writers Inc, 2019). Their aim is to promote writing and literature in the Geelong and Surf Coast regions, for both emerging writers and seasoned professionals. Geelong Writers Inc have published collections of memoirs, poetry and short stories and ‘flash fiction’ pieces called Touches of Resilience (2018), edited by Justine Stella. Other small owner-operated publishing businesses have emerged, distributing publications electronically through services such as Issuu. Drop of a Hat Publishing is run by Colin Mockett an ‘award-winning journalist, experienced at writing, editing (including illustrating) and creating attractive page layouts through to whole book design, proofreading and publishing’ (Mockett, 2019). There are a number of retail book shops operating in Geelong, with two Dymocks stores and the independently-owned The Book Bird founded in 2015 (The Book Bird, 2019). QBD Books operate in the arts precinct in Malop St, and Bookgrove which opened in 2009, hosts author events using social media channels for promotion (Bookgrove, 2019). Independent Artists – ceramicists, potters, painters and illustrators There are several visual artists working full-time in the region who make an income selling locally from studios and on Instagram, and teaching their craft (Wright and McGinn, iv 20 June 2019). Timothy White is an Australian ceramicist with 25 years’ experience. Lynne Tae is an impressionist and paints semi-abstract landscapes that evoke a sense of atmosphere (Houzz, 2019). Bahman Vahab carves stone and has been involved in sculpture for 30 years. Sisko Designs is a creative studio that specialises in custom art pieces for interior and exterior. Sandie Copland is a freelance Illustrator focused on the beauty of natural forms who has exhibited widely across Australia and who consolidated her love of illustration by completing the Natural History Illustration degree at the University of Newcastle (Houzz, 2019). StudioMade pottery studio, retail space and café was launched by artist Darren McGinn in 2018 as ‘a gallery collective with an emphasis on community engagement’ and ‘a way of sustaining [his] own craft and sculpture ambitions’ (McGinn iv, 20 June 2019). McGinn’s daughter runs the café attached to the studio as well as its Instagram page and his wife, a nurse, looks after the website and administration. McGinn prefers face-to-face client contact to online sales, which are ‘too much work for too little return’ but does use Instagram to promote his and his students’ work (McGinn iv, 20 June 2019). McGinn teaches in the studio with casual tutors and produces ceramics for high-end clients such as Aaron Turner’s Restaurant, Igni and Rakumba Lighting and others which gives them ‘extra income to help pay a huge mortgage’ (McGinn iv, 20 June 2019). He occasionally exhibits in Geelong galleries (McGinn iv, 20 June 2019). Instagram is the main way multimedia artist Amy Wright sells her work, including to international clients in the US and Berlin. Wright sells her works for between $300 and $5,500 and provides free shipping within Australia because ‘the cost of shipping would deter people’ (Wright iv, 20 June 2019). Non-fiction literary festival The annual Word for Word Festival is hosted at Geelong Library and Heritage Centre and is ‘the only exclusively non-fiction focused literary festival in the country’ (Smith iv, 17 June 2019). It started as a collaboration between the library, the City of Greater Geelong and Deakin University, and was hosted by the university for the first two years, moving to the library four years ago. Featuring in the order of 76 presenters and 45 sessions across three days, with masterclasses and panel discussions moderated by Deakin academics, it is a ‘significant literary festival that attracts a very high calibre of non-fiction authors’ (Smith iv, 17 June 2019). Past authors, across the genres of Final 25 November 2020 25 memoir, biography, true crime, sport and politics, have included Geelong-based Helen Garner, Peter Carey (from Bacchus Marsh, who attended Geelong Grammar), Anthony Albanese, Chris Masters, Stan Grant, Gillian Triggs, Julian Burnside, Jelena Dokic and Kon Karapanagiotidis. Festival topics, such as asylum seekers, can be confronting. Controversy attracts local and national media interest and plays out on social media. Writers and audiences interact on Twitter, and Instagram now gives a national and an international reach. The library has partnered with Express Media (at Melbourne’s Wheeler Centre), Scribe Publishing, and the nib Awards, to provide prizes for young writers, and Victoria Writers assists with curating a program of workshops. Festival director Rochelle Smith said it was well regarded: ‘We have a strong following of audiences and are very highly commended by the participants, the authors themselves as well’ (Smith iv, 17 June 2019). Manolis said the Word For Word festival is ‘the heart of what public libraries are all about creating a freedom of information and a safe space for encouraging debate’ (Manolis iv, 17 June 2019). Local writers are more likely to be promoted through the library’s usual program than the festival, which carries prestige (Smith iv, 17 June 2019). Geelong Gallery Established in 1896, Geelong Gallery is one of Australia’s oldest art galleries. It holds a collection of Australian and European paintings, sculptures, printmaking and decorative arts dating from the 18th century to today, notably Frederick McCubbin’s 1890 A Bush Burial, and Eugene von Guerard’s 1856 View of Geelong. In the year to June 2019, almost 160,000 people visited the gallery, a 36% increase on the previous year (Geelong Gallery, 2019). The gallery has space to display only 3% of its 6,000 works and is seeking to expand into the historic City Hall, with plans under the Cultural Precinct Master Plan of the broader Revitalising Central Geelong policy – a joint venture of the Victorian Government and the City of Greater Geelong, which owns the gallery building and is a ‘core funding partner’ (Smith iv, 17 June 2019). Geelong Gallery director Jason Smith said the gallery now has a ‘physical connection and shared exhibition space’ with Geelong Library and is ‘opening up the gallery’ to promote ‘inclusion’ (iv, 17 June 2019). Audience engagement is ‘a big deal’. More than 16,000 people took part in nearly Final 25 November 2020 26 300 public programs in 2018, ‘everyone from bubs up through to people with dementia and all ages in between’, and 4,500 students took part in the schools’ program. A structured partnership with Deakin University helps art students, and an events space is activated by ‘Talking Art’ sessions, with artists such as Geelong industrial designer and Melbourne exhibitor Dale Hardiman, who spoke about his practice during Design Week. Funding from the City of Greater Geelong and the Victorian Government (under the Regional Partnerships Agreement) and the Geelong Gallery Foundation is boosted by the work of gallery fundraising groups Geelong Conservation and Geelong Contemporary, and by corporate and community donations and bequests. The gallery’s business model also now includes one ticketed exhibition a year, which boosts revenue and, when it’s the Archibald, ‘is great for capacity building’ (Smith iv, 17 June 2019). More than 76,000 people visited the gallery during the 58 days of the 2018 Archibald Exhibition, the second year the gallery has shown the popular portraiture prize. Of the visitors to the Kylie on Stage exhibition from Art Centre Melbourne, which featured Kylie Minogue costumes, 30% ‘had never been to a gallery before’ (Smith iv, 17 June 2019). Ashmore Arts - the sum is greater than the parts Ashmore Arts is a notable and innovative commercial hub for Surf Coast artists of all genres. It houses 28 active artists and crafts businesses, who collectively employ 12 people in various capacities. Ashmore Arts offers studio spaces in sheds, shipping containers and small workshop spaces and affords artists a conducive environment to work, display and create pieces for private and commercial clients. Ashmore Arts began in 2006 and has an approximate turnover of $2,916,000, based on staff, truck movements and discussions with resident artists. Owned and run by artist Stewart Guthrie, Ashmore Arts has a collective attitude: ‘Being amongst like-minded people has an amazing effect on attitude and self esteem’ (iv 29 June 2020) There is a lot of direct financial Ashmore Arts is a notable and innovative commercial hub for Surf Coast artists of all genres. It houses 28 active artists and crafts businesses, who collectively employ 12 people in various capacities. Ashmore Arts offers studio spaces in sheds, shipping containers and small workshop spaces and affords artists a conducive environment to work, display and create pieces for private and commercial clients. Ashmore Arts began in 2006 and has an approximate turnover of $2,916,000, based on staff, truck movements and discussions with resident artists. Owned and run by artist Stewart Guthrie, Ashmore Arts has a collective attitude: ‘Being amongst like-minded people has an amazing effect on attitude and self-esteem’ (iv, 29 June 2020). There is a lot of direct financial collaboration, Guthrie explained, ‘where one artist will get a job for a timber sculptor and that sculptor collaborates with the blacksmith for the steel component. This happens a lot on all different levels, again part of being amongst like minded people’ (iv, 29 June 2020). Guthrie described the success at Ashmore Arts as being based around choice, lifestyle, finances, community and environment: ‘These artists are “rock stars”, and with that comes personal business branding. The development of their social persona … is something they are and should be very protective about. Online sales mean no shop front costs, no gallery fees and a far wider and more specific audience’ (Guthrie iv, 29 June 2020). The large range of expertise includes fine art, large art, illustration, animation, photography, graphic design, weaving, leather book binding, calligraphy, furniture making in wood and concrete, blacksmithing, sculpture in wood and stone, pottery, knife making, picture framing, home interior design, and music production. Geelong Gallery The gallery has recently acquired the work of two local artists, while engagement with local artists is mainly through events. Jason Smith from Geelong Gallery indicates there is some concern locally that the gallery might not ‘reflect local content’ but maintains that ‘quality has to be there before it gets in the door’ (Smith iv, 17 June 2019). Ashmore Arts - the sum is greater than the parts Studio spaces are sought- after, and artists are on a waiting list. Ashmore Arts has applied to Council to double its size, and ‘the neighbours have applied for a permit to build 10 new studios; they have seen the growth of Ashmore Arts and want to be a part of growing the industry in Torquay’ (Guthrie iv, 29 June 2020). Success stories include award-winning knife maker Metal Monkey Knives and stone tableware maker Takeawei Ceramics. Wathaurong Aboriginal Cooperative The Wathaurong Aboriginal Cooperative has a commercial enterprise, Wathaurong Glass, making art glass, such as Deadly Awards plaques, and architectural glass, such as kitchen splashbacks, using traditional designs and motifs from traditional stories (Ryan iv, 18 June 2019). Boom Gallery and co-working spaces Boom Gallery is an eclectic art gallery with a cafe in Geelong's progressive Rutland Street (Boom Gallery, 2019). Boom Gallery is run by former art teachers Ren Inei and Kate Jacoby. They started small, in 2011, so their fledgling business could fit in around their families. Leasing the building from a family who was ‘very supportive of the arts.’ This enabled them to establish financially as they also set about subleasing smaller spaces within. Now Boom Gallery and a second building, Factory 21, is ‘filled with creatives’; working artists on the lower floor and creative services such as web designers and media businesses upstairs (Jacoby iv, 18 June 2019). Art is sold on consignment and Boom takes a commission when it sells, in addition to asking artists for a $300 monthly fee, which gives them ‘buy-in’ (Jacoby iv, 18 June 2019). It took eight years to build up ‘profile and reputation’ for the commercial gallery (Jacoby iv, 18 June 2019) and for sales to be consistent. Achieving this means that Boom can now employ a gallery manager and bookkeeper, and outsource events management, marketing, design, website and social media. Local artists are the focus, as ‘there’s been a lot of resentment in the Geelong community from artists and also art lovers that the [Geelong] Gallery does not really represent regional artists very well’ (Inei iv, 18 June 2019). Success stories include textile artist Ingrid Daniell and painter Chelsea Gustafsson, who was picked up by Flinders Lane Gallery in Melbourne (Inei iv, 18 June 2019). Interstate artists are increasing, and their first international was a digital collage artist based in France that Jacoby contacted through Instagram. Anglesea Art House Anglesea Art House, a former scout hall renovated by the Council, is now ‘a beautiful workspace with two glass kilns [and] two clay kilns’ that is used for classes, workshops and exhibitions. Its members put on the annual Anglesea Art Show (Gaffney iv, 19 June 2019). Surf Coast Shire Arts Development Officer, Harriet Gaffney, says Final 25 November 2020 27 the Shire has two groups of artists: those working on ‘large scale works that they ship out of Torquay, so they don’t have any visible presence’ and the hobbyists, ‘an extraordinarily prolific and vocal group’ (Gaffney iv, 19 June 2019). However, as with other ‘little coastal towns hugging this extraordinary road’, the artists ‘become quite isolated; many don’t go outside to keep developing their skills. They hit a benchmark, they become technically very proficient, but it’s quite insular’ (Gaffney iv, 19 June 2019). Harriet Gaffney wants to ‘break down the barriers’ between ‘siloed’ groups of artists; painters, ceramicists, sculptors, glass artists, musicians, performers and writers: ‘That makes much more exciting and invigorated art’ (Gaffney iv, 19 June 2019). Surf Coast co-working spaces Collaboration is occurring more broadly in the Creative Industries, with three co-worker spaces in Torquay alone: the Workers Hut in Baines Crescent; Happy Spaces in Gilbert Street, and the old Quiksilver building. The Quicksilver building is home to designers. There’s a ‘strong affiliation with Bali’ because surf brands used to manufacture there ‘so a lot of the designers that have come from there have contacts there already’. They are now ‘selling online and at the markets’, having ‘gone out on their own’ (Gaffney iv, 19 June 2019). The Shire has also attracted ‘young entrepreneurs’, ‘creative entrepreneurs’ who are ‘doing so much’. These are surfers who become ‘attached to this exquisite environment, but they have the freedom to go overseas for three months in the middle of the year, so they don’t have to get cold when they are surfing’ (Gaffney iv, 19 June 2019). Creative Geelong’s Makers Hub The Makers Hub, part of Creative Geelong, was founded with funding through the City of Greater Geelong and Creative Victoria, in ‘a pretty fractured area’ of the CBD (Cromarty iv, 18 June 2019). The Makers Hub began in 2017 and offers freelancers and small creative start-ups a co-working environment, with maker spaces, and multimedia, exhibition and workshop spaces (Creative Geelong, 2019). Housed in an abandoned 1980s shopping arcade, successful start-ups have flourished here, including animation studio Pillowfort Creative, game developer Hanalei Studios, and creative media services provider Video Confidence Coach. Makers Hub Co-ordinator Robert Menaul said they have also created a ‘social enterprise model … working with different organizations including GenU, The Barwon Adolescent TaskForce, Good Cycles, Department of Justice and other agencies to find ways to help people who may have fallen through the cracks, develop more internal life skills and coping skills in creative ways’ (Menaul iv, 18 June 2019). To combat slow NBN speeds at the Makers Hub, Creative Geelong installed ‘expensive routers’ to provide Wi-Fi throughout the arcade (Cromarty iv, 18 June 2019). Renew Geelong There are more than 100 vacant ground-level shops in Geelong’s CBD, and Renew Geelong’s concept space popped up in one of those in 2018. Like all Renew projects, it has brought to life an empty commercial space. Thw work of Renew Geelong has been made possible through relationships with the City of Greater Geelong, Bluebird Foundation, Creative Victoria and two local private investors Batman Investments and David Hamilton Group. These investors are ‘really active supporters of enlivening the CBD’ (Bruce iv, 17 June 2019). Renew Geelong also Final 25 November 2020 28 has connections with Creative Geelong, Central Geelong Marketing, Diversitat, Pako Festa and Gordon TAFE, and Geelong Illustrators group is an active participant (Bruce iv 17 June 2019). Renew Geelong has a 12-person advisory committee with representatives from Tourism Geelong, Bellarine, Market Square shopping centre, City of Greater Geelong’s Economic Development Unit and their Arts and Culture Partner. Renew Geelong has been funded through Creative Victoria and the City of Greater Geelong, each contributing $75,000 for two years (Bruce iv, 17 June 2019). A part-time coordinator, Andrea Bruce, is charged with recruiting participants and Geelong landlords. Bruce has pursued un-leased premises that could be co-opted into the Renew model, in which no rent is charged for 30 day. At the time of the interview, Renew Geelong had secured 15 vacant retail or commercial spaces, engaged 24 participants and hosted more than 40 workshops. However, local real estate agents have not been willing to cooperate, and this may mean Renew cannot continue beyond its two-year funding (Bruce iv 17 June 2019). Federation Mills – Pivot City Innovation District David Hamilton Property Group (DHPG) purchased the derelict Federation Mills site in 2013. The reinvigoration of these historic buildings won a National Trust Heritage Restoration award in 2016. There are two more re- developments that will complete the Pivot City Innovation District, The Power Station and the Glass House with both still in the planning stages. The refurbished Federal Mills site is a growing innovation ecosystem in Geelong. Along with Runway Geelong, and global education techs Tribal and Civica, tenants including State Government offices, Wathaurong Aboriginal Co-Operative and Popcultcha, a retail and online outlet. ‘Fed Mills’ as it is known, has 37 tenants in total who have created 1,000 on-site jobs; the aim is to have 3,500 on-site jobs by 2021, fuelling Geelong’s tech and innovation sector. One of the attractors is Australia’s fastest available internet, 10 times faster than the NBN, with 1,000Mbps fibre to all tenants (Pivot City, 2020). One international software company who has delayed their promised arrival is LiveTiles. The Victorian Government gave them $1.5 million because they were to create 500 jobs, but their arrival has been delayed by 18 months. LiveTiles intends to partner with ‘local universities, TAFEs and secondary schools … to develop new opportunities for recent technology graduates and researchers’ (LiveTiles, 2019). 29 Final 25 November 2020 Strategic theme 2: The relationship of cultural and creative activity to the wider economy In Software and Digital Content, Pivot City Innovation Precinct is an exemplar for Geelong. This precinct has attracted global software companies working cross-sectorally in health and education because of its extremely fast and reliable internet and provides more than 1,000 local jobs in CI and other economic sectors. Asia Pacific Marketing Manager for Tribal educational software, Liam Banks, said the last two Geelong businesses he has worked for ‘had employed people that had moved from Melbourne to Geelong for work’ (Banks iv, 18 June 2019). Another example is Runway Geelong, also located at Pivot City. It has been funded through the state body Launch Vic, which is committed to investing in Victorian’s ‘start-up ecosystems’. Census and Australian Business Register data at SA2 level also identified clusters of Creative Industries operatives working as part of the wider economy, and interviews revealed operational details. Supporting Regional Development in Geelong While Regional Development Victoria does not target Creative Industries per se, it supports industries that are ‘creative in nature’ in a variety of ways (Ryan iv, 18 June 2019). For example, a software or games developer may be looking for a regional Victorian location, and RDV could help them with relocation support (Ryan iv, 18 June 2019). The government’s Regional Skills Fund may support a Creative Industries company looking to upskill its workers or fill a skills shortage ‘and they can partner with other companies or universities’ on a proposal that RDV could support with funding (Ryan iv, 18 June 2019). A key role is referral, to ‘identify the needs of a business and speak to other agencies and refer them on to where they’ll receive the best support’ (Ryan iv, 18 June 2019). This might be to Small Business Victoria or Economic Development Geelong (the Council’s economic agency) for workshops on developing business acumen, or to Creative Victoria for advice on funding programs, as RDV did for Creative Geelong. RDV does not have specific events and festival funds but may support an event that is large enough to have ‘a significant economic impact’ (Ryan iv, 18 June 2019). That support might be through funding infrastructure, such as that used by the Queenscliff Music Festival, or the redevelopment of the Safe Harbour Precinct, an investment that is ‘opening up public access but also supporting the Royal Geelong Yacht Club in terms of the significant sailing events that they attract’ (Ryan iv, 18 June 2019). Event organisers may be referred to Council’s Creative Communities Grant Program for small grants, or at the other end of the scale the government’s Regional Jobs and Infrastructure Fund, set up to build stronger communities through ‘community capacity building and population attraction’ (Ryan iv, 18 June 2019). Tribal – global education software Tribal is a global cloud-enabled educational software business with its Asia-Pacific headquarters in Geelong’s Pivot City Innovation Precinct at Federal Mills. Tribal offers software solutions for public, private and alternative education institutions. Tribal is Australia and New Zealand’s largest provider of student management software systems, servicing 35% of Australian schools, 30% of Australian universities, 100% of NSW TAFE, and a high percentage of New Zealand vocational institutions (Banks iv, 18 June 2019). Tribal employs more than 800 people globally; more than 100 in Geelong. Tribal has grown through acquisition. It acquired three Australian companies; one from Melbourne, Human Edge Software, and two from Geelong, Sky Software, and Callista (Banks iv, 18 June 2019). Callista originated from Deakin University and many of Tribal’s employees have worked or studied at Deakin: ‘A lot of people that leave here, unfortunately, we hear they're going back to Deakin’ to work on cutting edge projects such as Artificial Intelligence (AI) and virtual assistant software (Banks iv, 18 June 2019). Runway Geelong Runway Geelong accelerates the start-up process by providing support for business ideas that are innovative and entrepreneurial (Dostis & Burns iv, 17 June 2019). While they realise physical incubators and accelerators are critical components of creative ecosystems, Runway also leverages the effect ‘quality mentoring, access to capital and access to networks made on the relative success of startups’ (Runway Geelong, 2019). Located at The Mills, it received $1.25 million in 2017 from Launch Vic to act as ‘a catalyst for innovation in the region, creating new businesses and jobs through the provision of mentoring, networks, training and access to venture capital’ (Launch Vic, 2020). Started by Nick Stanley and Peter Dostis, Runway is a not-for-profit and aims to customise each start- up idea by developing and delivering a specific entrepreneurial program through four stages: bootcamp, incubator, Runway virtual, and masterclass. Runway participants have access to co-working spaces, mentoring, networking between entrepreneurs and a range of discounted professional services. Runway’s Partners include Launch Vic, Harwood Andrews, Adroit, West Carr Harvey, Fluid, Strathouse, Geelong Angel Investor Group, PwC, and BTS (Global Victoria, 2019). Among 19 alumni, there does not appear to be specific industry focus, with a Final 25 November 2020 30 range of organisations and businesses including health, wellbeing and Creative Industries such as fashion, apps development, marketing and events. Runway connects with a high school; their multi-media VET students ‘film an event, edit video, record a podcast and do graphic design for social media’ (Burns iv, 17 June 2019). range of organisations and businesses including health, wellbeing and Creative Industries such as fashion, apps development, marketing and events. Runway connects with a high school; their multi-media VET students ‘film an event, edit video, record a podcast and do graphic design for social media’ (Burns iv, 17 June 2019). FE Technologies – Radio Frequency Identification labels Software and Digital Content specialists are in demand across the economy, and in 2016 were slightly more likely to be employed (embedded) in other industries than in sector-specialist firms. FE Technologies is a Geelong business that specialises in creating library management software and hardware. This business supports the GLAM sector, by employing embedded creatives in IT technology, software development and marketing and sales. CEO Andrew Powell said their library products are installed in more than 900 libraries in Australia, New Zealand, the United States, Malaysia and China. With more than 200 international clients, products are available in 30 languages (Powell iv, 17 June 2019). FE Technologies creates the radio frequency identification tags (RFDI) used on every library book, CD and DVD. The technology is designed, manufactured, installed and serviced by FE Technologies and allows ‘libraries to speed up their processes of borrowing and returns’ (Powell iv, 17 June 2019). China-based parent company Invengo has other markets that cover anti-counterfeit measures. Invengo employs 30 people in Geelong (Powell iv, 17 June 2019). Fortek Geelong Ford automotive company sold its sites at Geelong and Broadmeadows in 2019 to Melbourne developer and builder Pelligra Group, which plans to develop high-end business and industrial parks that could generate thousands of jobs in manufacturing industries (Pelligra, 2019). The parks will be named Fortek Geelong and Assembly Broadmeadows. Under the deal, Ford will keep its existing research, design and engineering facilities in Geelong and Broadmeadows and its You Yangs proving ground at Lara as part of the redevelopment. Parts of the distinctive brick plant at Geelong are covered by heritage restrictions, and the structures will be restored and turned into innovation and research hubs tied to universities (Pelligra, 2019). The Committee for Geelong Jen Cromarty is the CEO for the Committee for Geelong, which is focused on Geelong’s economic transformation (Committee for Geelong, 2020). Cromarty believes the Creative Industries sector ‘needs to put the case for investment’ (Cromarty iv, 18 June 2019); to set aside the ‘thinking that it is just about basket weavers’ and ‘struggling artists’ and to ‘tap into commercial money, because it’s out there’ (Cromarty iv, 18 June 2019). For Cromarty, collaboration is key, and the ‘future of work’, a key theme of the Committee of Geelong’s work, demands it. Cromarty says ‘connecting and joining’ are more important than ‘hierarchical models and empire- building’ and that organisations, which are ‘organisms not machines’ must be responsive: ‘We have self- sabotaged our organisations [by] having policies and bureaucracy [that] absolutely smash creative-thinking innovation’ (Cromarty iv, 18 June 2019). On her wish list for Geelong is ‘an edgy arts kind of creative industries festival’ such as Dark Mofo in Tasmania, and an experience that could ‘transform the CBD’ through ‘experimental activation of buildings through art and tech’ (Cromarty iv, 18 June 2019). TAFE and Tertiary Education Creative Industry courses are available at Deakin University and Gordon Institute of TAFE. ‘The Gordon’, as it is known, offers courses in graphic design, visual arts, interior design and decoration, introduction to Adobe photoshop, applied fashion design and merchandising, library and information services, and information technology (Gordon TAFE, 2019). Deakin’s waterfront campus at Corio Bay houses the School of Architecture and Built Environment. At the same location The School of Communication and Creative Arts offers a ‘diverse curriculum in the artistic and communicative fields, with courses in dance, drama, photography, visual arts, advertising, digital and social media, journalism and public relations, film, television, animation, and design (both visual and technological), literature and creative writing’ (Deakin University, 2019). Deakin champions the idea that university research has commercial applications and that it benefits from industry partners. An example is Tribal-Callista. Innovation involves ‘creativity through research [and] creativity through application’ (Cairns iv, 17 June 2019). Through the University, Geelong is now home to Imagine Intelligent Materials, a company seeking to develop a commercial application for graphene. They were attracted to the region by Geelong’s can-do manufacturing attitude. ‘They’ve come to a community where they think that they can get workers and people who can help them solve their problems to create this product, which will become their global hub’ (Cairns iv, 17 June 2019). Carbon fibre industrial design Carbon Revolution supplies lightweight wheel technology to automotive manufacturers around the world. It has successfully moved from an industrial design prototyping phase through to innovation and commercialisation. It is ‘an example of the kind of world-leading high-tech firm that Innovation and Science Australia chairman Bill Ferris says Australia needs more of to prosper in the global market after the closure of local car assembly and the waning of the mining boom’ (Potter, 2018). Lightweight carbon fibre wheels are selling as an option on ‘Ford GT supercar in 2016 and as standard equipment on the Mustang Shelby GTR in 2015’ (Potter, 2018). Carbon Revolution has ‘incredible technology, incredible design, but at a very practical level’ (Menaul iv, 18 June 2019). The manufacturing plant is at Waurn Ponds. Revenue growth for 2020 ‘was very strong, with full-year revenue of $38.9 million ($23.8 million or 158% growth over FY19) (Carbon Revolution, 2020). This was a result of the company’s ability to ‘industrialise its manufacturing processes and reduces costs’ (Potter, 2018). The pandemic has affected the business, with a July 2020 announcement that ‘sales will be negatively impacted … pushing out the launch of a new program and lack of promotional activities … including the cancellation of a major motor show’ (Carbon Revolution, 2020). Sources of raw material have also been affected, increasing costs (Carbon Revolution, 2020). Final 25 November 2020 31 Fortek Geelong Oxygen – Creative Arts College Oxygen College is a private creative arts college in Geelong offering music, photography, digital media, visual arts, film and design vocational programs supported through the Victorian Government’s Skills First program. Founded in 2006 by musician brothers Tony, Joe and Dominic Monea, it grew out of a music school they started in 1986 as a way ‘to supplement their performance income via regular teaching’ (Monea iv, 17 June 2019). The school continues, with private instrument tuition for 100 students, and the brothers also have the retail outlet, Oxygen Music. Oxygen College has a focus on ‘contemporary music’ with ‘industry application’ and encourages original composition (Monea iv, 17 June 2019). It grew out of a need they identified in that ‘students who wanted to take Final 25 November 2020 32 the next step and make music a vocation … had to travel to Melbourne’. They talk to their students about developing ‘multiple income streams, because there’s a journey for them, it’s not an instant destination’ (Monea iv, 17 June 2019). the next step and make music a vocation … had to travel to Melbourne’. They talk to their students about developing ‘multiple income streams, because there’s a journey for them, it’s not an instant destination’ (Monea iv, 17 June 2019). Figure 11 Tourism activity, Greater Geelong and Surf Coast Another challenge for Bellarine tourism and the Council’s Queenscliff Economic Advisory Panel has been to ‘seamlessly integrate arts and culture into tourism and business’ (Armstrong iv, 20 June 2019). The Surf Coast hosts major events, bringing more than $105 million to the region and reaching global audiences of 32 million: including the Falls Festival, Cadel Evans Great Ocean Road Race, Amy’s Gran Fondo, Bells Beach Rip Curl Pro, Surf Coast Century and the Great Ocean & Otway Classic Ride (Surf Coast Shire 2017, p. 31). This calendar ‘attracts visitors from across the globe and plays a vital role in sustaining businesses throughout the off-peak periods’ (Surf Coast Shire 2017, p. 31). The popularity of the Great Ocean Road and the volume of vehicular traffic it brings has led to a less-than-optima visitor experience, and planned facility and infrastructure upgrades ‘will provide a safer, more enjoyable visitor experience while better protecting the environment and coastline’ (G21, 2020, p. 9). Another challenge for Bellarine tourism and the Council’s Queenscliff Economic Advisory Panel has been to ‘seamlessly integrate arts and culture into tourism and business’ (Armstrong iv, 20 June 2019). The Surf Coast hosts major events, bringing more than $105 million to the region and reaching global audiences of 32 million: including the Falls Festival, Cadel Evans Great Ocean Road Race, Amy’s Gran Fondo, Bells Beach Rip Curl Pro, Surf Coast Century and the Great Ocean & Otway Classic Ride (Surf Coast Shire 2017, p. 31). This calendar ‘attracts visitors from across the globe and plays a vital role in sustaining businesses throughout the off-peak periods’ (Surf Coast Shire 2017, p. 31). Figure 11 Tourism activity, Greater Geelong and Surf Coast Figure 11 Tourism activity, Greater Geelong and Surf Coast Figure 11 Tourism activity, Greater Geelong and Surf Coast Figure 11 Tourism activity, Greater Geelong and Surf Coast The popularity of the Great Ocean Road and the volume of vehicular traffic it brings has led to a less-than-optimal visitor experience, and planned facility and infrastructure upgrades ‘will provide a safer, more enjoyable visitor experience while better protecting the environment and coastline’ (G21, 2020, p. 9). Another challenge for Bellarine tourism and the Council’s Queenscliff Economic Advisory Panel has been to ‘seamlessly integrate arts and culture into tourism and business’ (Armstrong iv, 20 June 2019). The Surf Coast hosts major events, bringing more than $105 million to the region and reaching global audiences of 32 million: including the Falls Festival, Cadel Evans Great Ocean Road Race, Amy’s Gran Fondo, Bells Beach Rip Curl Pro, Surf Coast Century and the Great Ocean & Otway Classic Ride (Surf Coast Shire 2017, p. 31). This calendar ‘attracts visitors from across the globe and plays a vital role in sustaining businesses throughout the off-peak periods’ (Surf Coast Shire 2017, p. 31). Figure 11 Tourism activity, Greater Geelong and Surf Coast The popularity of the Great Ocean Road and the volume of vehicular traffic it brings has led to a less-than-optimal visitor experience, and planned facility and infrastructure upgrades ‘will provide a safer, more enjoyable visitor experience while better protecting the environment and coastline’ (G21, 2020, p. 9). Another challenge for Bellarine tourism and the Council’s Queenscliff Economic Advisory Panel has been to ‘seamlessly integrate arts and culture into tourism and business’ (Armstrong iv, 20 June 2019). The Surf Coast hosts major events, bringing more than $105 million to the region and reaching global audiences of 32 million: including the Falls Festival, Cadel Evans Great Ocean Road Race, Amy’s Gran Fondo, Bells Beach Rip Curl Pro, Surf Coast Century and the Great Ocean & Otway Classic Ride (Surf Coast Shire 2017, p. 31). This calendar ‘attracts visitors from across the globe and plays a vital role in sustaining businesses throughout the off-peak periods’ (Surf Coast Shire 2017, p. 31). The popularity of the Great Ocean Road and the volume of vehicular traffic it brings has led to a less-than-optimal visitor experience, and planned facility and infrastructure upgrades ‘will provide a safer, more enjoyable visitor experience while better protecting the environment and coastline’ (G21, 2020, p. 9). Tourism and cultural policy The Surf Coast Shire is one of Australia’s top tourist destinations, with the Great Ocean Road drawing more than two million visitors in 2016, ‘directly expending over $430 million’ (Surf Co The Surf Coast Shire is one of Australia’s top tourist destinations, with the Great Ocean Road and hinterland drawing more than two million visitors in 2016, ‘directly expending over $430 million’ (Surf Coast Shire, 2017, p. 31). The summer period accounts for more than 40% of annual visitation, and the volume of tourist traffic in this period is the reason the Surf Coast Shire prioritises tourism and economic development (Surf Coast Shire, 2017, p. 31). p. 31). The summer period accounts for more than 40% of annual visitation, and the volume of tourist traffic in this period is the reason the Surf Coast Shire prioritises tourism and economic development (Surf Coast Shire, 2017, p. 31). The G21 City Deal suggested that visitor facilities and infrastructure upgrades will provide a $3.7 million boost to the potential tourism in Geelong, Bellarine and the Great Ocean Road (G21, 2020 p. 9). The G21 City Deal suggested that visitor facilities and infrastructure upgrades will provide a $3.7 million boost to the potential tourism in Geelong, Bellarine and the Great Ocean Road (G21, 2020 p. 9). Falls Festival on the Surf Coast There are four Falls Festival events held each year from December 28 to January 6, in Lorne (Victoria), Marion Bay (Tasmania), Byron Bay (NSW) and Fremantle (WA). The traditional four-day camping festival at Lorne is the first in the calendar and attracts 16,500 visitors each year. About 500 tickets are set aside for locals; attendees travel Final 25 November 2020 33 from NSW and SA and elsewhere in Victoria. The company announced in July that Tasmania’s event will not go ahead this year because of the COVID-19 pandemic, similar announcements may follow for other sites. Bands play across the four locations and the focus is on ‘art-based’, ‘theatre’ and ‘carnival-type’ music, with the line-up ‘a great blend of local upcoming or old-school Aussie talent’, and ‘international flavour’ (Burton iv, 19 June 2019). Aside from a handful of salaried staff, festival marketing, ticketing, sponsorship, IT and production are outsourced to ‘90% local’ sub-contractors (Burton iv, 19 June 2019). The festival has permanent infrastructure installed onsite including stages, roads, fences and amenities; the production, PA and lighting comes from Melbourne and Geelong-Ballarat-region suppliers provide marquees, tents and bars: ‘Local trade is a very important aspect of my role’, explains Chris Burton, the Falls Festival site manager based in Lorne. An army of local volunteers includes football clubs who work in return for financial support and a sizable group of Burmese refugees who have ‘become part of the furniture’, working in waste management, and many have gone on to find employment out of it. Burton says Falls works because of local ‘collaboration’; ‘the Falls Festival as a brand, as a company, has a fantastic working relationship with the Surf Coast Shire’ and ‘the relationships are second to none’ (Burton iv, 19 June 2019). Falls is ‘such a valued event on the coast’ that ‘everybody is so supportive of us’ (Burton iv, 19 June 2019). Victorian Government support was also demonstrated when Falls had to relocate at short notice a few years ago because of bushfires; ‘it certainly was evident that the powers that be from state level were behind us with that move’ (Burton iv, 19 June 2019). Burton says the NBN is critical to the event, as is ‘a good network of accountability as far as phone [and] the internet’ (Burton iv, 19 June 2019). Mountain to Mouth Walk Two cultural events came about a decade ago when Geelong was undergoing ‘the greatest upheaval … that it’s seen in its history in terms of industries challenged, large-scale industry going out of business or offshore’ and ‘changing demographics and shifts in the built and urban landscape’ (Esler iv, 18 June 2019). The Mount to Mouth Arts Walk was ‘a platform for community to explore, with support of artists through their artwork “what is Geelong? What is unique about Geelong?”’ (Esler iv, 18 June 2019). The ‘walk across land in the tradition of Indigenous peoples, and songline, a journey from the mountain top at the You Yangs to the mouth of the Barwon River’ created ‘a connected pathway for exploration of local identity through art and culture’, delving into ‘culture in its art context and culture in its collective community meaning context’ (Esler iv, 18 June 18 2019). Although the first walk attracted a small number ‘there were hundreds and hundreds of community members involved, so the great strength of that artwork was actually largely invisible. It happened in the community developments, in the endorsement of artists and exploration of ideas on the ground’ which were then fed ‘back into civic processes’ (Esler iv, 18 June 2019). Falls Festival on the Surf Coast He said, ‘we put a lot of work and time and effort into having Telstra COWS [cells on wheels] and Vodafone and Optus on-site’ (Burton iv, 19 June 2019) to make sure festival goers have good connections, especially for social media. Australian National Surfing Museum The Australian National Surfing Museum – the legacy of pioneering Torquay surfers Peter Troy, Vic Tantau and Alan Reid – was formed in 1993 and is operated by Surf Coast Shire Council. The museum’s mission is ‘to take the stories of surfing and share them with as many people as possible’ (Baird iv, 19 June 2019). The museum attracts a diverse crowd and anecdotally half of visitors have never surfed; they are ‘just curious’ (Dunne iv, 19 June 2019). Through social media, particularly their Surfworld TV YouTube channel, the museum is ‘getting to share stories with hundreds of thousands of people’, something that would ‘take decades’ if they had to visit in person (Baird iv, 19 June 2019). Only 10% of the collection is on show, and the Museum has participated in external exhibitions at the National Sports Museum at the MCG and initiated an oral history program asking ‘significant surfers’ to ‘tell their stories’ (Baird iv 19 June 2019). The museum exhibits material from the creative industries in the form of evolving surfing magazines, iconic images of surfers taken by professional and innovative photographers, posters created by exceptional graphic designers, books written on surfing and films exhibited as a part of the revolution in film making, exhibition and distribution that the surfing community engaged in as it developed its early professional stance. The museum’s co-ordinator, Craig Baird, sees his role as a ‘content specialist’ (Baird iv, 19 June 2019). Drawing on past lives as a surfer, manager of a surf shop, tee-shirt designer and surfboard painter, Baird can communicate to visitors ‘how evolution in technology and design’ has affected surfing history (Baird iv, 19 June 2019). Gary Dunne is the Project and Tourism Officer at the Australian National Surfing Museum. Space is at a premium, and Dunne says the Council is ‘looking at getting state and federal funding to help us with a bigger physical space’ and to maintain Museums Australia accreditation. ‘There is an onus on us to develop that cataloguing of material and tell more stories from the history of surfing’s development in Australia’ (Dunne iv, 19 June 2019). And with the museum operated as a not-for-profit currently, there’s ‘not a lot of availability of resource funding’ (Dunne iv, 19 June 2019). Australian National Surfing Museum Now part of surfing culture, surf retailers Rip Curl, Billabong, and Quiksilver originated on the Surf Coast, a region that relies on these businesses which represent significant local employment prospects. Almost 27% of all jobs and 26.5% ($217 million) of all ‘Value Add’ activity occur as a result of what the surfing industry does (Surf Coast Shire, 2017 p. 31). They employ fashion designers in-house, and rely on working photographers, advertising and marketing specialists, dedicated surfing writers, website designers and many other creative people to help promote not only their businesses but the core image of surfing itself. Given that many are no longer Australian- owned, local jobs have been lost. That iconic Australian surf brand Quiksilver, for example, has ‘shipped off all their creative people to France’ (Daniell, iv 19 June 2019). Geelong After Dark Festival By contrast Geelong After Dark was about ‘discovering’ and ‘recovering the night scape of Central Geelong at a time when people had concerns about the safety of going into places, and about looking at Geelong through different eyes, and having the artists take over the city’ (Esler iv, 18 June 2019). The 2020 festival has been cancelled in 2020 because of COVID-19 but the 2019 Geelong After Dark festival included 70 art-projects that transformed central Geelong through music, street performances, installations, exhibitions, dance and projections’ (Cartwright, 2019). All Geelong’s cultural venues welcomed a broad variety of performance, artistic and musical genres, attracting, ‘20,000 attendees ... and contributing around $2.1 million’ to the local economy (ibid). The City of Greater Geelong survey on Geelong After Dark showed people ‘feel really strongly about [Council’s] investment in our local artists’ and ‘that growing sense of Geelong’s cultural identity’ (Cramer iv, 18 June 2019). After Dark offers a ‘familiar festival framework’ that the community and artists embrace (Esler iv, 18 Final 25 November 2020 34 June 2019). Broad community support is essential to a strong creative sector, and the City of Greater Geelong realises the community ‘are looking to see themselves in culture and arts around them’, with people ‘more inclined to want intimacy rather than spectacle’ (Esler iv, 18 June 2019). National Wool Museum Established in 1988, the National Wool Museum presents the story of the Australian wool industry. Their collection consists of 7,500 objects, including 4,300 photographs, with permanent exhibitions on the region’s heritage, endangered birds, a designers’ showcase, and a maker-in-residence showcase. The Museum was closed temporarily because of COVID-19 restrictions (National Wool Museum, 2020). Using virtual reality technology, the Little L dinosaur project was a partnership between the Wool Museum, and Deakin and Monash universities (Fisher iv, 18 June 2019). This project was shortlisted for both the Museums Australasia Multimedia and Publication Design Awards (MAPDA) and Museums and Galleries National Awards (MAGNA) (Geelong Independent, 2019 ). National Wool Museum Director Padraic Fisher explained: ‘We had a story to tell’ about a small polar dinosaur that lived in that area of Victoria. Working with the palaeontologist who discovered it and virtual reality technology, the scanned skeleton was ‘brought to virtual life’ in what was considered a world first, combining ‘virtual reality, 3D printing [of dinosaur ‘skin’] and paleontology’(Fisher iv, 18 June 2019) in an impressive interactive and creative project. Surf Coast Arts Trail The Surf Coast Arts Trail, which has been running for eight years, involved 67 venues and 201 artists across the Shire and generated $77,000 worth of sales in 2018 (Gaffney iv, 19 June 2019). Surf Coast Shire Arts Development Officer Harriet Gaffney said it’s ‘the big event in the arts on the Surf Coast’ (Gaffney iv, 19 June 2019). The Surf Coast Shire has a shopfront in Anglesea called Surf Coast Art Space, where the community holds exhibitions. It is ‘booked solid’ by ‘hobbyists’ who run their own exhibitions, some making sales of $8,000 in two weeks. Final 25 November 2020 35 Lorne Sculpture Biennale Lorne Sculpture Biennale, Victoria’s premier sculpture festival, is a free three-week event held every two years or so in which the Lorne foreshore is transformed by large sculptural works. Run as a not-for-profit under the direction of Qdos Gallery owner Graeme Wilkie OAM, Lorne Sculpture Biennale relies on volunteers, memberships, sponsorships, donations, fundraisers and grants. In its seventh iteration, a decision was taken in December 2019 to postpone the festival scheduled for October-November 2020 for a year, so the next Lorne Sculpture Biennale will be held in October-November 2021. In July, the Federal Government announced $17,500 funding through the Festivals Australia program for the event. The part-time paid manager of Lorne Sculpture Biennale, Leon Walker, said the festival attracts locals and regional visitors, but at least half come from Melbourne (Walker iv, 19 June 2019). Sixteen ‘mostly Victorian’ artists will be invited to exhibit in the next festival; and artists are asked to make ‘something that’s dynamic’ as well as works for sale, on which the festival takes a commission. In order to increase ‘dwell time’, so people ‘hang around more’, the festival is held out of peak tourist season as an ‘immersion’ not just ‘a static display’, and to increase revenue and enhance the experience, they are planning to develop an app (Walker iv, 19 June 2019). Strategic theme 3: Hotspot Comparisons In contrast to areas such as La Trobe, where ‘economies have been singly focused on their manufacturing’, Ballarat, Geelong and Bendigo, have developed economies that are ‘much broader and that has allowed for that broadening of infrastructure across a whole gamut of professions to emerge’ (White iv, 26 June 2019). While it is a common theme that the liveability of regional cities makes up for lower wages, it also remains a fact that active agents, that is individuals who ‘get things done’, are vitally important as one of the drivers of the Creative Industries. In all these communities, Creative Industries clusters have exhibited a patterned set of demographic movements, with sea changes and tree changers taking advantage of the regional lifestyle and real estate prices, commuters moving daily between Melbourne, Geelong and Ballarat, and from surrounding regions to the Surf Coast at holiday times. It is notable that the relationship between innovation and start-up culture within the Creative Industries, which is very strong in Ballarat and Geelong, has become more entrenched and interconnected, while there is an increasingly wide array of approaches to gaining an income, particularly in micro businesses where highly skilled creatives sell their services or products. Ballarat has Federation University, and with State Government investing over decades into tech parks here and in other regions, Software and Digital Content is a key area of growth for the broader economy. Geelong has a very large urban area including the Bellarine Peninsula, and there has been significant State Government investment in the Geelong Arts Centre the Library and the Gallery. Geelong also has Deakin University with a number of Creative Industries programs on offer, including architecture, which has massively increased capacity. In Bendigo, the Art Gallery works very closely with Bendigo Tourism and also has a developed relationship with La Trobe University’s Bendigo campus and the La Trobe Art Institute, a higher education partner with Bendigo Art Gallery. Cultural tourism was strongly featured in the four sites, with an increasing importance placed on strengthening the connection between Creative Industries and cultural tourism. The economic injection from out-of-town visitations means these regional centres can prosper. Wodonga delivers a strong cultural program including events staged at The Cube Wodonga, the Bonegilla Migrant Experience, and Gateway Village creating a vibrant group of cultural organisations that attract visitors to Murray Arts, HotHouse Theatre and the Jazz Basement. Strategic theme 3: Hotspot Comparisons There is no doubt that Victoria has stolen a march on all other states in attracting the Creative Industries. Across all Victorian regions in the study, there was evidence gathered of ecological interdependence, where the regional Creative Industries are interconnected across sectors, and networked within and between each other, exhibiting complementary activity at all scales. Geelong and Ballarat, as well as Ballarat-Bendigo-Wodonga, are connected through rail and road infrastructure that has developed over a long period of time, proving beneficial to each region as it has allowed Creative Industries workers to commute between regions and to Melbourne, a national hub of Creative Industries. The constraining and enabling effects of policy actions were also observed in all regions. Each exhibited a very deep connection between digitisation and the ability for regional players to operate competitively in both the local and the global environments. While it is worth noting that Wodonga has the poorest internet quality of the four Victorian sites, a variety of CI initiatives were supported in all hotspots by Regional Development Victoria, Launch Vic, and Creative Victoria. Final 25 November 2020 36 The Historic Urban Landscape (HUL) is strong in both Bendigo and Ballarat. A view is held that the City of Bendigo has been a leader in heritage and town planning, and there exists some rivalry between Ballarat and Bendigo in this regard. Greater Bendigo occupies a broad physical area with 15-17% of the population living in rural parts of Bendigo, whereas, Ballarat is virtually an urban area, ‘so if there is a lower proportion of people working in the Creative Industries in Bendigo it’s because of the rural areas, that actually drags down our percentage, while Geelong is the urban area plus the Bellarine Peninsula, which is a pretty wealthy creative area’ (Budge iv, 24 June 2019). While generalisations between the creative capacity of regional Victorian towns can be made, looking at the specialties of each in this study highlights each region’s strengths many of which have developed over time. When examined systematically the complexities and networks that typify the Creative Industries tend to benefit not only each region but the Victorian state as a whole. Strategic theme 3: Hotspot Comparisons Bendigo has the Bendigo Easter Festival, Bendigo Blues and Roots Music Festival and Bendigo Art Gallery, which are impressive visitor attractors for Bendigo’s economy. For Ballarat, there are museums and family experiences to be found at Sovereign Hill, Eureka Centre, and the Gold Museum, and the Archibald Prize has been hosted by the Art Gallery of Ballarat. Other visual and performing arts events are the Ballarat International Foto Biennale, The Royal South Street Eisteddfod held at Her Majesty’s Theatre, White Night and the Meredith Music Festival. In Geelong. there is the Mount to Mouth Arts Walk, Geelong After Dark and National Wool Museum, and the Surf Coast has Falls Festival, Lorne Sculpture Biennale, Surf Coast Arts Trail and The Australian National Surfing Museum. Final 25 November 2020 37 37 Table 2 Victoria hotspot comparisons Table 1 Victoria hotspot comparisons Table 1 Victoria hotspot comparisons Greater Geelong & Surf Coast LGAs Ballarat LGA Greater Bendigo LGA Albury & Wodonga LGAs ASGS remoteness category Inner regional Australia Inner regional Australia Inner regional Australia Inner regional Australia RAI region type Regional city / Connected lifestyle region Regional city Regional city Regional city / Industry & service hub Resident population, 2016a 262,828 101,689 110,479 90,427 Average annual growth 2011-2016 2.1% 1.7% 1.9% 1.6% Employed persons, 2016b 103,752 45,794 45,051 43,915 Average annual growth 2011-2016 3.8% 3.3% 3.5% 3.0% Total creative employment, 2016 b 4,316 2,137 1,367 1,113 Average annual growth 2011-2016 5.7% 2.0% 2.9% 0.3% Total earnings from creative employment, 2016 b $260.2m $119.1m $77.0m $64.1m Average annual growth 2011-2016 4.8% 4.4% 4.7% 3.8% Total businesses, 2016 66,897 23,499 22,709 21,361 Average annual growth 2011-2016 2.3% 2.9% 1.7% 1.0% Total creative businesses, 2016 5,060 1,668 1,444 1,125 Average annual growth 2011-2016 4.7% 4.1% 3.0% 2.9% Proportion of all businesses registered for GST, 2016 49.4% 52.0% 51.2% 51.4% Proportion of creative businesses registered for GST, 2016 38.0% 40.5% 38.0% 39.9% Regional domestic product, 2017-18 $13,852m $5,632m $5,305m $5,192m Average annual growth 5.0% 8.4% 0.8% 0.1% Mean age a 40.1 39.0 39.5 38.7 Unemployment ratea 6.5% 7.6% 6.8% 6.9% Youth unemployment ratea 13.2% 15.0% 14.0% 13.3% Youth unemployment ratioa 45.3% 46.7% 44.8% 45.3% Indigenousa 2.6% 1.5% 1.7% 2.6% Volunteera 17.4% 17.8% 18.7% 17.3% Note a. These statistics are provided by place of residence, and b. are by place of work Source: Australian Bureau of Statistics (2016), ABR (2019), .idcommunity (2019), Regional Australia Institute (201 These statistics are provided by place of residence, and b. are by place of work References .id. (2019). National economic indicators series. Retrieved from https://economic-indicators.id.com.au Australian Bureau of Statistics. (2016). Census of Population and HousingFindings based on use of ABS TableBuilder data. Retrieved from https://www.abs.gov.au/websitedbs/censushome.nsf/home/tablebuilder .id. (2019). National economic indicators series. Retrieved from https://economic-indicators.id.com.au Australian Bureau of Statistics. (2016). Census of Population and HousingFindings based on use of ABS TableBuilder data. Retrieved from https://www.abs.gov.au/websitedbs/censushome.nsf/home/tablebuilder Final 25 November 2020 38 Final 25 November 2020 Australian Bureau of Statistics. (2018a). 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Home – School of Communication and Creative Arts. Retrieved from https://www.deakin.edu.au/communication-creative-arts Deakin University. (2019). Home – School of Communication and Cre https://www.deakin.edu.au/communication-creative-arts Doran, M. (2020). News Corp to cut jobs in restructure towards digital-only community and regional newspapers, viewed Retrieved from https://www.abc.net.au/news/2020-05-28/news-corp-to-cut-jobs-in-restructure- towards-digital-newspapers/12294970 Doran, M. (2020). News Corp to cut jobs in restructure towards digital-only community and regional newspapers, viewed Retrieved from https://www.abc.net.au/news/2020-05-28/news-corp-to-cut-jobs-in-restructure- towards-digital-newspapers/12294970 G21. (2019). The G21 Arts, Heritage & Culture Pillar. Retrieved from Geelong https://www.g21.com.au/arts- heritage-culture-pillar G21. (2019). The G21 Arts, Heritage & Culture Pillar. Retrieved from Geelong https://www.g21.com.au/arts- heritage-culture-pillar Gee, M. (2020). Australian Media Guide. Retrieved from https://connectweb-com- au.ezproxy.newcastle.edu.au/view-media-guide.aspx?oid=5353&eid=5353 Gee, M. (2020). Australian Media Guide. Retrieved from https://connectweb-com- au.ezproxy.newcastle.edu.au/view-media-guide.aspx?oid=5353&eid=5353 39 Final 25 November 2020 Geelong City Council. (2020). Creative Communities Grant Program. Retrieved from https://www.geelongaustralia.com.au/grants/article/item/8d38f990f9c3be4.aspx Geelong Gallery. (2019). Annual Report Retrieved from Geelong https://www.geelonggallery.org.au/cms_uploads/docs/gg Geelong Gallery. (2019). Annual Report Retrieved from Geelong https://www.geelonggallery.org.au/cms_uploads/docs/gg15538_2018-19annualrepo Geelong Independent. (2019 ). Wool museum to bring dino might to national awards. Geelong Independent. Retrieved from https://geelongindy.com.au/news/24-04-2019/wool-museum-to-bring-dino-might-to- national-awards/ Geelong Regional Libraries. (2019). 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Retrieved from http://peterwinklerarchitect.com.au/about/ Appendix A Census data Appendix A.1 Creative employment: counts, growth rates, intensities and heat maps Appendix A.2 Creative employment earnings: totals, growth rates, intensities and heat maps Appendix A.3 Creative employment incomes: mean incomes, growth rates, comparative ratios and heat maps Appendix A.4 Creative trident employment heat maps by sector Appendix A.5 Creative ANZSIC4 industry hotspot comparison by state Appendix A.6 Creative ANZSCO4 occupation hotspot comparison by state Appendix A.7 Employment and business comparison Appendix A.8 Cultural grant funding by government and type Appendices Data tables and heat maps are available via the following hyperlinks: d_Regional_Arts_Centre.pdf Paper presented at the Safe Cities Conference, Melbourne. Gray, F., & Novacevski, M. (2017). Zombie urbanism and the city by the bay: What’s really eating Geelong? Journal of Urban Cultural Studies, 4(3), 309–332. Greater City of Geelong. (2017). Live Music Action Plan - Central Geelong. Retrieved from https://www.geelongaustralia.com.au/arts/documents/item/8d505b20302c7ae.aspx Groves, D. (2019). Luke Saliba, Tessa de Josselin and Gary Sweet headline ‘Paper Champions’. IF Magazine. Retrieved from https://www.if.com.au/luke-saliba-tessa-de-josselin-and-gary-sweet-headline-paper- champions/ Holden, K. (2016, October 1–7,). Behind the scenes with Back to Back Theatre. The Saturday Paper, . Retrieved from https://www.thesaturdaypaper.com.au/2016/10/01/behind-the-scenes-with-back-back- theatre/14752440003793 Holden, K. (2016, October 1–7,). Behind the scenes with Back to Back Theatre. The Saturday Paper, . Retrieved from https://www.thesaturdaypaper.com.au/2016/10/01/behind-the-scenes-with-back-back- theatre/14752440003793 Houzz. (2019). Geelong, Victoria Artists & Artisans. Retrieved from https://www.houzz.com.au/professionals/artists-and-artisans/c/Geelong--Victoria Launch Vic. (2020). Round 1 Funding. Retrieved from https://launchvic.org/rounds/round- LiveTiles. (2019). LiveTiles to open its APAC HQ in Geelong, Victoria. Retrieved from https://www.livetiles.nyc/livetiles-open-apac-hq-geelong-victoria Media Entertainment and Arts Alliance. (2020). Standing up for regional and community media. Retrieved from https://www.meaa.org/news/take-action-to-reverse-the-closure-of-regional-newspapers/ Millar, R. (2007). Barwon Heads bridge wins its own sea change. The Age. Retrieved from https://www.theage.com.au/national/barwon-heads-bridge-wins-its-own-sea-change-20070316- ge4fo9.html Millar, R. (2007). Barwon Heads bridge wins its own sea change. The Age. Retrieved from https://www.theage.com.au/national/barwon-heads-bridge-wins-its-own-sea-change-20070316- ge4fo9.html Mockett, C. (2019). Drop of a Hat Publishing Geelong Community Directory. Retrieved from https://www.geelongaustralia.com.au/directory/item/643.aspx National Wool Museum. (2020). Exhibtions. Retrieved from https://www.geelongaustralia.com.au/nwm/exhibitions/article/item/8cc98f1d2801625.aspx National Wool Museum. (2020). Exhibtions. Retrieved from https://www.geelongaustralia.com.au/nwm/exhibitions/article/item/8cc98f1d2801625.aspx Pace. (2019). Home. Retrieved from http://www.pace.com.au/ Pace. (2019). Home. Retrieved from http://www.pace.com.au/ Pelligra. (2019, 17 May 2019). Pelligra confirmed as buyer for Ford’s iconic manufacturing site https://www.pelligra.com/pelligra-ford/ Pobjie, B. (2019, June 11). These Aussie ‘retail wage slaves’ hit pay dirt with underdog show. Sydney Morning Herald. Retrieved from https://www.smh.com.au/entertainment/tv-and-radio/will-success-spoil-the-diy- sitcom-about-wage-slaves-in-retail-hell-20190610-p51wah.html Pobjie, B. (2019, June 11). These Aussie ‘retail wage slaves’ hit pay dirt with underdog show. Sydney Morning Herald. Retrieved from https://www.smh.com.au/entertainment/tv-and-radio/will-success-spoil-the-diy- sitcom-about-wage-slaves-in-retail-hell-20190610-p51wah.html Final 25 November 2020 40 Potter, B. (2018). Carbon Revolution inks Ferrari deal, mulls $100m IPO. Financial Review. Retrieved from https://www.afr.com/politics/carbon-revolution-inks-ferrari-deal-mulls-100m-ipo-20180307-h0x48r Regional Australia Institute. (2014). Foundations of Regional Australia. Retrieved from http://www.regionalaustralia.org.au/wp-content/uploads/2014/12/Foundations-of-regional-Australia- FINAL-.pdf Renew Geelong. (2018). Renew Projects: Renew Geelong. Retrieved from https://www.renewaustralia.org/project/renew-geelong Renew Geelong. (2018). Renew Projects: Renew Geelong. Retrieved from https://www.renewaustralia.org/project/renew-geelong Robot Army Productions (Writer). (2014). Geelong Reinvented. In. Australia: Geelong: Tourism Greater Geelong and the Bellarine,. Robot Army Productions (Writer). (2014). Geelong Reinvented. In. Australia: Geelong: Tourism Greater Geelong and the Bellarine,. Rodgers, J. (2015). Appendix B Australian Business Register data Appendix B.1 Creative businesses: counts, growth rates, intensities and heat maps (forthcoming) online Final 25 November 2020 Final 25 November 2020 41